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Blackwell Publishing Ltd

Arabidopsis thaliana acyl-CoA-binding protein ACBP2 interacts with heavy-metal-binding farnesylated protein AtFP6

Wei Gao1, Shi Xiao1, Hong-Ye Li1, Sai-Wah Tsao2 and Mee-Len Chye1,3

1School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China; 2Department of Anatomy, Li Ka Shing Faculty of Medicine,

The University of Hong Kong, Pokfulam Road, Hong Kong, China; 3State (China) Key Laboratory of Agrobiotechnology, Chinese University of Hong Kong,

Shatin, Hong Kong, China

Summary

• Arabidopsis thaliana acyl-CoA-binding protein 2 (ACBP2) was observed to interactwith farnesylated protein 6 (AtFP6), which has a metal-binding motif (M/LXCXXC).Their interaction and expression in response to heavy metals were investigated.• Yeast two-hybrid analysis and in vitro assays showed that an ACBP2 derivativelacking ankyrin repeats did not interact with AtFP6, indicating that the ankyrinrepeats mediate protein–protein interaction. Autofluorescence-tagged ACBP2 andAtFP6 transiently co-expressed in tobacco (Nicotiana tabacum) were both targetedto the plasma membrane.• Reverse transcriptase polymerase chain reaction and northern blot analysesrevealed that AtFP6 mRNA was induced by cadmium (Cd(II)) in A. thaliana roots.Assays using metal-chelate affinity chromatography demonstrated that in vitrotranslated ACBP2 and AtFP6 bound lead (Pb(II)), Cd(II) and copper (Cu(II)). Con-sistently, assays using fluorescence analysis confirmed that (His)6-AtFP6 boundPb(II), like (His)6-ACBP2.• Arabidopsis thaliana plants overexpressing ACBP2 or AtFP6 were more tolerantto Cd(II) than wild-type plants. Plasma membrane-localized ACBP2 and AtFP6probably mediate Pb(II), Cd(II) and Cu(II) transport in A. thaliana roots. Also,(His)6-ACBP2 binds [14C]linoleoyl-CoA and [14C]linolenoyl-CoA, the precursors forphospholipid repair following lipid peroxidation under heavy metal stress at theplasma membrane. ACBP2-overexpressing plants were more tolerant to hydrogenperoxide than wild-type plants, further supporting a role for ACBP2 in post-stressmembrane repair.

Author for correspondence:Mee-Len ChyeTel: +852 22990319Fax: +852 28583477Email: mlchye@hkucc.hku.hk

Received: 21 June 2008Accepted: 5 August 2008

New Phytologist (2009) 181: 89–102 doi: 10.1111/j.1469-8137.2008.02631.x

Key words: Arabidopsis thaliana acyl-CoA-binding protein (ACBP), AtFP6, farnesylated protein, heavy metals, metal-binding motif, plasma membrane, protein–protein interaction.

Introduction

Arabidopsis thaliana acyl-CoA-binding proteins (ACBPs),encoded by a gene family of six members (designated ACBP1to ACBP6 ), range in size from 10.4 to 73.1 kDa (Leung et al.,2004). They are conserved at the acyl-CoA-binding domainto which long-chain acyl-CoA esters bind (Engeseth et al.,1996; Chye, 1998; Chye et al., 1999, 2000; Leung et al., 2004,2006). ACBP1 and ACBP2, plasma membrane-localizedproteins, probably maintain a membrane-associated acyl-CoApool (Chye et al., 1999; Li & Chye, 2003). Other than anAgave americana L. ACBP (Guerrero et al., 2006), which has89% homology to ACBP1, homologues of ACBP1 to ACBP5have not been characterized. ACBP3 is extracellularly targeted

and recombinant ACBP3 binds [14C]arachidonyl-CoA with highaffinity in comparison to [14C]palmitoyl-CoA and [14C]oleoyl-CoA (Leung et al., 2006). In contrast, recombinant ACBP4and ACBP5 preferentially bind [14C]oleoyl-CoA (Leunget al., 2004). The larger ACBPs contain conserved domainsthat can mediate protein–protein interactions, ankyrin repeats(ACBP1 and ACBP2) or kelch motifs (ACBP4 and ACBP5)(Leung et al., 2004; Li & Chye, 2004). The smallest memberof the gene family, 10-kDa ACBP6, shows 57% aminoacid identity to the well-characterized bovine 10-kDa ACBP(Engeseth et al., 1996).

Human 9-kDa ACBP binds the heavy metal lead (Pb(II))with high affinity and is a molecular target of Pb(II) (Smithet al., 1998). Heavy metals participate in reactions that

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generate oxygen radicals which cause oxidative damage to DNA,RNA, proteins and lipids, the latter via lipid peroxidation(Stohs & Bagchi, 1995). As A. thaliana ACBPs also containthe acyl-CoA-binding domain, we have investigated whetherACBP6 and the plasma membrane-associated A. thalianaACBPs bind Pb(II) (Xiao et al., 2008). In vitro-translatedACBP1 and ACBP2 and (His)6-ACBP1 and (His)6-ACBP2were observed to bind Pb(II) better than ACBP6, and theexpression of ACBP1 and ACBP2 (but not ACBP6) mRNAswas Pb(II)-inducible (Xiao et al., 2008). Further, transgenicA. thaliana plants overexpressing ACBP1 were more tolerantto Pb(II) treatment than wild-type plants, which showedPb(II) accumulation in shoots (Xiao et al., 2008).

Transgenic A. thaliana plants overexpressing the ATP-binding cassette protein genes AtPDR8 (pleiotropic drugresistance 8) and AtPDR12 show enhanced tolerance to Pb(II)and cadmium (Cd(II)) (Kim et al., 2007; Lee et al., 2005).Arabidopsis thaliana P1B-type heavy metal ATPases (HMA1 toHMA8) potentially interact with heavy metals at heavy-metal-binding motifs (Baxter et al., 2003). While HMA2 andHMA4, which possess CC dipeptide heavy-metal-bindingmotifs, function in zinc (Zn(II)) translocation (Baxter et al.,2003; Hussain et al., 2004), HMA5, which contains heavy-metal-associated domains (MXCXXC), is involved in copper(Cu(II)) detoxification (Baxter et al., 2003; Andrés-Coláset al., 2006). M/LXCXXC domains also occur in A. thalianafarnesylated proteins (ATFPs) which are characterized by aC-terminal isoprenylation site (CaaX); for example, recom-binant ATFP3 binds Cu(II), nickel (Ni(II)) and Zn(II)(Dykema et al., 1999). The A. thaliana farnesylated proteinCd induced 19 (CdI19) directly interacts with Cd(II) at itsCXXC motif and its mRNA is induced by Cd(II) (Suzukiet al., 2002).

ACBP2 was observed here, in a yeast two-hybrid analysis,to interact with another member of the ATFP family, AtFP6.As ACBP2 has C-terminal ankyrin repeats (Chye et al., 2000;Li & Chye, 2004), we investigated whether these repeatsmediate its interaction with AtFP6. We also tested the bindingof ACBP2 and AtFP6 with various heavy metals and examinedtheir effects on ACBP2 and AtFP6 expression.

