Molecular Identification and Characterization of the ... - Plant Physiology

Molecular Identification and Characterization of the Arabidopsis D3,5,D2,4-Dienoyl-Coenzyme A Isomerase, a Peroxisomal Enzyme Participating in the b-Oxidation Cycle of Unsaturated Fatty Acids1 Simon Goepfert, Charles Vidoudez, Enea Rezzonico2, J. Kalervo Hiltunen, and Yves Poirier* De´partement de Biologie Mole´culaire Ve´ge´tale, Baˆtiment de Biologie, Universite´ de Lausanne, CH–1015 Lausanne, Switzerland (S.G., C.V., E.R., Y.P.); and Biocenter Oulu and Department of Biochemistry, University of Oulu, FIN–90570 Oulu, Finland (J.K.H.)

Degradation of unsaturated fatty acids through the peroxisomal b-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core b-oxidation cycle. The auxiliary enzyme D3,5,D2,4-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) D3,5,D2,4-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have D3,5,D2,4-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the b-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the D3,5,D2,4-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has D3,5,D2,4-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.

Catabolism of fatty acids occurs primarily through the b-oxidation cycle. In plants, b-oxidation is located in peroxisomes, while in animal cells, it occurs in both peroxisomes and mitochondria (for review, see Gerhardt, 1992; Graham and Eastmond, 2002; Hooks, 2002). b-Oxidation is of primary importance for seedling establishment following germination since it allows the breakdown of fatty acids stored in triacylglycerides into acetyl-CoA, which is subsequently converted to Glc via the glyoxylate cycle and gluconeogenesis (Hayashi et al., 1998; Germain et al., 2001). Although b-oxidation is very active during germination, this cycle is also present in mature photosynthetic tissues, as well as in developing seeds (Poirier et al., 1999; Graham and Eastmond, 2002).

1 This work was supported by the Etat de Vaud, the Universite´ de Lausanne, and grants from the Fonds National Suisse (grant no. 3100A0–105874) and the Herbette Foundation (to Y.P.), and by the Academy of Finland and the Sigrid Juse´lius Foundation (to J.K.H.). 2 Present address: Nutrition and Health Department, Functional Microbiology Group, Nestle´ Research Center, P.O. Box 44, CH–1000 Lausanne 26, Switzerland. * Corresponding author; e-mail [email protected]; fax 41–21– 692–4195. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.064311.

The peroxisomal core b-oxidation cycle in plants is composed of four enzymatic activities located on three proteins. The first enzyme is acyl-CoA oxidase, converting acyl-CoA into 2-trans-enoyl-CoA. The second is a multifunctional enzyme that harbors two activities in a single polypeptide, namely, an enoyl-CoA hydratase and a 3-hydroxyacyl-CoA dehydrogenase, catalyzing the successive conversion of 2-trans-enoyl-CoA into 3-hydroxyacyl-CoA and 3-ketoacyl-CoA, respectively. The final enzyme is the 3-ketothiolase and is responsible for cleavage of 3-ketoacyl-CoA to form acetyl-CoA and an acyl-CoA that is two carbons shorter and that can reenter the b-oxidation spiral. In Arabidopsis (Arabidopsis thaliana), at least four genes have been characterized to encode for acyl-CoA oxidases with different chain-length specificities, two genes encode for multifunctional enzymes, and four genes encode 3-ketothiolases (Graham and Eastmond, 2002). In contrast to the degradation of saturated fatty acids, which can be mediated completely by the four enzymatic activities of the core b-oxidation cycle, the degradation of several types of unsaturated fatty acids has been shown to require the presence of auxiliary enzymes (Kunau et al., 1995; Hiltunen et al., 2003; van Roermund et al., 2003). This is because the core b-oxidation functions through a 2-trans-enoyl-CoA intermediate while many fatty acids have unsaturated bonds either on an odd-numbered carbon or in the

Plant Physiology, August 2005, Vol. 138, pp. 1947–1956, www.plantphysiol.org Ó 2005 American Society of Plant Biologists

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cis-configuration, leading to the generation of enoyl-CoA intermediates that cannot be directly metabolized via the core b-oxidation enzymes. Three auxiliary enzymes have thus been described to be involved in the metabolism of unsaturated fatty acids. The D3,D2-enoyl-CoA isomerase converts a 3-cis- or 3-trans-enoyl-CoA into 2-trans-enoyl-CoA. In many organisms, a D3,D2-enoylCoA isomerase activity has been found to be associated with the multifunctional protein of the core b-oxidation cycle as well as with monofunctional enzymes. The 2,4dienoyl-CoA reductase catalyzes, in eukaryotes, the conversion of either 2-trans,4-cis-dienoyl or 2-trans,4trans-dienoyl-CoA to 3-trans-enoyl-CoA. The D3,D2enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase have been found to be essential in yeast (Saccharomyces cerevisiae) for the metabolism of fatty acids having cis-double bonds on even-chain carbons, such as petroselinic acid (6-cis-octadecenoic acid), catalyzing the stepwise conversion of the intermediate 2-trans,4-cisdienoyl-CoA to 3-trans-enoyl-CoA and 2-trans-enoylCoA, the latter intermediate being a normal substrate for the core b-oxidation cycle. Both D3,D2-enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase activities have been detected in plants, but the corresponding genes have not been characterized (Behrends et al., 1988; Engeland and Kindl, 1991). The third auxiliary enzyme is the D3,5,D2,4-dienoylCoA isomerase catalyzing the conversion of 3,5-dienoyl-CoA to 2,4-dienoyl-CoA. This enzyme is thought to contribute to the degradation of conjugated fatty acids, such as 9-cis-11-trans-octadecadienoic acid, in animal cells (Ren and Schulz, 2003). It also has been implicated in one of the alternative pathways for the degradation of fatty acids with cis-double bonds on odd-numbered carbons (Shoukry and Schultz, 1998; Gurvitz et al., 1999). For example, degradation of oleic acid (9-cis-octadecenoic acid) generates the intermediate 2-trans,5-cis-dienoyl-CoA, which can be metabolized to the conjugated intermediate 3-trans,5cis-dienoyl-CoA by the D3,D2-enoyl-CoA isomerase, and subsequently converted to 2-trans,4-trans-dienoylCoA by the D3,5,D2,4-dienoyl-CoA isomerase. This latter intermediate is further metabolized to 2-transenoyl-CoA by the combined action of the 2,4-dienoylCoA reductase and the D3,D2-enoyl-CoA isomerase. Although the D3,5,D2,4-dienoyl-CoA isomerase has been characterized and the corresponding gene cloned in yeast and mammals, little information is available on this enzyme in plants at the biochemical and molecular level. In this work, we present the identification and the molecular characterization of the D3,5,D2,4-dienoyl-CoA isomerase from Arabidopsis. RESULTS Identification of an Arabidopsis Gene Coding for a Putative D3,5,D2,4-Dienoyl-CoA Isomerase

