Peroxisomal ATP Import Is Essential for Seedling Developmentin Arabidopsis thaliana W

Nicole Linka,a,b,1 Frederica L. Theodoulou,c Richard P. Haslam,c Marc Linka,a Jonathan A. Napier,c

H. Ekkehard Neuhaus,d and Andreas P.M. Webera,b

a Institut fur Biochemie der Pflanzen, Heinrich-Heine Universitat Dusseldorf, D-40225 Dusseldorf, Germanyb Department of Plant Biology, Michigan State University, East Lansing, Michigan, 48824-1312c Biological Chemistry Department, Rothamsted Research, Harpenden, AL5 2JQ, United Kingdomd Pflanzenphysiologie, Technische Universitat Kaiserslautern, D-67653 Kaiserslautern, Germany

Several recent proteomic studies of plant peroxisomes indicate that the peroxisomal matrix harbors multiple ATP-

dependent enzymes and chaperones. However, it is unknown whether plant peroxisomes are able to produce ATP by

substrate-level phosphorylation or whether external ATP fuels the energy-dependent reactions within peroxisomes. The

existence of transport proteins that supply plant peroxisomes with energy for fatty acid oxidation and other ATP-dependent

processes has not previously been demonstrated. Here, we describe two Arabidopsis thaliana genes that encode

peroxisomal adenine nucleotide carriers, PNC1 and PNC2. Both proteins, when fused to enhanced yellow fluorescent

protein, are targeted to peroxisomes. Complementation of a yeast mutant deficient in peroxisomal ATP import and in vitro

transport assays using recombinant transporter proteins revealed that PNC1 and PNC2 catalyze the counterexchange of

ATP with ADP or AMP. Transgenic Arabidopsis lines repressing both PNC genes were generated using ethanol-inducible

RNA interference. A detailed analysis of these plants showed that an impaired peroxisomal ATP import inhibits fatty acid

breakdown during early seedling growth and other b-oxidation reactions, such as auxin biosynthesis. We show conclusively

that PNC1 and PNC2 are essential for supplying peroxisomes with ATP, indicating that no other ATP generating systems

exist inside plant peroxisomes.

The b-oxidation of fatty acids, a process that exclusively occurs

within peroxisomes in plants and yeast, plays an important role in

storage oil mobilization to support seedling establishment

of oilseed plants, such as Arabidopsis thaliana (Graham and

Eastmond, 2002; Baker et al., 2006; Graham, 2008). Upon ger-

mination, fatty acids are released from storage oil triacylglycerol

(TAG) by lipolysis, degraded via b-oxidation in specialized peroxi-

somes, termed glyoxysomes, and subsequently converted to

sucrose, which drives growth and development until seedlings

become photoautotrophic (Graham and Eastmond, 2002; Baker

et al., 2006; Graham, 2008). Before the fatty acids can enter

b-oxidation, they are imported into peroxisomesby a peroxisomal

ATP binding cassette (ABC) transporter, variously known as CTS

(COMATOSE), At PXA1 (Arabidopsis peroxisomal ABC trans-

porter), or PED3 (peroxisomal defective 3) and hereafter referred

to as CTS (Zolman et al., 2001; Footitt et al., 2002; Hayashi et al.,

2002). Subsequently, the imported fatty acids are activated by

esterification to CoA. This ATP-dependent reaction within per-

oxisomes is catalyzed by long-chain acyl-CoA synthetases 6 and

7 (LACS6 and LACS7, respectively), which are named according

to their substrate specificity for long-chain fatty acids, which are

significant components of seed storage oil in Arabidopsis (Fulda

et al., 2002, 2004).

In Saccharomyces cerevisiae, two mechanisms exist for im-

port and activation of fatty acids, depending on chain length

(Hettema et al., 1996). Long-chain fatty acids (C16 and C18) are

converted to acyl-CoA esters in the cytosol prior to transport by

the heterodimeric peroxisomal ABC transporter, Pxa1p/Pxa2

(Hettema et al., 1996). By contrast, short- and medium-chain

fatty acids (#C14) that enter the peroxisomes by passive diffu-

sion or by an unknown transport protein are activated within

peroxisomes (Hettema et al., 1996). The possibility cannot be

excluded, though, that CTS imports the corresponding CoA

derivatives, as is the case for the yeast Pxa1p/Pxa2p hetero-

dimer (Hettema et al., 1996; Verleur et al., 1997), implicating a

cytosolic activation of the fatty acids, catalyzed by a hitherto

unknown enzyme. The actual substrates transported by CTS

in Arabidopsis have not yet been experimentally determined

(Theodoulou et al., 2006). However, the sucrose-dependent

seedling growth phenotype of the lacs6 lacs7 double knockout

mutant demonstrated that peroxisomal activation is essential

for lipid mobilization to provide energy for early seedling growth

(Fulda et al., 2004). The lacs6 lacs7 mutant is impaired in the

degradation of fatty acids, leading to growth arrest shortly after

germination (Fulda et al., 2004).