Materials and Methods

Plant treatments

Arabidopsis thaliana (L.) Heynh. wild-type Columbia (ecotypeCol-0) was grown in a growth chamber (8 : 16 h; 21 : 23°Cdark:light). Seeds were surface-sterilized in 20% Cloroxsupplemented with 0.1% Tween-20 for 30 min and grownfor 3 wk on Murashige & Skoog (1962) (MS) mediumcontaining 2% sucrose, before 24 h of treatment in a 1 mMsolution of Pb(NO3)2 (Aldrich, St. Louis, MO, USA), CdCl2(Aldrich), CuCl2 (Aldrich) or ZnCl2 (Aldrich) (Lee et al.,2005). Shoot and root samples were collected for total RNA

extraction. For Cd(II) sensitivity tests, 3-d-old seeds grown onMS medium containing 2% sucrose, with or without 0.15 mMCdCl2, were transferred to fresh identical medium for verticalgrowth for 2 wk (Suzuki et al., 2002). To distinguish the effectsof Cd(II) on germination from post-germination development,5-d-old seeds grown on MS medium were transferred forvertical growth for 2 wk to MS medium containing 2%sucrose with or without heavy metals (Xiao et al., 2008).For hydrogen peroxide (H2O2) sensitivity testing (Jasinskiet al., 2008), seeds were grown vertically for 2 wk on MSmedium containing 2% sucrose, with or without 1 mM H2O2(BDH AnalaR, Poole, UK).

Yeast two-hybrid analysis

The 1.1-kb ACBP2 coding region was cloned in the baitvector pBUTE, a pGBDU-C1 derivative with a transcriptionfactor of galactose gene cluster (GAL4) DNA-binding domain(GAL4(BD)) (James et al., 1996). The resultant plasmid wasintroduced into Saccharomyces cerevisiae mating type strainPJ69-4A (James et al., 1996). Library screening carried out atthe Molecular Interaction Facility (University of Wisconsin,Madison, WI, USA) used an A. thaliana library collectionrepresenting cDNAs from flowering A. thaliana, in the preyvector pGADT7 (Clontech) with a GAL4 activation domain(GAL4(AD)) (James et al., 1996). This library was used totransform S. cerevisiae harbouring the pGAL4(BD)-ACBP2bait vector. About 18 million independent transformants wereobtained on [SD-leu-ura] medium. Twenty-two putative positivesselected on [SD-leu-ura-his] medium were tested usingcolony filter β-galactosidase assays. Prey plasmids rescued fromputative positives were introduced into PJ69-4A, harbouringthe ACBP2 bait vector, for mating and selection assays followedby nucleotide analysis (http://www.ncbi.nlm.nih.gov/cgibin/BLAST).

An ankyrin-lacking region encoding the first 229 (of 354)amino acids of ACBP2, generated by PCR using primersML993 (5′-GGATCCATGGGTGATTGGGCTCAAC-3′;BamHI site underlined) and ML994 (5′-CTGCAGTTAT-TCGGACTCCTCATCATAAA-3′; PstI site underlined), wascloned into the pGEM-T Easy vector (Promega, Madison, WI,USA). The resultant plasmid pAT458, digested with BamHIand SalI, yielded a 0.7-kb derived fragment that was cloned inpBUTE to generate bait pGAL4(BD)-ACBP2ΔANK.

Expression of baits GAL4(BD)-ACBP2ΔANK andGAL4(BD)-ACBP2 in yeast was confirmed by western blotanalysis. Yeast cells were harvested and total proteins extractedfor sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS-PAGE) analysis, followed by western blot analysis usingECL membranes (Amersham, Little Chalfont, UK), anti-GAL4(BD) monoclonal antibodies (Clontech, Mountain View,CA, USA), and goat anti-mouse immunoglobulin G (IgG)(whole molecule)–alkaline phosphatase conjugate (Sigma,St. Louis, MO, USA) to detect bait proteins.

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In vitro protein-binding assays

In vitro protein-binding assays (Li & Chye, 2004) were usedto confirm the interaction between AtFP6 and ACBP2. ThepKS-GAL4(TA)-ACBP2 construct and pKS-Brassica napuslysophosphatidic acid acyltransferase (BnLPAAT) construct(control; gift of Dr M. Frentzen; GenBank accession numberZ95637) have been described previously (Li & Chye, 2004).The AtFP6 coding region, generated by reverse-transcriptasepolymerase chain reaction (RT-PCR) using primers ML776(5′-ATGGGTGTTCTTGATCATGTCTC-3′) and ML777(5′-TCACATGACCACACAAGCCG-3′), was cloned intothe pGEM-T Easy vector. The 639-bp ACBP2 fragment(amino acids 2–214, lacking ankyrin repeats) was similarlygenerated using primers ML904 (5′-CAAGAATTCGGTG-ATTGGGCTCAACTTG-3′; EcoRI site underlined) andML905 (CAAAGATCTGGTTCCTCTTGAGTTGGAG-3′; BglII site underlined). Following EcoRI and BglII digestion,the fragment was cloned into vector pKS-GAL4(TA) (Li &Chye, 2004) to produce pKS-GAL4(TA)-ACBP2ΔANK.

[35S]Methionine-labelled proteins were produced by invitro transcription/translation using the TNT® Wheat GermExtract System (Promega) to investigate binding (Li & Chye,2004). GAL4(TA)-ACBP2 or GAL4(TA)-ACBP2ΔANK wasimmobilized to protein A/agarose beads using monoclonalantibodies against GAL4(TA), incubated with the candidateprotein on a rotating wheel for 6 h and washed four timeswith 1 ml of phosphate-buffered saline (PBS) washing buffer;the washed pellet was then analysed by SDS-PAGE, followedby autoradiography.

Construction of autofluorescence-tagged fusions and transient expression

A 0.46-kb AtFP6 fragment encoding amino acids 1–153,amplified by RT-PCR using primers ML845 (5′-CACCTCGAGTTATGGGTGTTCTTGATCATGTC-3′;XhoI site underlined) and ML846 (5′-CATGGATCCTCAC-ATGACCACACAAGCCG-3′; BamHI site underlined), wascloned into the pGEM-T Easy vector. The XhoI-BamHIderived fragment was subsequently cloned into vector pGDG(Goodin et al., 2002) to fuse AtFP6 to the C-terminus ofthe green fluorescent protein (GFP). The Discosoma sp. redfluorescent protein (DsRed):ACBP2 fusion has been describedpreviously (Li & Chye, 2004). The DsRed:ACBP2ΔANKfusion was constructed by cloning a BamHI-SalI fragmentfrom plasmid pAT458 into pGDR (Goodin et al., 2002).

The GFP:AtFP6 construct was introduced into onion(Allium cepa L.) epidermal cells with a Biolistic PDS-1000/Hesystem (BioRad, Hercules, CA, USA). About 360 µg of goldparticles (1.0-µm diameter) coated with 0.9 µg of DNA wasused per shot. Onion pieces were kept in darkness (22°C) for15 h following bombardment. For cell plasmolysis followingbombardment, the layer of epidermal cells was lifted and

incubated in 1 M sucrose solution for 20 min (Li & Chye,2003). Cells were examined under a Zeiss LSM 510 invertedconfocal laser-scanning microscope (Zeiss, Hamburg, Germany)equipped with helium/neon lasers (Li & Chye, 2004). Foreach plasmid construct, 20–30 cells were imaged with similarresults.