The search for a D3,5,D2,4-dienoyl-CoA isomerase in Arabidopsis was first performed through homology 1948

searches using the BLASTP program available on The Arabidopsis Information Resource (TAIR) database (www.arabidopsis.org) with protein sequences for the D3,5,D2,4-dienoyl-CoA isomerase from yeast and rat (Rattus norvegicus). The Arabidopsis genome contained only one protein of unknown function, encoded by At4g16210, showing low, but significant, homology to the yeast Dci1p (E 5 2 3 1026). In contrast, analysis with the rat protein sequence revealed the existence of seven Arabidopsis proteins with E-values higher than 5 3 10210. These proteins are, in decreasing order, At5g43280 (E 5 1 3 10260), At4g16800 (E 5 1 3 10219), At4g16210 (E 5 3 3 10216), At1g60550 (E 5 1 3 10212), At3g06860 (E 5 2 3 10211), At2g30650 (E 5 3 3 10211), and At4g29010 (E 5 8 3 10210). All these proteins contain regions with homology to the enoyl-CoA hydratase/isomerase fingerprint (Interpro domain IPR001753; www.ebi.ac.uk/InterProScan). Genes At3g06860 and At4g29010 encode the previously characterized peroxisomal multifunctional enzymes, while At2g30650 codes for a peroxisomal hydroxybutyryl-CoA hydratase (Richmond and Bleecker, 1999; Eastmond and Graham, 2000; Zolman et al., 2001). Of the remaining proteins, only At4g16210 and At5g43280 possess a peroxisomal targeting sequence (PTS), namely a PTS1 at the C terminus, indicating their potential localization in the peroxisome, and only At5g43280 showed correct alignment with the rat D3,5,D2,4-dienoyl-CoA isomerase with regard to the Asp and Glu residues defined to be important for catalytic activity (Modis et al., 1998; Zhang et al., 2001; Fig. 1). The protein encoded by At5g43280 was thus assigned the name AtDCI1 and analyzed in further detail. AtDCI1 encodes a protein of 278 amino acids with a predicted pI of 7.6 and a calculated Mr of 29,920. Figure 1 shows the alignment of the Arabidopsis protein with human (Homo sapiens), rat, and yeast D3,5,D2,4dienoyl-CoA isomerases. Based on the Blosum62 matrix, AtDCI1 exhibits 39%, 37%, and 18% amino acid identity to rat, human, and yeast D3,5,D2,4-dienoylCoA isomerases, respectively. The Arabidopsis protein is thus more closely related to the mammalian enzyme than to the yeast counterpart. AtDCI1 shares with other D3,5,D2,4-dienoyl-CoA isomerases the hydratase/ isomerase fingerprint found between amino acids 118 and 138, but does not contain a predictable mitochondrial transit peptide as found in the mammalian homologs. Localization of Enhanced Yellow Fluorescent Protein Fusion Protein

The protein AtDCI1 contains the tripeptide Ala-LysLeu at the carboxy end, a major PTS1 in plants conforming to the consensus sequence Ser-Lys-Leu (Reumann et al., 2004). In order to confirm the peroxisomal nature of AtDCI1, the localization of a fusion protein between an enhanced yellow fluorescent protein (EYFP) at the N terminus and AtDCI1 protein at the C terminus was analyzed by a transient Plant Physiol. Vol. 138, 2005

Peroxisomal-Dienoyl-CoA Isomerase from Arabidopsis

Figure 1. Sequence alignment of the protein sequence from At5g43280 (AtDCI1) with the D2,4,D3,5 dienoyl-CoA isomerase from human (HsECH1), rat (RnECH1), and yeast (ScDci1). Amino acids important for the catalytic activity of the rat D2,4,D3,5 dienoylCoA isomerase are indicated by a star. Peroxisome targeting sequences for all proteins are underlined, and mitochondrial signal peptides for the human and rat proteins are boxed (shading has been omitted). Shading threshold for identity/similarity is 50%.