Besides fatty acid mobilization, b-oxidation is also involved in

generation of signaling molecules, such as the phytohormones

auxin and fatty acid–derived jasmonic acid (JA) (Zolman et al.,

2000; Schaller et al., 2004; Delker et al., 2007). By analogy to fatty

1 Address correspondence to nicole.linka@uni-duesseldorf.de.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Nicole Linka(nicole.linka@uni-duesseldorf.de).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.108.062042

The Plant Cell, Vol. 20: 3241–3257, December 2008, www.plantcell.org ã 2008 American Society of Plant Biologists

acids released from storage oil, the precursors of these signaling

molecules require CoA esterification before they can enter

b-oxidation (Baker et al., 2006; Goepfert and Poirier, 2007).

While the enzymes responsible for ATP-dependent activation of

natural auxin (indole butyric acid [IBA]) and proherbicide 2,4-

dichlorophenoxybutyric acid (2,4-DB) are currently unknown,

several enzymes belonging to the acyl-activating enzyme (AAE)

family have been implicated in jasmonate biosynthesis (Schneider

et al., 2005; Koo et al., 2006; Kienow et al., 2008). Moreover,

several as yet uncharacterized members of the large AAE family

carry a putative peroxisome targeting signal (PTS) and thusmight

be good candidates to activate the additional b-oxidation sub-

strates within peroxisomes (Shockey et al., 2002, 2003).

In the case where activation of fatty acids or other substrates

takes place within peroxisomes, the question arises as to how

these ATP-dependent reactions are supplied with ATP. It is

currently unknown whether plant peroxisomes are able to pro-

duce ATP by substrate-level phosphorylation or whether they

depend on external ATP to supply energy-dependent reactions

within peroxisomes. So far, transport proteins that supply plant

peroxisomes with energy for fatty acid oxidation have not been

characterized. However, in bakers’ yeast, a peroxisomal adenine

nucleotide transporter, ANT1, that is required for the ATP-

dependent activation of medium-chain fatty acids inside peroxi-

somes has been characterized (Palmieri et al., 2001).

ATP transport proteins play an important role in the distribution

of the primary agent coupling endergonic and exergonic reac-

tions in every cellular compartment (Winkler andNeuhaus, 1999).

In Arabidopsis and other plants, various adenine nucleotide

carriers have been identified at the molecular level. The mito-

chondrial ADP/ATP carrier mediates the export of ATP that is

synthesized in the mitochondrion to provide energy for cellular

metabolism (Heimpel et al., 2001; Haferkamp et al., 2002). The

plastidial ATP/ADP transporter (nucleotide transporter) is in-

volved in ATP uptake by both chloroplasts and heterotrophic

plastids, to enable the nocturnal ATP supply required for chloro-

phyll biosynthesis (Reiser et al., 2004; Reinhold et al., 2007), as

well as byheterotrophicplastids todrive starch biosynthesis (Batz

et al., 1992; Tjaden et al., 1998). Yet another ATP/ADP antiporter

located in the endoplasmic reticulum (ER) membrane provides

energy by importing ATP into the ER for the accumulation of ER-

related storage lipids and proteins (Leroch et al., 2008).

In this study, we identified two novel peroxisomal adenine

nucleotide carrier proteins (PNC1 and PNC2) from Arabidopsis.

Colocalization studies demonstrated that these proteins are

targeted to peroxisomes. Yeast complementation and in vitro

ATP uptake assays showed that both PNC1 and PNC2 catalyze

the counterexchange of ATP with AMP. Using an inducible RNA

interference (RNAi) repression strategy, we further established

several transgenic Arabidopsis lines with reduced expression

levels of bothPNC1 andPNC2. Our results showed that import of

ATP into peroxisomes that is catalyzed by PNC1 and PNC2 is

essential for activation of fatty acids during seedling germination

and plays a role in other b-oxidation reactions in peroxisomes,

such as auxin metabolism. Analysis of PNC1 and PNC2 repres-

sion lines further indicates that no other ATP generating systems

exist inside plant peroxisomes and that ATP import is the only

way to supply the peroxisomal matrix with ATP.