Agrobacterium tumefaciens LBA4404 derivatives containingautofluorescence-tagged constructs were grown at 28°C over-night in Luria Bertani medium (LB) supplemented withkanamycin (50 µg ml−1) and streptomycin (25 µg ml−1).Following agroinfiltration of glasshouse (22°C)-grown 6-wk-old tobacco (Nicotiana tabacum var. Xanthi) leaves (Li & Chye,2004), leaf epidermal cells were examined by confocal micro-scopy. For each plasmid construct, 10–15 cells were imagedwith similar results.

Fluorescence resonance energy transfer (FRET) pairs ofGFP/DsRed were analysed using the confocal laser-scanningmicroscope Zeiss LSM 510 META. FRET measurements ofDsRed emission with zero contribution from GFP were obtainedas described by Erickson et al. (2003), using the followingsettings: excitation, 488 nm; emission filter, bandpass 505/30 nmfor GFP and bandpass 600/37 nm for DsRed.

Northern blot analysis

Total RNA (20 µg) separated on a 1.5% agarose gel containing6% formaldehyde was transferred to Hybond N membranes(Amersham). An ACBP2-specific probe was prepared byrandom-primed [32P]dCTP-labelling a 0.5-kb 5′-end ACBP2cDNA fragment. Hybridization was carried out in a solutioncontaining 50% formamide at 42°C (Chye et al., 2000).Digoxigenin probes (Xiao et al., 2008) for induced expressionwere labelled using the PCR Digoxigenin Probe SynthesisKit (Roche, Mannheim, Germany) on a 0.89-kb ACBP2cDNA generated by RT-PCR using primers ML194 (5′-TCAAGGGGAGAGTTTCC-3′) and ML205 (5′-CGTCA-CCCAGAGGAGTC-3′) and a 0.46-kb AtFP6 cDNAgenerated using primers ML868 (5′-CACGGATCCAT-GGGTGTTCTTGATCATGTC-3′; BamHI site underlined)and ML869 (5′-CATCTCGAGTCACATGACCACACAA-GCC-3′; XhoI site underlined). Hybridization and detec-tion were performed following procedures described byRoche.

RT-PCR analysis

Total RNA was isolated using TRIzol reagent (Invitrogen,Carlsbad, CA, USA) from 3-wk-old A. thaliana shoots or roots.First-strand synthesis was carried out using the SuperscriptTM

First-Strand Synthesis system (Invitrogen). AtFP6 was amplifiedusing primers ML868 and ML869, and 18S rDNA usingprimers 18S-F (5′-CACCGCTTAACCCGAA-3′) and 18S-R(5′-GTGAGGTCACGACCAG-3′), with denaturation at95°C for 3 min, followed by 25 cycles of 94°C for 30 s, 55°C

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for 30 s and 72°C for 1 min, and an extension at 72°C for10 min.

Metal-chelate affinity chromatography

RT-PCR-derived coding regions of ACBP2, AtFP6 andACBP6 were cloned into the pGEM-T Easy vector for in vitrotranscription/translation (Xiao et al., 2008). Primers used were:ACBP2 (ML902, 5′-ATGGGTGATTGGGCTCAACT-3′ andML903, 5′-TTAGTCTGCCTGCTTTGCAG-3′), AtFP6(ML776 and ML777) and ACBP6 (ML812, 5′-TTCTCC-GTCTTACACCGATT-3′ and ML751, 5′-GACATGGTGT-GATCTCTTAAAAAAGCTTGA-3′). [35S]Methionine-labelledproteins were produced by in vitro transcription/translationusing the TNT® Wheat Germ Extract System (Promega).

Pb(II)-, Cd(II)- and Cu(II)-equilibrated matrices for metal-binding assays were prepared by stripping Ni-NTA agarose(Qiagen, Valencia, CA, USA) of nickel, and re-equilibratingwith 0.1 M Pb(NO3)2, CdCl2 or CuCl2 according to Xiao et al.(2008). The metal-binding assays were performed accordingto Xiao et al. (2008). To determine if binding is dependenton divalent cations, ethylenediaminetetraacetic acid (EDTA)was added to the reaction at a final concentration of 50 mM.

Expression and purification of (His)6-tagged proteins for Pb(II)-binding fluorescence analysis

Batch extractions of (His)6-ACBP1 (Chye, 1998) and (His)6-ACBP2 (Chye et al., 2000) were prepared using Ni-NTAagarose. Proteins were concentrated using Centricon-10(Amicon, Billerica, MA, USA) spin-columns.

The 0.46-kb AtFP6 coding region, RT-PCR generatedusing primers ML857 (5′-CACCTCGAGCATGGGTGT-TCTTGATCATGTC-3′; XhoI site underlined) and ML858(5′-CATGAATTCTCACATGACCACACAAGCCG-3′;EcoRI site underlined) followed by XhoI and EcoRI digestion,was cloned into vector pRSET B (Invitrogen). Escherichia coliBL21(DE3)Star pLysS (Invitrogen) transformants were culturedto an optical density (OD600nm) of 0.4 and induced with1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3 h.Cells were harvested and soluble and insoluble proteins wereextracted for SDS-PAGE, followed by western blot analysisusing ECL membranes (Amersham) and the QIAexpressNi-NTA AP Conjugate (Qiagen) to detect (His)6-taggedprotein. Batch extraction of (His)6-AtFP6 was carried outunder native conditions using an anity column of Ni-NTAagarose. The protein, eluted with a native elution buffer(50 mM NaH2PO4, pH 8.0, 300 mM NaCl and 250 mMimidazole), was dialysed overnight using Spectra/Por® molecularporous membrane tubing (Spectrum Laboratory, RanchoDominguez, CA, USA).

Fluorescence analysis was used to quantify the bindingof (His)6-AtFP6 and (His)6-ACBP2 to Pb(II) (Xiao et al.,2008).

Generation of 35S::ACBP2 and 35S::AtFP6 overexpressing A. thaliana

The 1.5-kb full-length ACBP2 cDNA was cloned into theXhoI-SpeI sites of binary vector pSMB (Mylne & Botella, 1998)to produce pAT377. Plasmid pSa7 (Xu et al., 2004), a derivativeof pBI121 (Clontech), was digested with SalI to remove a 0.5-kbfragment to produce vector pSa13. The 0.46-kb AtFP6 cDNAfragment was cloned into the BamHI-XhoI sites of vector pSa13to produce pAT394. Plasmids pAT377 and pAT394 wereused to transform A. tumefaciens LBA4404 for subsequentA. thaliana ‘floral dip’ transformation (Clough & Bent, 1998).In ACBP2 transformation, T0 seedlings germinated on MSmedium containing 50 µM glufosinate ammonium (Basta) wereconfirmed by PCR using a 35S promoter-specific primer, 35SB(5′-CAATCCCACTATCCTTCGCAAGACC-3′), and anACBP2-specific primer, ML194. In AtFP6 transformation,T0 seedlings germinated on MS medium containing kanamycin(50 µg ml−1) were confirmed by PCR using the 35SB primerand the AtFP6-specific primer ML777. Putative transformantswere analysed by northern blotting.