transformation assay of onion (Allium cepa) epidermal cells with the corresponding gene fusion. As a control, a second plasmid carrying a fusion gene between an enhanced cyan fluorescent protein (ECFP) and the peroxisomal malate dehydrogenase (MDH) from cucumber (Cucumis sativus) was used. The EYFP:AtDCI1 and ECFP:MDH gene constructs were transformed either individually or together in onion epidermal cells and the fluorescence was examined by confocal microscopy after 12 h. Control experiments have determined that fluorescence of the EYFP and ECFP could be detected without cross-interference when expressed in the same cells (data not shown). Cells expressing the EYFP:AtDCI1 have a punctate fluorescence pattern that was expected for proteins located in the peroxisomes (Fig. 2A). In cells cotransformed with the EYFP:AtDCI1 and ECFP:MDH gene constructs, the fluorescence pattern observed with the EYFP:AtDCI1 matched precisely the pattern observed with the ECFP:MDH, revealing that AtDCI1 is a peroxisomal protein (Fig. 2, B and C). Bacterial Expression and Enzymatic Assay of the Recombinant Protein

The ability of the AtDCI1 protein to catalyze the conversion of D3,5-dienoyl-CoA to D2,4-dienoyl-CoA in an in vitro assay was tested. The AtDCI1 gene was modified to add a His-tag at the N terminus of the protein and the final construct cloned in an Escherichia coli vector allowing gene expression and protein synthesis under the control of a T7 promoter (see ‘‘Materials and Methods’’). Enzyme assays were performed either with crude extracts from cells expressing Plant Physiol. Vol. 138, 2005

AtDCI1, with AtDCI1 protein purified from the bacterial extract on a nickel affinity column, or from crude extracts from cells transformed with the expression plasmid without the AtDCI1 gene (negative control plasmid). Isomerization of a D3,5-dienoyl-CoA to a D2,4-dienoylCoA can be followed by monitoring changes of the UV absorption spectrum (Filppula et al., 1998; Zhang et al., 2001). The D3,5-dienoyl-CoA has a maximum absorption peak at 240 nm, whereas the D2,4-dienoyl-CoA has a maximum absorption peak at 260 nm and a shoulder peak at 300 nm. The difference of absorption at 300 nm is thus used to monitor the formation of the product of the D3,5,D2,4 isomerization reaction. A diagram showing the in vitro reaction cascade used is presented in Figure 3A. Synthesis of the substrate for the isomerization assay is achieved from the reaction of arachidonoyl-CoA (5,8,11,14-eicosatetraenoyl-CoA) with the yeast acyl-CoA oxidase, which also possesses an intrinsic D3,D2-enoyl-CoA isomerase activity, resulting in the formation of 3,5,8,11,14-eicosapentaenoyl-CoA. This reaction can be followed by an increase in the absorption at 240 nm (Filppula et al., 1998). Enzyme assays with crude extracts from bacteria expressing the AtDCI1 protein were compared to extract from bacteria transformed with the negative control plasmid and grown under the same conditions. The conversion of 5,8,11,14-eicosatetraenoyl-CoA to 2,4,8,11,14-eicosapentaenoyl-CoA via the isomerization of the intermediate 3,5,8,11,14-eicosapentaenoyl-CoAwas shown to be strictly dependent on both the expression of the AtDCI1 protein in E. coli (Fig. 3B, comparison of the black and white circles) and the prior incubation of the starting substrate 5,8,11,14-eicosatetraenoyl-CoA 1949

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D3,5,D2,4-dienoyl-CoA isomerase (Dci1p), and expressing a bacterial polyhydroxyalkanoate (PHA) synthase targeted to the peroxisome. Synthesis of PHA in the peroxisome of yeast and Arabidopsis has previously been shown to be a valuable tool to monitor the in

Figure 2. Subcellular localization of EYFP:AtDCI1 fusion protein in onion epidermal cells. Confocal microscopy projections of onion cells transiently coexpressing the fusion proteins EYFP:AtDCI and ECFP:MDH (glyoxisomal MDH). A, Fluorescence acquired in yellow channel (EYFP). B, Fluorescence acquired in the cyan channel (ECFP). C, Overlay of A and B. D, Light transmission image. Bar 5 10 mm.

with the yeast acyl-CoA oxidase (Fig. 3B, white diamonds). No increase in A300 was observed if extracts from bacteria transformed with the negative control plasmid were used or if the acyl-CoA oxidase was omitted from the reaction cascade. The reaction cascade from 5,8,11,14-eicosatetraenoyl-CoA to 2,4,8,11,14-eicosapentaenoyl-CoA was also monitored by the 220- to 340-nm absorbance spectra (Fig. 3C). Thus, the conversion of 5,8,11,14-eicosatetraenoyl-CoA to 3,5,8,11,14-eicosapentaenoyl-CoA by the yeast acylCoA oxidase and its intrinsic D3,D2-enoyl-CoA isomerase activity is shown by the increase in A250 (Fig. 3C, white square and black diamond), while the isomerization of 3,5,8,11,14-eicosapentaenoyl-CoA to 2,4,8,11,14-eicosapentaenoyl-CoA was followed by the dramatic increase in A300 (Fig. 3C, white triangle). The specific activity of the extracts measured in the linear range of activity using 100 mM arachidonoyl-CoA were 5.32 6 0.26 mmol min21 mg21 (n 5 3) for crude extracts and 16.63 6 6.72 mmol min21 mg21 (n 5 5) for nickel column affinity-purified fractions. The potential presence of a D3,D2-enoyl-CoA isomerase activity associated with the AtDCI1 protein was assayed as described by Palosaari et al. (1990) with crude and purified extracts. No such activities could be detected (data not shown). Complementation of the dci1D Mutant

The D3,5,D2,4-dienoyl-CoA isomerase activity of the AtDCI1 protein was tested in vivo through the expression of the plant protein in a yeast mutant having a deletion in the Dci1 gene (YOR180c), encoding the 1950

Figure 3. In vitro D3,5,D2,4-dienoyl-CoA isomerase activity of AtDCI1. A, Diagram of the enzymatic cascade used for detection of D2,4,D3,5 dienoyl-CoA isomerase activity. B, Kinetics of 5,8,11,14-eicosatetraenoyl-CoA conversion to 2,4,8,11,14-eicosapentaenoyl-CoA were monitored at 300 nm. White and black circles, 50 mM 5,8,11,14eicosatetraenoyl-CoA was preincubated for 10 min with 0.2 units of yeast acyl-CoA oxidase, and 12 mg of protein extract from control bacteria (black circles) or bacteria expressing AtDCI1 (white circles) was added at time point I (arrow I). White diamonds, addition to 50 mM 5,8,11,14-eicosatetraenoyl-CoA of 3.6 mg of crude extract from bacteria expressing AtDCI1 at time point I (arrow I) and 0.2 units of yeast acyl-CoA oxidase at time point II (arrow II). All baselines have been normalized to 0. C, UV absorption spectra of 50 mM 5,8,11,14eicosatetraenoyl-CoA at time 0 (white boxes), after 10-min incubation with 0.2 units of yeast acyl-CoA oxidase (black diamonds), followed by an additional 10-min incubation with 3.6 mg of crude extract from AtDCI1-expressing bacteria (white triangles). Plant Physiol. Vol. 138, 2005


vivo carbon flux through the b-oxidation pathway (Mittendorf et al., 1998, 1999; Allenbach and Poirier, 2000; Poirier et al., 2001). In this system, PHA is synthesized from the polymerization of 3-hydroxyacyl-CoA intermediates generated by the b-oxidation of fatty acids via the activity of a PHA synthase from Pseudomonas aeruginosa modified for peroxisomal targeting (Poirier, 2002). The D3,5,D2,4-dienoyl-CoA isomerase activity can be monitored in yeast synthesizing peroxisomal PHA from the b-oxidation of the fatty acid 10-cis,13-cisnonadecadienoic acid (H.M. Bogdawa, Y. Poirier, and S. Marchesini, unpublished data). Based on current knowledge of the activity of yeast auxiliary enzymes involved in b-oxidation of unsaturated fatty acids, degradation of 10-cis,13-cis-nonadecadienoic acid gives the intermediate 2-trans-5-cis-undecadienoylCoA (Gurvitz et al., 1999; Hiltunen et al., 2003). This intermediate can be further metabolized via several pathways that are distinguishable by the nature of the 11-carbon 3-hydroxyacyl-CoA generated. Thus, in one pathway independent of the Dci1p, the intermediate 3-hydroxy,5-cis-undecenoyl-CoA is generated, while the intermediate 3-hydroxyundecanoyl-CoA and 3-hydroxy,4-trans-undecenoyl-CoA are generated by two other pathways requiring the participation of the Dci1p (Fig. 4). Whereas the intermediates 3-hydroxy, 5-cis-undecenoyl-CoA and 3-hydroxyundecanoylCoA are substrates for the bacterial PHA synthase, 3-hydroxyacyl-CoAs with an unsaturated bond on the fourth carbon, such as 3-hydroxy,4-trans-undecenoylCoA, are known to be very poor substrates for the PHA synthase (Mittendorf et al., 1998, 1999; Robert et al., 2005). Thus, the monomer 3-hydroxy,5-cisundecenoic acid and 3-hydroxyundecanoic acid in

PHA synthesized from cells degrading the fatty acid 10-cis,13-cis-nonadecadienoic acid are markers of the carbon flux through the pathways that are independent or dependent, respectively, of the D3,5,D2,4-dienoylCoA isomerase. The gas chromatography-mass spectrometry profiles of the 11-carbon monomers present in PHA synthesized in yeast cells grown in media containing 10-cis,13-cis nonadecedienoic acid are shown in Figure 5. Hydroxyacid monomers present in PHA are identified with the prefix H, followed by the number of carbons and the number of unsaturated bonds. While both the H11:1 and H11:0 monomers are present in wild-type cells, only the H11:1 monomer is detectable in the dci1D mutant, thus confirming that the generation of the intermediate 3-hydroxyundecanoyl-CoA is dependent on the presence of the D3,5,D2,4-dienoyl-CoA isomerase encoded by Dci1 (Fig. 5, A and B). Expression of the Arabidopsis AtDCI1 gene in the dci1D strain resulted in a PHA containing both the H11:1 and H11:0 monomers (Fig. 5C), thus revealing that AtDCI1 possesses D3,5,D2,4-dienoyl-CoA isomerase activity in vivo. Expression Pattern of AtDCI1

The temporal and spatial expression profile of AtDCI1 was determined by northern-blot analysis. Transcript level of AtDCI1 in whole seedlings strongly increases upon germination, decreasing slightly from day 2 to day 4, and increasing again at day 5 and day 7 (Fig. 6A). A similar pattern of expression was also observed for the AtAIM1 gene encoding the peroxisomal multifunctional enzyme, with the exception of the high expression level of AtAIM1 present in hydrated seeds at 4°C (0 d after imbibition [DAI];