Acyl-CoA-binding assays using (His)6-ACBP1 and (His)6-ACBP2

In vitro binding assays (Leung et al., 2004) with [14C]linoleoyl-CoA (American Radiolabelled Chemicals, St. Louis, MO, USA),[14C]linolenoyl-CoA (American Radiolabelled Chemicals) and[14C]palmitoyl-CoA (Amersham) were carried out usingLipidexTM-1000 (PerkinElmer, Boston, MA, USA). Purifiedrecombinant ACBP (at a final concentration of 0.2 µM)was mixed with [14C]linoleoyl-CoA, [14C]linolenoyl-CoA or[14C]palmitoyl-CoA, at final acyl-CoA concentrations rangingfrom 0.2 to 4 µM, and incubated for 30 min at 37°C, and400 µl of ice-cold 50% slurry of LipidexTM-1000 in bindingbuffer was added. Samples were centrifuged at 12 000 g for5 min at 4°C and a 200-µl aliquot of the supernatant was takenfor analysis of radioactivity counts using an LS 6500 liquidscintillation counter (Beckman, Fullerton, CA, USA). Assayswere performed in triplicate, with blanks, at each con-centration of acyl-CoA.

Results

Yeast two-hybrid analysis

To confirm the interaction between AtFP6 and ACBP2,the prey vector pGAL4(AD)-AtFP6 was introduced intoS. cerevisiae PJ69-4A harbouring the pGAL4(BD)-ACBP2bait vector. Positive transformants selected on [SD-leu-ura-his]medium were tested using colony filter β-galactosidase assays.Unlike the control (Fig. 1a), consisting of yeast harbouringthe pGAL4(BD)-ACBP2 bait vector and control prey vectorpGAD-C1 containing GAL4(AD) (James et al., 1996), in

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which the bait vector did not activate β-galactosidase activity,a blue colour was observed with the prey vector pGAL4(AD)-AtFP6 (Fig. 1b), indicating that AtFP6 interacts with ACBP2to produce significant levels of β-galactosidase.

To determine whether the ankyrin repeats of ACBP2mediate the interaction between ACBP2 and AtFP6, theprey vector pGAL4(AD)-AtFP6 was introduced into yeastharbouring the ankyrin-lacking bait vector pGAL4(BD)-ACBP2ΔANK. First, yeast transformed with pGAL4(BD)-ACBP2ΔANK and pGAD-C1 was tested to ensure that the baitvector itself did not activate β-galactosidase activity (Fig. 1c).The lack of a blue colour in β-galactosidase assays when preypGAL4(AD)-AtFP6 was introduced into yeast harbouringpGAL4(BD)-ACBP2ΔANK suggested that the protein–proteininteraction was abolished when the ankyrin repeats wereremoved (Fig. 1d), suggesting that ACBP2 interacts withAtFP6 through these repeats. Expression of GAL4(BD)-ACBP2ΔANK and GAL4(BD)-ACBP2 proteins in transformedyeast was shown as cross-reacted bands of 41.6 and 55.4 kDa,respectively, in western blot analysis (Fig. 1e).

ACBP2 and AtFP6 interact in vitro

In vitro protein-binding assays confirmed the interactionbetween AtFP6 and ACBP2. SDS-PAGE followed byautoradiography of [35S]methionine-labelled in vitro-translatedGAL4(TA)-ACBP2, AtFP6 and BnLPAAT (negative control)revealed that these proteins were translated as predicted fromtheir calculated molecular masses (50.0, 19.6 and 43.0 kDa,respectively, in Fig. 1f). An autoradiography of the gel frombinding assays showed that the GAL4(TA)-ACBP2 fusion,immobilized to protein A/agarose beads using monoclonalantibodies against GAL4(TA), bound AtFP6 in vitro, incontrast to the negative control (Fig. 1f).

To verify that the ankyrin repeats of ACBP2 mediate protein–protein interactions, the ankyrin-lacking 39.5-kDa GAL4(TA)-ACBP2ΔANK fusion and AtFP6 were tested in in vitroprotein-binding assays. Weaker binding of AtFP6 withGAL4(TA)-ACBP2ΔANK than with GAL4(TA)-ACBP2(Fig. 1f) suggested that removal of these repeats affected theinteraction between AtFP6 and ACBP2.

DsRed:ACBP2 and GFP:AtFP6 co-localize at the plasma membrane

AtFP6 is predicted (http://psort.nibb.ac.jp) to be localizedat the plasma membrane with high certainty (0.650). Toexperimentally verify the localization of AtFP6 in onionepidermal cells, we tagged AtFP6 to GFP. With GFP as acontrol (Fig. 2a), representative confocal images showedGFP:AtFP6 expression at the plasma membrane (Fig. 2b).When bombarded onion epidermal cells were subjected toplasmolysis, fluorescence was associated with the retractedplasma membrane (Fig. 2c,d).

Fig. 1 Interaction of Arabidopsis thaliana acyl-CoA-binding protein 2 (ACBP2) and farnesylated protein 6 (AtFP6) analysed using colony filter β-galactosidase assays and in vitro protein-binding assays. (a–e) Interaction of ACBP2 and AtFP6 analysed using colony filter β-galactosidase assays. The prey vector pGAL4(AD)-AtFP6 or the control prey vector pGAD-C1 containing GAL4(AD) alone was introduced into Saccharomyces cerevisiae PJ69-4A harbouring pGAL4(BD)-ACBP2 and pGAL4(BD)-ACBP2ΔANK. (a) Yeast carrying the pGAL4(BD)-ACBP2 bait vector and the control prey vector pGAD-C1. (b) Yeast carrying the pGAL4(BD)-ACBP2 bait vector and the prey vector pGAL4(AD)-AtFP6. (c) Yeast carrying the pGAL4(BD)-ACBP2ΔANK bait vector and the control prey vector pGAD-C1. (d) Yeast carrying the pGAL4(BD)-ACBP2ΔANK bait vector and the prey vector pGAL4(AD)-AtFP6. (e) Western blot analysis of yeast PJ69-4A harbouring pGAL4(BD)-ACBP2ΔANK and pGAL4(BD)-ACBP2. (f) In vitro protein-binding assays for ACBP2 and AtFP6. The left panel (Input) shows input [35S]methionine-labelled proteins generated by in vitro transcription/translation. The right panel (Binding) shows in vitro protein binding between GAL4(TA)-ACBP2, GAL4(TA)-ACBP2ΔANK and AtFP6. The GAL4(TA)-ACBP2 fusion protein or GAL4(TA)-ACBP2ΔANK fusion protein was immobilized to protein A/agarose beads using monoclonal antibodies against GAL4(TA). Beads were incubated with the candidate binding protein and washed four times with phosphate-buffered saline (PBS), and the washed pellet was analysed on a 15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gel followed by autoradiography. Arrows indicate the location of proteins.