Figure 4. Degradation pathway of 10-cis,13-cis-nonadecadienoic acid in yeast. The alternative pathways involved in the metabolism of the intermediate 2-trans,5-cis-undecadienoyl-CoA are highlighted. I, Conversion of 10-cis,13-cis-nonadecadienoic acid to the intermediate 2-trans,5-cis-undecadienoyl-CoA requires three turns through the core b-oxidation cycle to generate 2-trans,4-cis,7-cis-tridecatrienoyl-CoA, followed by the successive action of the D3,D2-enoyl-CoA isomerase and 2,4dienoyl-CoA reductase leading to the intermediate 2-trans,7-cis-tridecadienoic acid, which then undergoes one more cycle of b-oxidation. II, Action of the enoyl-CoA hydratase activity of the Fox2p multifunctional enzyme on enoyl-CoAs to generate various 3-hydroxyacyl-CoAs of 11 carbons. The nomenclature used is position and conformation of the double bond (t for trans and c for cis) or position of the hydroxy group (OH), followed by the number of carbons of the aliphatic chain of the CoA ester (thus 2t,5c-C11 is 2-trans,5-cis-undecadienoyl-CoA and 3OH,5c-C11 is 3-hydroxy,5-cis-undecenoyl-CoA). The 11-carbon substrates for the PHA synthase are underlined. Eci1p, D3,D2-enoyl-CoA isomerase; Dci1p, D3,5,D2,4-dienoyl-CoA isomerase; Sps19p, 2,4-dienoyl-CoA reductase. Plant Physiol. Vol. 138, 2005

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sion does not appear to contain any targeting information to a subcellular compartment (data not shown).

DISCUSSION

Searches of the Arabidopsis genome for proteins with homology to the mammalian D3,5,D2,4-dienoyl-CoA isomerase gave two potential candidate genes coding

Figure 5. Complementation of the yeast dci1D mutant with AtDCI1. Gas chromatography-mass spectrometry spectra (m/z 103) of the monomers 3-hydroxyundecanoic acid (H11:0) and 3-hydroxy,5-cisundecenoic acid (H11:1) present in PHA synthesized in yeast wild type (A), dci1D mutant (B), and dci1D mutant expressing AtDCI1 (C) and grown in media containing 0.05% 10-cis,13-cis-nonadecadienoic acid are shown.

Richmond and Bleecker, 1999). The AtDCI1 gene appears to be expressed at a similar level in green leaves, senescing leaves, flowers, and floral stems, but at lower level in roots. Identification of Plant Homologs of AtDCI1

The sequences available in public databases were used to identify homologs to AtDCI1 in a large spectrum of plants, including both a gymnosperm (Pinus taeda) and several angiosperms belonging to the Monocotyledon (Oryza sativa, Zea mays) or Dicotyledon (Nicotiana benthamiana, Lycopersicon esculentum, Citrus sinesis, Medicago truncatula) classes. An alignment of the sequences revealed a high level of amino acid conservation distributed throughout the protein with the exception of a region of low homology corresponding to amino acids 74 to 93 in AtDCI1 (Fig. 7). Furthermore, DCI1 from monocots have an additional 12- to 14-amino acid extension at the N terminus compared to dicots. This N-terminal exten1952

Figure 6. Expression pattern of AtDCI1. A, Total RNA of entire seedlings was extracted 0, 1, 2, 3, 4, 5, and 7 DAI. Membrane was hybridized with probes for AtDCI1 and AtAIM1. Graph represents the relative intensity of the autoradiograph signals over the signals from the UV/ethidium bromide RNA. B, Plants were grown in soil for 7 weeks. Total RNA was extracted from whole plant (P); flower buds (F); senescing leaves (sL; approximately 30% to 50% chlorotic leaf); green leaves (gL); stems (St); and roots (R). Membrane was hybridized with a probe for AtDCI1. Histogram presented is the relative intensity of the autoradiograph signals over the signals from the UV/ethidium bromide RNA. Plant Physiol. Vol. 138, 2005


Figure 7. Sequence alignment of AtDCI1 with homologs in other plants. At, Arabidopsis; Pt, Pinus taeda; Nb, Nicotiana benthamiana; Le, Lycopersicon esculentum; Cs, Citrus sinenis; Mt, Medicago truncatula; Os, Oryza sativa cv japonica; Zm, Zea mays. Stars, Putative important residues for catalytic activity by similarity to rat dienoyl-CoA isomerase. Shading threshold for identity/similarity is 75%.

for previously uncharacterized proteins, namely At5g43280 and At4g16210. These proteins had two characteristics expected to be present in a plant D3,5,D2,4-dienoyl-CoA isomerase, namely the presence of an enoyl-CoA hydratase/isomerase fingerprint and the existence of a peroxisomal targeting peptide sequence. Alignment of these proteic sequences with the rat D3,5,D2,4-dienoyl-CoA isomerase revealed that only At5g43280 contained the conserved aspartic and glutamic residues shown to be important for catalytic activity of the rat enzyme (Modis et al., 1998; Zhang et al., 2001). Analysis of the enzymatic activity of the protein encoded by At5g43280 was first performed by an in vitro assay via the expression of the plant protein in E. coli, a host that does not contain an endogenous D3,5,D2,4-dienoyl-CoA isomerase. D3,5,D2,4-Dienoyl-CoA isomerase activity was clearly detected in both the crude extracts of bacteria expressing At5g43280 as well as with purified protein from the same extracts, while extracts from bacteria transformed with negative control vector were devoid of the same activity. Proof that At5g43280 encodes a protein with a D3,5,D2,4-dienoyl-CoA isomerase activity in vivo was provided by the complementation of the yeast dci1D mutant. Previous studies showed that the dci1D mutant, although deficient in D3,5,D2,4-dienoyl-CoA isomerase activity, could grow as well as wild type in media containing unsaturated fatty acid and was essentially symptomless (Gurvitz et al., 1999). Complementation of the dci1D mutant was thus assayed by a method relying on the synthesis of PHA from the polymerization of the b-oxidation intermediates 3-hydroxyacylCoAs by a bacterial peroxisome-targeted PHA synthase. Plant Physiol. Vol. 138, 2005

Although synthesis of PHA in plant and yeast peroxisomes can be employed for the production of biodegradable polymers with plastics properties, this system has also been successfully exploited to learn fundamental aspects of fatty acid metabolism in the peroxisome, including the pathways involved in the degradation of unsaturated fatty acids (Allenbach and Poirier, 2000). In a recent study aimed at understanding the contribution of the yeast Dci1p protein to the degradation of 10-cis,13-cis-nonadecadienoic acid, it was demonstrated that the pathway requiring the participation of Dci1p for the conversion of 3-trans,5cis-undecadienoyl-CoA to 2-trans,4-trans-undecadienoyl-CoA could be distinguished by the generation of the b-oxidation intermediate 3-hydroxyundecanoylCoA, which is itself a substrate for PHA synthase (H.M. Bogdawa, S. Marchesini, and Y. Poirier, unpublished data). Thus, while wild-type yeast cells expressing a peroxisomal PHA synthase and grown on 10-cis,13-cis-nonadecadienoic acid produce a PHA containing both the monomers H11:1 and H11:0, the isogenic dci1D strain synthesized a PHA containing the H11:1 monomer, but not the H11:0 monomer (Fig. 4). Functional complementation of the dci1D mutant with the At5g43280 gene was shown by the presence of the H11:0 monomer in PHA. Based on the demonstration of D3,5,D2,4-dienoyl-CoA isomerase activity by in vitro and in vivo assays, At5g43280 was thus named AtDCI1. AtDCI1 terminates at the carboxy terminus with the tripeptide Ala-Lys-Leu, which has been defined as major PTS1 for plants (Reumann, 2004). Since the presence of a PTS1-like C-terminal tripeptide can also be 1953

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found in nonperoxisomal proteins, we confirmed the peroxisomal localization of AtDCI1 experimentally. This was achieved by showing that a fusion protein between the EYFP and AtDCI1 was colocalized with the fusion protein between the peroxisomal MDH and the ECFP. Furthermore, complementation of the yeast dci1D mutant with AtDCI1 showed that the plant protein was adequately targeted to the peroxisomes in yeast, since b-oxidation in this host occurs exclusively in the peroxisomes. Such cross-species targeting is not unexpected, as peroxisomal targeting sequences are known to be generally well conserved between insect, mammals, fungi, and plants (Gould et al., 1990; Subramani, 1993). The mammalian D3,5,D2,4-dienoylCoA isomerase contains both a C-terminal peroxisomal targeting signal and an N-terminal mitochondrial targeting signal, and the protein was shown to be present in both organelles (Filppula et al., 1998). In contrast, no evidence of an N-terminal mitochondrial targeting sequence was found on AtDCI1. These results are in agreement with the plant b-oxidation occurring primarily, if not exclusively, in peroxisomes, while in animal cells the cycle occurs in both mitochondria and peroxisomes. AtDCI1 shows greater homology to the mammalian D3,5,D2,4-dienoyl-CoA isomerases than to the yeast counterpart, both at the whole protein level, as well as for the aspartic and glutamic residues found to be important for catalysis in the rat D3,5,D2,4-dienoyl-CoA isomerase (Modis et al., 1998; Zhang et al., 2001). The divergence of the yeast D3,5,D2,4-dienoyl-CoA isomerase to the mammalian homologs has been noted in previous studies (Gurvitz et al., 1999). Another distinctive feature of the fungi D3,5,D2,4-dienoyl-CoA isomerase is the presence of a D3,D2-enoyl-CoA isomerase activity within the protein, while no such activity has been found with the mammalian enzymes (Gurvitz et al., 1999; Zhang et al., 2001). No D3,D2-enoyl-CoA isomerase activity could be detected associated to AtDCI1 either with in vitro enzyme assays with purified protein or with in vivo assays via complementation of the yeast eci1D mutants (data not shown). BLAST searches identified another gene, namely At4g16210, encoding a protein with relatively weak homology to Dci1p of yeast. The same Arabidopsis protein also showed some weak homology to the rat D3,5,D2,4-dienoyl-CoA isomerase. This homology is largely based on the presence of an enoyl-CoA hydratase/isomerase fingerprint in all three proteins. The function of the protein encoded by At4g16210 is at present unknown. A recent analysis of putative peroxisomal proteins in Arabidopsis based on bioinformatic tools had tentatively grouped At5g43280 with the D3,5,D2,4-dienoyl-CoA isomerase clade, while At4g16210 was grouped in a different clade with other peroxisomal proteins of undefined function (Reumann et al., 2004). No functional complementation of the yeast dci1D or eci1D mutants was achieved through expression of the At4g16210 gene, indicating that, at least in yeast, the protein encoded by At4g16210 does 1954