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Fig. 2 Localization of the green fluorescent protein:farnesylated protein 6 (GFP:AtFP6) and Discosoma sp. red fluorescent protein:acyl-CoA-binding protein 2 (DsRed:ACBP2) fusions transiently expressed in epidermal cells of onions (Allium cepa) and tobacco (Nicotiana tabacum) leaves. Representative confocal images show GFP:AtFP6 expressed in onion epidermal cells by particle bombardment (a–d) and GFP:AtFP6 and DsRed:ACBP2 in tobacco leaf epidermal cells by agroinfiltration (e–p). (a) GFP control; (b) GFP:AtFP6; (c) GFP:AtFP6 after plasmolysis; (d) differential interference contrast image of cell in (c); (e) GFP control; (f) DsRed; (g) GFP:AtFP6; (h) DsRed:ACBP2; (i) DsRed:ACBP2ΔANK; (j–l) control fluorescence resonance energy transfer (FRET) detection in tobacco leaf epidermal cells expressing GFP:AtFP6; (j) the green channel shows GFP:AtFP6; (k) the red channel shows zero contribution from GFP:AtFP6; (l) merged images of (j) and (k); (m–p) FRET detection in tobacco leaf epidermal cells co-expressing GFP:AtFP6 and DsRed:ACBP2; (m) differential interference contrast; (n) the green channel shows GFP:AtFP6; (o) the red channel shows DsRed:ACBP2; (p) co-localization of two signals is indicated by a yellow colour in merged images of (n) and (o). Bars, 20 µm.

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Subsequently, GFP:AtFP6 and DsRed:ACBP2 were tran-siently expressed in tobacco leaf epidermal cells by agroinfiltration.While controls GFP (Fig. 2e) and DsRed (Fig. 2f) were expressedthroughout the cell, fluorescence from GFP:AtFP6(Fig. 2g), DsRed:ACBP2 (Fig. 2h) and DsRed:ACBP2ΔANK(Fig. 2i) was observed at the plasma membrane.

In FRET analysis, green fluorescence (Fig. 2j) could beseen in control tobacco leaf cells expressing GFP:AtFP6alone, and no emission contributed by GFP was detected inthe red channel (Fig. 2k). However, for cells co-expressingGFP:AtFP6 and DsRed:ACBP2, not only GFP:AtFP6fluorescence (Fig. 2n) but also DsRed:ACBP2 fluorescence(Fig. 2o), which overlapped with the GFP signals (Fig. 2p),was detected, indicating that FRET occurred betweenGFP:AtFP6 and DsRed:ACBP2.

AtFP6 and ACBP2 show similar spatial expression

Northern blot analyses showed higher expression of ACBP2and AtFP6 in roots, stems and flowers (Fig. 3a, lanes R, St andF), and lower expression in siliques and leaves (Fig. 3a, lanesSi and L). Their similar spatial expression patterns suggestthat they can potentially interact in the co-expressed organs.High expression in root and stem is particularly relevant to theirpossible roles in metal binding and translocation.

AtFP6 expression is induced by Cd(II) and Zn(II)

RT-PCR analysis was used to investigate AtFP6 expression inresponse to heavy metals. Following treatment of 3-wk-oldA. thaliana with 1 mM of different heavy metal ions for 24 h,AtFP6 expression was up-regulated by Cd(II) in roots and toa lesser extent in shoots (Fig. 3b). Northern blot analysisindicated that AtFP6 expression, which was not induced byPb(II) and Cu(II), was induced by Cd(II) and Zn(II) in bothroots and shoots. Following Zn(II) treatment, AtFP6 mRNAwas rapidly degraded for as yet unknown reasons (Fig. 3c).ACBP2 expression in roots is Pb(II)-inducible (Xiao et al.,2008) and was used as a positive control (Fig. 3c).

In vitro-translated AtFP6 and ACBP2 bind Pb(II), Cd(II) and Cu(II)

We previously used metal-chelate affinity chromatographyto demonstrate that ACBP2 binds Pb(II) in vitro (Xiao et al.,2008), and applied the same method here to test whether invitro-translated AtFP6, which has a metal-binding motif (M/LXCXXC), binds Pb(II), Cd(II) and Cu(II). ACBP6, whichdoes not bind Pb(II) as well as ACBP1 (Xiao et al., 2008), wasused as a control. SDS-PAGE followed by autoradiography of[35S]methionine-labelled proteins (Fig. 4a) indicated that AtFP6(19.6 kDa) binds Pb(II), Cd(II) and Cu(II) better thanACBP6 (15.5 kDa). ACBP2 (41.1 kDa) also bound Cd(II)and Cu(II). Binding of AtFP6 and ACBP2 to Pb(II), Cd(II) and

Cu(II) was inhibited by the metal chelator EDTA, suggestingthat binding is dependent on divalent cations (Fig. 4a).

(His)6-AtFP6 binds Pb(II) in vitro

In western blot analysis, the 21-kDa (His)6-AtFP6 expressedin E. coli was induced in both soluble and insoluble fractions

Fig. 3 Expression of farnesylated protein 6 (AtFP6) and acyl-CoA-binding protein 2 (ACBP2) in Arabidopsis thaliana. (a) Northern blot analysis using an AtFP6-specific cDNA probe and total RNAs from roots (R), siliques (Si), stems (St), leaves (L), and flowers (F). The membrane was re-probed with ACBP2 cDNA, and rRNA was stained with ethidium bromide (bottom). (b) Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of AtFP6 expression. Total RNA was from roots and shoots of 14-d-old seedlings treated for 24 h with 1 mM Pb(NO3)2, CdCl2 or ZnCl2. The 0.46-kb AtFP6 and 0.26-kb 18S rDNA (control) bands are indicated. (c) Northern blot analysis of AtFP6 and ACBP2 expression. Total RNA was from roots and shoots of 14-d-old A. thaliana seedlings treated for 24 h with 1 mM Pb(NO3)2, CdCl2, CuCl2 or ZnCl2. rRNA was stained with ethidium bromide (bottom).

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after induction by IPTG for 2 and 3 h (Fig. 4b), respectively,in contrast to E. coli bearing vector pRSET B (Fig. 4b). Thebinding between (His)6-AtFP6 and Pb(II) was examined byfluorescence analysis using dansylated proteins incubatedwith different concentrations of Pb(II). As (His)6-ACBP2binds Pb(II) in fluorescence analysis (Xiao et al., 2008), it

served as a positive control. The fluorescence of dansylatedAtFP6 increased in the presence of Pb(II) (Fig. 4c), suggestingthat it binds Pb(II). The dissociation constants (Kd values) ofPb(II)-binding derived from the Scatchard plot (not shown)for (His)6-AtFP6 and (His)6-ACBP2 were 10.1 and 12.4 µM,respectively.

Fig. 4 In vitro heavy metal binding assays. (a) In vitro gel-binding assays of in vitro-translated acyl-CoA-binding protein 2 (ACBP2), farnesylated protein 6 (AtFP6) and ACBP6 with lead (Pb(II)), cadmium (Cd(II)) and copper (Cu(II)) by metal-chelate affinity chromatography. The first panel on the left (Input) shows equal loadings of [35S]methionine-labelled ACBP2, AtFP6 and ACBP6. The next panels show in vitro binding between radiolabelled proteins and heavy metals as indicated. The Pb(II)-, Cd(II)- or Cu(II)-equilibrated matrix was incubated with [35S]methionine-labelled proteins and washed three times, and the binding protein was eluted with an imidazole elution buffer followed by extraction with 2% sodium dodecyl sulphate (SDS) and 50 mM dithiothreitol (DTT). The eluted protein was analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Binding of ACBP2 and AtFP6 to Pb(II), Cd(II) and Cu(II) was inhibited by the metal chelator ethylenediaminetetraacetic acid (EDTA) (at a final concentration of 50 mM), suggesting that binding is dependent on divalent cations. (b) Western blot analysis of (His)6-AtFP6 expression. Soluble and insoluble proteins were extracted from Escherichia coli at 0, 2, and 3 h after isopropyl-β-D-thiogalactopyranoside (IPTG) induction. Total protein from the pRSET B transformant was used as a control. The (His)6-AtFP6 cross-reacting 21-kDa band (arrowed) was detected using the QIAexpress Ni-NTA conjugate. (c) Fluorescence analysis of Pb(II) binding to dansyl aziridine-labelled (His)6-AtFP6. (His)6-ACBP2 was used as a positive control. (His)6 proteins were labelled with 2 µl of 200 mM dansyl aziridine for 2 h at room temperature. Dansylated proteins were incubated with various concentrations (0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 µM) of Pb(NO3)2 at room temperature for 1 h and the fluorescence was measured. The relative fluorescence intensity of each sample was derived using the average of three readings versus the maximum reading for fluorescence (100%). The experiment was repeated three times and average values are shown. Bars represent SE (n = 3).

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A. thaliana AtFP6 overexpressors show enhance tolerance to Cd(II)

Northern blot analysis was used to confirm the overexpressionof AtFP6 mRNA in eight putative A. thaliana AtFP6overexpressors. All eight lines overexpressed the AtFP6mRNA, and results for two lines (AtFP6 ox-4 and AtFP6 ox-7)are shown (Fig. 5a). Five of these eight AtFP6-overexpressinglines showed an approx. 3 : 1 (resistant:sensitive) segregationratio in the T2 population when grown on kanamycin-containing medium, indicative of one copy of the 35S::AtFP6transgene. Subsequently, T3 homozygous transgenic plantsfrom two lines (AtFP6 ox-4 and AtFP6 ox-7) were used forCd(II) treatment. Root lengths of wild type and both AtFP6overexpressors (AtFP6 ox-4 and AtFP6 ox-7) were similar17 d after germination on MS medium (Fig. 5b) but AtFP6overexpressors grew better than wild type on MS mediumcontaining 0.15 mM CdCl2 (Fig. 5b). The relative rootlengths of the two transgenic lines AtFP6 ox-4 and AtFP6ox-7 grown on Cd(II)-containing medium were 36.1 ± 2.7%and 36.7 ± 1.8%, respectively, of those of similar plants grownin MS medium (Fig. 5c). These values were significantly(P < 0.01) higher than those of wild type (28.9 ± 2.4%).

To distinguish the effects of Cd(II) stress on seed germinationfrom those of post-germination development, A. thaliana seedswere germinated on MS medium for 5 d and transferred toeither MS or MS medium containing 0.15 mM CdCl2 for2 wk. Although no differences in root length were observedbetween wild type and AtFP6 overexpressors on MS medium,the root lengths of AtFP6 overexpressors were longer thanthose of wild type on Cd(II)-containing medium (Fig. 5d).The relative root lengths of AtFP6 ox-4 and AtFP6 ox-7grown on Cd(II)-containing medium were 47.9 ± 1.1% and45.2 ± 1.0%, respectively, of those of similar plants grown inMS medium (Fig. 5e). These values were significantly (P < 0.01)higher than those of wild type (36.9 ± 1.0%).

Arabidopsis thaliana ACBP2 overexpressors show enhanced tolerance to Cd(II)

All seven putative A. thaliana lines overexpressing ACBP2 testedwere shown to accumulate ACBP2 mRNA in northern blotanalysis. Results from two such lines (ACBP2 ox-3 and ACBP2ox-6) are shown in Fig. 5f. Four of seven lines showed an approx.3:1 (resistant:sensitive) segregation ratio in the T2 populationwhen grown on Basta-containing medium, indicative ofone copy of the 35S::ACBP2 transgene. Subsequently, T3homozygous transgenic plants from two lines (ACBP2 ox-3and ACBP2 ox-6 ) were used for Cd(II) treatment. Root lengthsof wild type and ACBP2-overexpressing lines were similar 17 dafter germination on MS medium (Fig. 5g). Both ACBP2-overexpressing lines (ACBP2 ox-3 and ACBP2 ox-6) grew betterthan wild type on MS medium containing 0.15 mM CdCl2(Fig. 5g). The relative root lengths of ACBP2 ox-3 and ACBP2

ox-6 grown on Cd(II)-containing medium were 38.3 ± 0.9%and 38.8 ± 0.5%, respectively, of those of similar plants grownin MS medium (Fig. 5h). These values were significantly(P < 0.01) higher than those of wild type (29.3 ± 0.5%).

To distinguish the effects of Cd(II) stress on seed germinationfrom those of post-germination development, A. thalianaseeds were germinated on MS medium for 5 d and transferredto either MS or MS medium containing 0.15 mM CdCl2 for2 wk. Although no differences in root length were observedbetween wild type and ACBP2 overexpressors on MS medium,their root lengths were longer than those of wild type grownon Cd(II)-containing medium (Fig. 5i). The relative rootlengths of ACBP2 ox-3 and ACBP2 ox-6 grown on Cd(II)-containing medium were 49.5 ± 3.1% and 53.1 ± 3.7%,respectively, of those of similar plants grown in MS medium(Fig. 5j). These values were significantly (P < 0.01) higherthan those of wild type (35.3 ± 1.8%).

(His)6-ACBP binds linoleoyl-CoA and linolenoyl-CoA in vitro

To investigate a possible role for ACBP2 in phospholipidrepair following lipid peroxidation caused by heavy metalstress at the plasma membrane, we tested whether ACBP2binds linoleoyl-CoA and linolenoyl-CoA. We previouslyshowed, in Lipidex assays, that (His)6-ACBP2 binds[14C]palmitoyl-CoA but not [14C]oleoyl-CoA, while thereverse applies for ACBP1 (Chye et al., 2000). Here, linoleoyl-CoA and linolenoyl-CoA were selected because linoleic acidand linolenic acid are the major fatty acids found in plants.Further, linoleoyl-CoA is the major acyl-CoA involved inacylation by lysophosphatidylcholine acyltransferase in safflower(Carthamus tinctorius L.) (Stymne & Stobart, 1984), yeast(Tamaki et al., 2007) and mammals (Soupene et al., 2008;Zhao et al., 2008). We also examined the binding of (His)6-ACBP1 to linoleoyl-CoA and linolenoyl-CoA, as ACBP1binds Pb(II) in vitro and confers Pb(II) tolerance whenoverexpressed in transgenic A. thaliana (Xiao et al., 2008).