not have a D3,5,D2,4-dienoyl-CoA isomerase or a D3,D2enoyl-CoA isomerase activity (data not shown). The AtDCI1 gene is expressed in a broad range of tissues, including various photosynthetic tissues (leaves, stems) and to a lower, but detectable, extent in roots, but is particularly enhanced during seed germination. Germination is a period of high b-oxidation requirement in oleaginous plants. The similarity between the expression pattern of AtDCI1 and that of other genes coding for peroxisomal proteins involved in b-oxidation is in accord with the involvement of AtDCI1 in fatty acid catabolism (Hooks et al., 1999; Eastmond and Graham, 2000). The physiological contributions of the D3,5,D2,4dienoyl-CoA isomerase to the degradation of unsaturated fatty acids in eukaryotes remains unclear in both yeast and mammals. Deletion of Dci1 in yeast was shown to be associated with no deficiencies in growth or utilization of oleic acid (Gurvitz et al., 1999). Furthermore, degradation of the conjugated fatty acid 9-cis-11-trans-octadecadienoic acid in yeast was recently shown to be largely mediated by a pathway that was independent of Dci1p (Bogdawa et al., 2005). Studies with mitochondrial extracts from rat heart revealed that the degradation of oleic acid was also largely mediated by a pathway not requiring the participation of the D3,5,D2,4-dienoyl-CoA isomerase (Ren and Schulz, 2003). Similar conclusions were also reached for the degradation of 10-cis,13-cis-nonadecadienoic acid in yeast (H.M. Bogdawa, S. Marchesini, and Y. Poirier, unpublished data). The apparent dispensable role of D3,5,D2,4-dienoylCoA isomerase in the degradation of unsaturated fatty acids in yeast and mammals appears paradoxical considering that the conservation of this enzyme across the microbial, plant, and animal kingdoms suggests a positive selective pressure. Proteins with high homology to AtDCI1 are found in a broad range of plants, including gymnosperms and angiosperms, suggesting an important physiological role for the D3,5,D2,4-dienoyl-CoA isomerase in plants. Several recent studies have revealed the involvement of b-oxidation enzymes into developmental processes and stress responses in plants (Eastmond and Graham, 2000; Rylott et al., 2003; Castillo et al., 2004). Thus, the study of D3,5,D2,4-dienoyl-CoA isomerase in Arabidopsis may provide novel valuable information on the physiological role of this enzyme.

MATERIALS AND METHODS Sequence Analysis Searches against the Arabidopsis (Arabidopsis thaliana) sequence database were done using the BLASTP algorithm available from the TAIR database (http://www.arabidopsis.org/Blast). The characterized D3,5,D2,4-dienoyl-CoA isomerases cited in this article are from yeast (Saccharomyces cerevisiae; ScDci1p; YOR180c; Geisbrecht et al., 1999; Gurvitz et al., 1999), rat (Rattus norvegicus; RnECH1; NP_072116; FitzPatrick et al., 1995), and humans (Homo sapiens; HsECH1; AAH17408; FitzPatrick et al., 1995). Protein sequences of the D3,5,D2,4-dienoyl-CoA isomerase from Arabidopsis, rat, human, and yeast were

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aligned using ClustalW (http://www.ebi.ac.uk/clustalw). Genes homologous to the Arabidopsis D3,5,D2,4-dienoyl-CoA isomerase in other plants were searched by TBLASTN using the AtDCI1 sequence and performed against Oryza sativa and Nicotiana benthamiana expressed sequence tag (EST) collections available on GenBank. The DCI1 in O. sativa and N. benthamiana are encoded by the EST XM_463670 and EST CK294507, respectively. For other plants, genes homologous to AtDCI1 were found by TBLASTN using sequences available on the plant genome database (http://www.plantgdb.org; Dong et al., 2004). Accession references for the corresponding EST or Genome Survey Sequence (GSS) contigs are Zea mays ZmGSStuc04-27-04.27328; Lycopersicon esculentum LeTUC02-10-21.12451; Citrus sinensis CsTUC04-08-17.4127; and Medicago truncatula MtTUC04-08-17.5308. For Pinus taeda, the deduced PtDCI1 gene was derived from the assembly of ESTs CF470462 and CF400188.

Bacterial Growth, Protein Purification, and Enzymatic Assays Escherichia coli DH5a was used to maintain and propagate all plasmids, according to Sambrook and Russell (2001), with the exception of Gateway native plasmids (pDEST17, pDONR207), which were maintained in E. coli DB3.1 (Invitrogen, Brenda, The Netherlands). For experiments aimed at protein expression in E. coli, the strain BL21 (DE3) pLysS was used. The bacterial expression vector pDEST17-AtDCI was constructed by Gateway recombination. High-fidelity (Roche, Rotkreuz, Switzerland) PCR was performed on pYE352-AtDCI (see yeast section) using the primers AttB1-DCI 5#-(AttB1)TAATGACAATGGAGTCATAC-3# and AttB2-DCI 5#-(AttB2)TCAAAGTTTAGCAAACCG-3#. The product was recombined in a BP reaction to the pDONR207 plasmid (Invitrogen), resulting in the plasmid pDONR207-AtDCI. This entry clone was finally LR recombined with pDEST17 (Invitrogen) to result in the pDEST17-AtDCI. Bacteria carrying this plasmid were grown to saturation in selective Luria-Bertani medium at 37°C. This starter culture was used to initiate a selective NCYZM medium culture. Cells were grown at 29°C to an OD600 of approximately 0.6, induced with 1 mM isopropylthiob-galactoside, and grown for 4 to 8 additional hours. Cells were collected by centrifugation and resuspended in sodium phosphate buffer (50 mM, pH 8.0), 0.3 M NaCl, 20% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride. Cells were lysed by four cycles of 10-s sonication followed by 30 s on ice. Lysates were centrifuged at 16,000g for 30 min at 4°C, and the supernatant was adjusted to 50% glycerol and stored at 220°C. His-tagged proteins were purified by nickel affinity chromatography with His-select nickel affinity gel (Sigma, St. Louis), according to the manufacturer’s instructions. Enzymatic assays were performed essentially as previously described (Gurvitz et al., 1999). Arachidonoyl-CoA and yeast acyl-CoA oxidase were purchased from Sigma and 100 mM and 0.2 units were used, respectively, per assay in a volume of 500 mL of buffer containing 25 mM Tris-HCl, pH 8.0, and 5 mM EDTA. Optical density measurements were made with an Ultrospec 2000 (Amersham Biosciences, Piscataway, NJ).