(His)6-ACBP1 (Fig. 6a) and (His)6-ACBP2 (Fig. 6b) wereobserved to bind [14C]linoleoyl-CoA and [14C]linolenoyl-CoA, suggesting that they may participate in the repair of per-oxidized membrane lipids following heavy metal stress. TheKd values for [14C]linoleoyl-CoA and [14C]linolenoyl-CoAderived from the Scatchard plot (not shown) for (His)6-ACBP1were 1.1 and 1.2 µM, respectively. The Kd values for[14C]linoleoyl-CoA, [14C]linolenoyl-CoA and [14C]palmitoyl-CoA derived from the Scatchard plot (not shown) for (His)6-ACBP2 were 1.2, 3.3 and 20.2 µM, respectively.

Arabidopsis thaliana ACBP2 overexpressors show enhanced tolerance to H2O2

To further demonstrate that ACBP2 plays a role in phospholipidrepair following lipid peroxidation resulting from heavy metal

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stress, wild type and ACBP2 overexpressors were treatedwith the external oxidant H2O2. Although no differences inroot length were observed between wild type and ACBP2overexpressors on MS medium, their root lengths were greaterthan those of wild type when grown on MS mediumcontaining 1 mM H2O2 (Fig. 7a). The relative root lengths ofACBP2 ox-3 and ACBP2 ox-6 grown on H2O2-containingmedium were 37.8 ± 2.3% and 38.8 ± 2.9%, respectively, ofthose of similar plants grown on MS medium (Fig. 7b). Thesevalues were significantly (P < 0.01) higher than those of wildtype (25.9 ± 1.6%).

Discussion

We demonstrated here that ACBP2 interacts with AtFP6,a protein containing the heavy-metal-binding motif (M/LXCXXC). A deletion derivative of ACBP2 lacking ankyrinrepeats did not interact with AtFP6, confirming thesignificance of the repeats in mediating protein–proteininteractions. Confocal microscopy identified the plasmamembrane as a potential site for their subcellular interaction.AtFP6 resembles other isoprenylated proteins in the conservationof a carboxyl-terminal isoprenylation site (CaaX). Isoprenylation,which involves attachment of a hydrophobic isoprenoidpost-translationally, promotes membrane interactions andother prenylated proteins have been localized to the plasmamembrane (Willumsen et al., 1984; Zhang & Casey, 1996).The CaaX motif of CdI19 is responsible for plasmamembrane localization (Suzuki et al., 2002). This motif anda polybasic domain of six lysine residues at the C-terminusof the p21ras protein targeted it to the plasma membrane(Hancock et al., 1991). GFP:AtFP6 localization at the plasmamembrane is not only consistent with the localization of other

Fig. 5 Arabidopsis thaliana lines overexpressing farnesylated protein 6 (AtFP6) or acyl-CoA-binding protein 2 (ACBP2) are more tolerant to cadmium (Cd(II))-induced stress than wild type. (a) Northern blot analysis showing AtFP6 mRNA levels in wild type (Columbia (Col-0)) and AtFP6 overexpressors (AtFP6 ox-4 and AtFP6 ox-7). Total RNA (20 µg per well) was loaded for gel electrophoresis, blotting and hybridization to a digoxgenin-labelled AtFP6 probe. rRNA was stained with ethidium bromide (bottom). (b) Phenotypes of wild-type, AtFP6 ox-4 and AtFP6 ox-7 seedlings germinated and grown in either Murashige and Skoog (MS) medium or MS medium containing 0.15 mM CdCl2. Arabidopsis thaliana seeds were surface-sterilized and grown on MS medium containing 2% sucrose with or without 0.15 mM CdCl2 for 3 d, followed by transfer to fresh identical medium for vertical growth for 2 wk before photography. (c) Measurements of root lengths of plants shown in (b). Root lengths are expressed relative to the values obtained from seedlings grown on MS (100%). Bars represent SE (n = 15). **, P < 0.01 by Student’s t test. (d) Phenotypes of wild-type, AtFP6 ox-4 and AtFP6 ox-7 seedlings germinated and grown on MS medium for 5 d followed by transfer to either MS medium or MS medium containing 0.15 mM CdCl2 for 2 wk before photography. (e) Measurements of root lengths of plants shown in (d). Root lengths are expressed relative to the values obtained from seedlings grown on MS (100%). Bars represent SE (n = 15). **, P < 0.01 by Student’s t test. (f) Northern blot analysis showing ACBP2 mRNA levels in wild type (Col-0) and ACBP2 overexpressors (ACBP2 ox-3 and ACBP2 ox-6). Total RNA (20 µg per well) was loaded for gel electrophoresis, blotting and hybridization to a 32P-labelled ACBP2 probe. rRNA was stained with ethidium bromide (bottom). (g) Phenotypes of wild-type, ACBP2 ox-3 and ACBP2 ox-6 seedlings germinated and grown in either MS medium or MS medium containing 0.15 mM CdCl2. Arabidopsis thaliana seeds were surface-sterilized and grown on MS medium containing 2% sucrose with or without 0.15 mM CdCl2 for 3 d, followed by transfer to fresh identical medium for vertical growth for 2 wk before photography. (h) Measurements of root lengths of plants shown in (g). Root lengths are expressed relative to the values obtained from seedlings grown on MS (100%). Bars represent SE (n = 15). **, P < 0.01 by Student’s t test. (i) Phenotypes of wild-type, ACBP2 ox-3 and ACBP2 ox-6 seedlings germinated and grown on MS medium for 5 d followed by transfer to either MS medium or MS medium containing 0.15 mM CdCl2 for 2 wk before photography. (j) Measurements of root lengths of plants shown in (i). Root lengths are expressed relative to the values obtained for seedlings grown on MS (100%). Bars represent SE (n = 15). **, P < 0.01 by Student’s t test.

Fig. 6 Lipidex assays of (His)6-acyl-CoA-binding protein 1 (ACBP1) and (His)6-ACBP2 incubated with [14C]linoleoyl-CoA, [14C]linolenoyl-CoA or [14C]palmitoyl-CoA. (His)6-ACBP (at a final concentration of 0.2 µM) was incubated with [14C]linoleoyl-CoA, [14C]linolenoyl-CoA or [14C]palmitoyl-CoA, at final acyl-CoA concentrations ranging from 0.2 to 4 µM. The mixtures were mixed with LipidexTM-1000. Aliquots (200 µl) of the supernatant were taken for analysis of radioactivity. Assays were performed in triplicate, with blanks, at each concentration of acyl-CoA. Bars represent SE (n = 3). (a) (His)6-ACBP1 incubated with [14C]linoleoyl-CoA and [14C]linolenoyl-CoA. (b) (His)6-ACBP2 incubated with [14C]linoleoyl-CoA, [14C]linolenoyl-CoA and [14C]palmitoyl-CoA.

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prenylated proteins, but is consistent with our previous reportthat ACBP2 is targeted via the endoplasmic reticulum to theplasma membrane (Li & Chye, 2003).