Microscopy The plasmids pCAT-ECFP-MDH and pCAT-EYFP-Not have previously been described (Fulda et al., 2002). In-frame fusion of AtDCI with the EYFP was achieved by amplifying AtDCI1 from pYE352-AtDCI with the primers dci-YFP-5# 5#-TAAGGGCCCTGACAATGGAGTCA-3# and dci-YFP-3# 5#-TAATCTAGAGTATCAAAGTTTAGCAAA-3#, restricting the fragment with Bsp120I and XbaI and inserting the fragment into pCAT-EYFP-Not digested with NotI and XbaI. Plasmids were precipitated on gold beads and transformed into the epidermis of fresh onions (Allium cepa) as previously described (Mueller et al., 1997). Tissues were left 12 h in the dark on solid medium containing half-strength Murashige and Skoog salts. Images were acquired with a Leica TCS SP2 confocal scanning microscope (Leica, Heidelberg). Wavelengths were set on appropriate parameters for YFP and CFP. Images were analyzed and combined using Leica confocal software. They represent the flat projection of images acquired at maximal frequency with a step size of 0.6 mm on depth of 22 mm; colors are artificial.

Yeast Strains, PHA Production, and Analysis Wild-type yeast strain BY4742 (mata his3D1 leu2D0 lys2D0 ura3D0) and the isogenic dci1D mutant (YOR180c::kanMX4) were obtained from EUROSCARF (www.uni-frankfurt.de/fb15/mikro/euroscarf/index.html). Plasmids were


transformed into yeast strains by the lithium acetate procedure (Gietz et al., 1992). Yeast strains were propagated on selective media composed of 0.67% yeast nitrogen base without amino acids (DIFCO Laboratories, Detroit), 0.5% ammonium sulfate, 2% Glc, and appropriate dropout supplement (CLONTECH, Palo Alto, CA). The yeast shuttle plasmid Yiplac111-PHA containing the PHAC1 synthase from Pseudomonas aeruginosa modified for peroxisomal targeting by the addition, at the carboxyl end of the protein, of the last 34 amino acids of the Brassica napus isocitrate lyase, has previously been described (Marchesini and Poirier, 2003). The shuttle plasmid pYE352-AtDCI was constructed by replacing the catalase A gene from pYE352-CTA (Filppula et al., 1995) by AtDCI. This was achieved by amplification of AtDCI from the cDNA clone AI996999 (Genome Systems, St. Louis) with high-fidelity polymerase (Roche) using the primers 5#-CACGCGAGCTCATCATCACTGT-3# and 5#-CAACCTCGAGACTGTATCAAAG-3#, followed by restriction with SacI and XhoI restriction enzymes. The resulting plasmid was verified by sequencing. For experiments analyzing PHA synthesis in cells growing in media containing 10-cis,13-cis-nonadecadienoic acid, a stationary phase culture of cells grown in selective media containing 2% Glc was harvested by centrifugation, cells were washed once in water, and resuspended at a 1:10 dilution in fresh selective media containing 0.1% (w/v) Glc, 2% Pluronic-127 (w/v; Sigma), and 0.05% (v/v) 10-cis,13-cis-nonadecadienoic acid. Cells were grown for an additional 3 to 4 d before harvesting them for PHA analysis as previously described (Poirier et al., 2001). 10-cis,13-cis-Nonadecadienoic acid was purchased from Nu-Check-Prep (Elysian, MN).

Plant Culture, RNA Extraction, and Northern-Blot Analysis Seeds of Arabidopsis, accession Columbia (Col-0), were sterilized and plated on media containing half-strength Murashige and Skoog salts, 1% Suc, and 0.8% agar. Plates were first placed at 4°C for 48 h before placing them under constant illumination (70 mE m22) at 21°C (defined as 0 DAI). After 15 DAI, plants were transplanted into soil under the same light and temperature conditions. Whole plants were collected from plates from 0 to 7 DAI. Green leaves, senescent leaves (30%–50% chlorotic), stems, and flower buds were collected from plants grown in pots 40 DAI. Roots were harvested 7 DAI on plants grown in liquid half-strength Murashige and Skoog medium supplemented with 2% Suc. Total RNA was extracted with hot borate protocol (Wan and Wilkins, 1994) for seedlings from 0 to 5 DAI and with the LiCl protocol (http://www2.unil.ch/ibpv/WWWPR/Docs/rna_preparation.htm) for all other samples. Northern blots were made with 20 mg of total RNA, according to Sambrook and Russell (2001). [32P]dCTP-labeled probes were made by random priming using the prime-a-gene labeling system (Promega, Madison, WI) and purified on the ProbeQant kit (Amersham Biosciences). Prehybridization and hybridization were carried out in sodium phosphate buffer, 25 mM, 7% SDS, 1% bovine serum albumin, 0.372 g L21 EDTA at 65°C. Washes were done at 65°C, twice for 10 min with 23 SSC, 0.1% SDS, then twice for 10 min in 13 SSC, 0.1% SDS. Autoradiographic signals were quantified using ImageJ software (http://rsb.info.nih.gov/ij). For ratio calculations, the autoradiographic signals were divided by the negative intensity of the ethidium bromide signal of the corresponding sample. Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes.

ACKNOWLEDGMENT We thank Martin Fulda (Washington State University) for providing the pCAT-EYFP-Not and pCAT-ECFP:MDH plasmids. Received April 18, 2005; revised May 26, 2005; accepted May 30, 2005; published July 22, 2005.

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