Observations of higher ACBP2 and AtFP6 mRNA expressionin roots and stems support roles for ACBP2 and AtFP6 ininteractions with heavy metals. The initial step of root uptakeand translocation of heavy metals involves transfer acrossthe plasma membrane of root cells by membrane-associatedtransporter proteins (Clemens, 2006) such as A. thalianaCdI19 (Suzuki et al., 2002), AtHMA2 (Hussain et al., 2004),AtPDR8 (Kim et al., 2007) and AtPDR12 (Lee et al., 2005).As these proteins are predicted to bind heavy metals, whichhas been verified only for CdI19 (Suzuki et al., 2002), it waspertinent to establish heavy metal binding experimentally forAtFP6 and its protein partner, ACBP2. AtFP6 was observedto bind Pb(II), Cd(II) and Cu(II) in vitro, confirming the sig-

nificance of its M/LXCXXC motif. Previously, recombinantATFP3 was found to bind Cu(II), Ni(II) and Zn(II) in vitroin metal-chelate affinity chromatography (Dykema et al., 1999)and E. coli-expressed CdI19 directly interacted with Cd(II) atits CXXC motif in circular dichroism analysis (Suzuki et al.,2002). Further, CdI19 expression was induced by Cd(II) andother dicationic metals including mercury (Hg(II)), iron (Fe(II))and Cu(II), and CdI19-overexpressing transgenic A. thalianaplants were more resistant to Cd(II) treatment than wild type(Suzuki et al., 2002). Our results also showed that A. thalianaplants overexpressing AtFP6 or ACBP2 were more tolerant toCd(II) treatment than wild type, further supporting possibleroles of AtFP6 and ACBP2 in responses to Cd(II) stress.

Our results revealed that AtFP6 expression was induced byCd(II), but not Cu(II). In its ability to bind Cu(II), presumablyat its M/LXCXXC motif, AtFP6 resembles the A. thalianacopper chaperone (CCH) which is a functional homologue ofS. cerevisiae antioxidant 1 (Atx1; Himelblau et al., 1998). InA. thaliana, CCH is involved in Cu(II) trafficking to thesecretory pathway (Himelblau et al., 1998; Mira et al., 2001)and Cu(II) has been shown to be essential for ethylene bindingand perception (Hirayama et al., 1999; Woeste & Keiber,2000). Interestingly, in addition to AtFP6, ACBP2 has beenshown to interact with the ethylene-inding protein AtEBP viaits ankyrin repeats (Li & Chye, 2004). Like AtFP6, ACBP2also binds Pb(II), Cd(II) and Cu(II) in vitro but the ACBP2transcript was induced by Pb(II), but not Cd(II). Takentogether, our results suggest that the protein–protein interactorsACBP2 and AtFP6 probably participate in Pb(II), Cd(II)and Cu(II) transfer in A. thaliana and may perhaps affectCu(II)-mediated ethylene binding and signalling throughAtEBP. Using yeast two-hybrid analysis, Pufahl et al. (1997)showed that S. cerevisae Atx1, which contains an MXCXXCmotif, interacts in a Cu(II)-dependent manner with theCu(II)-binding domain (MTCXXC) of Ca2+-sensitive Cross-Complementer (Ccc2), a vesicular protein involved in coppertrafficking, suggesting that Atx1 donates Cu(II) to Ccc2. Theinteraction of AtFP6 with ACBP2, if reminiscent of the inter-action of Atx1 with Ccc2, would involve an exchange of metalions between them as they both bind Pb(II), Cd(II) and Cu(II).

Another explanation for the interaction between ACBP2and AtFP6 may be related to a possible role for ACBP2 inphospholipid repair following lipid peroxidation as a conse-quence of heavy metal stress. We have shown that ACBP2 andACBP1 bind [14C]linoleoyl-CoA and [14C]linolenoyl-CoAin vitro, supporting the possibility that they may play roles inbinding these acyl-CoAs as substrates for membrane repair(Nigam & Schewe, 2000; Soupene et al., 2008). In plants,lipoxygenases catalyse the oxygenation of predominantly linoleicacid and linolenic acid into reactive hydroperoxides (Vellosilloet al., 2007). Nigam & Schewe (2000) proposed that repair ofperoxidized membrane lipids can be achieved by the selectivecleavage of the peroxidized fatty acid residues and their subse-quent replacement by native fatty acids. Oxidized acyl chains

Fig. 7 Arabidopsis thaliana lines overexpressing acyl-CoA-binding protein 2 (ACBP2) are more tolerant to H2O2 stress than wild type. (a) Phenotypes of wild-type, ACBP2 ox-3 and ACBP2 ox-6 seedlings grown in either Murashige and Skoog (MS) medium or MS medium containing 1 mM H2O2. Arabidopsis thaliana seeds were surface-sterilized and grown on MS medium containing 2% sucrose with or without 1 mM H2O2 for vertical growth for 2 wk before photography. (b) Measurements of root lengths of plants shown in (a). Root lengths are expressed relative to the values obtained from seedlings grown on MS (100%). Bars represent SE (n = 15). **, P < 0.01 by Student’s t test.

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of phospholipid are rapidly removed by phospholipase A2action and reacylation of the resulting lysophospholipid iscatalysed by acyl-CoA:lysophosphatidylcholine acyltransferase(Soupene et al., 2008). In microsomal preparations fromsafflower, the preferred substrates of acyl-CoA:lysophosphati-dylcholine acyltransferase were linoleoyl-CoA > oleoyl-CoA> linolenoyl-CoA, with linolenoyl-CoA transfer at c. 2/3 therate of linoleoyl-CoA and oleoyl-CoA transfer (Stymne &Stobart, 1984). In human red blood cells, the highest transferrate by lysophosphatidylcholine acyltransferase was achievedusing linoleoyl-CoA (Soupene et al., 2008). The highestenzymatic activity for lysophosphatidylcholine acyltransferaseein metabolic tissues including liver, adipose and pancreas, asevaluated by maximum velocity/Michaelis constant (Vmax/Km),occurred when linoleoyl-CoA was used as substrate (Zhaoet al., 2008). The yeast lysophospholipid acyltransferase Lpt1also showed a preference for linoleoyl-CoA and linolenoyl-CoA(Tamaki et al., 2007), further suggesting the importance ofthese acyl-CoAs in membrane repair. When plants are exposedto Cd(II), lipid peroxidation and significant intracellularaccumulation of H2O2 occur because Cd(II) induces thedepletion of glutathione and inhibits the action of antioxidativeenzymes (Schützendübel & Polle, 2002). The observationsthat A. thaliana lines overexpressing ACBP2 were more tolerantto both H2O2 and Cd(II) stress than wild type indicate thatACBP2 may thus be involved in phospholipid repair followinglipid peroxidation triggered by Cd(II) stress.

Acknowledgements

We thank M. M. Goodin (University of California, Berkeley)for vectors pGDR and pGDG, M. Frentzen (Institut fürBiologie I, Aachen) for BnLPAAT cDNA and W. K. Yip forprovision of the Biolistic PDS-1000/He system. This workwas supported by the University Grants Committee of theHong Kong Special Administrative Region, China (Project No.AoE/B-07/99), the University of Hong Kong (10208034) anda Croucher Senior Research Fellowship awarded to MLC. WGand SX were supported by postgraduate studentships from theUniversity of Hong Kong and HL by the Croucher Foundation.

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New Phytologist (2009) 181: 89–102 © The Authors (2008).www.newphytologist.org Journal compilation © New Phytologist (2008)

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