RESULTS

Identification of Genes Encoding Putative Peroxisomal ATP

Carriers in Arabidopsis

To identify candidate genes encoding peroxisomal ATP carriers,

theArabidopsis genomewas searched using the sequence of the

S. cerevisiae peroxisomal ATP carrier, ANT1 (Palmieri et al.,

2001), and the BLAST search algorithm (Altschul et al., 1990).

The three candidate genes showing highest similarity to ANT1

were selected for further analysis.

At2g39970 encodes a protein of 331 amino acids with a

calculated molecular mass of 36.2 kD that shares 21% identity

with ANT1. The At3g05290 gene product consists of 322 amino

acid residues, has a molecular mass of 35.4 kD, and is 23%

identical to the yeast carrier. The 321–amino acid protein

encoded by At5g27520 has a predicted molecular mass of

35.2 kD and shows 22% identity to the yeast protein. A protein

alignment revealed a strong similarity between At3g05290 and

At5g27520 (86% identity) (Figure 1). Both proteins display a

weaker similarity to the corresponding protein of the third

Arabidopsis candidate, At2g39970 (22% identity).

All three Arabidopsis candidate genes belong to the so-called

mitochondrial carrier family (MCF) (Picault et al., 2004; Haferkamp,

2007). They possess characteristic sequence features typical

for MCF members: three repeats of a conserved mitochondrial

energy transfer signature, identified as PS50920 by the PROSITE

program (http://au.expasy.org/prosite), and six predicted trans-

membrane-spanning domains (http://aramemnon.botanik.

uni-koeln.de) (Figure 1). The MCF is widespread in microorgan-

isms, animals, and humans, and several members of the MCF

have been described as carriers with various functions and

subcellular localizations (Picault et al., 2004; Bedhomme et al.,

2005; Bouvier et al., 2006; Palmieri et al., 2006;Haferkamp, 2007;

Weber and Fischer, 2007).

One of the candidate genes, At2g39970, is annotated as

peroxisomal membrane protein 38 kD (PMP38) and was previ-

ously proposed to represent a peroxisomal ATP carrier (Fukao

et al., 2001). However, biochemical data investigating its trans-

port function or mutants deficient in this protein have not yet

been reported.

At3g05290 andAt5g27520Complement aYeastMutant That

Is Deficient in the Peroxisomal ATP Transporter

To investigate whether the three Arabidopsis candidate genes

identified in silico indeed encode peroxisomal ATP carriers, we

attempted genetic complementation of a yeast mutant deficient

in ANT1 function (Palmieri et al., 2001). This knockout strain,

called ant1D, is unable to grow on media containing medium-

chain fatty acids, such as lauric acid (C12), as the sole carbon

source (Palmieri et al., 2001). In Figure 2, we show that heterol-

ogous expression of At3g05290 or At5g27520 restored the

capability of ant1D to grow efficiently on lauric acid, similar to

mutant yeast cells expressing the endogenous ANT1 gene and

the wild-type strain transformed with an empty vector (positive

control). By contrast, the knockout yeast strain expressing

At2g39970 failed to form colonies, as did the ant1D mutant

3242 The Plant Cell

transformedwith the empty vector (negative control). These data

indicate that the proteins encoded by At3g05290 and At5g27520

are able to mediate ATP import into yeast peroxisomes, which is

required for growth on lauric acid.

At2g39970, At3g05290, and At5g27520 Are Targeted to

Peroxisomes in Yeast

To assess whether the gene products of the Arabidopsis perox-

isomal ATP carrier candidates were targeted to peroxisomes

in yeast, their subcellular localization was analyzed using fluo-

rescent protein tagging in combination with fluorescence

microscopy. The open reading frames (ORFs) of At2g39970,

At3g05290, and At5g27520 were fused in frame to the N or C

terminus of enhanced yellow fluorescent protein (EYFP) and

transformed into the yeast mutant ant1D. The EYFP fusion

proteins were coexpressed with a peroxisomal-targeted fluo-

rescent marker that consisted of the cyan fluorescent protein

(CFP) tagged with the peroxisomal target signal 1 (PTS1) at its C

terminus (Gould et al., 1989). Figure 3A shows that fluorescent

signals colocalized in yeast cells expressing both CFP-PTS1 and

EYFP fusions exhibited, forming a punctuate pattern. These data

indicate that all three candidate proteins are targeted to perox-

isomes in yeast. Importantly, the EYFP-tagged proteins encoded

by At3g05290 and At5g27520 complemented the mutant

growth phenotype (see Supplemental Figure 1 online), demon-

strating that these fluorescently tagged plant proteins were

functional in yeast peroxisomes. Jointly, complementation and

subcellular localization data strongly suggest that At3g05290

and At5g27520 represent peroxisomal ATP carriers and thus

Figure 1. Amino Acid Sequence Alignment of the Three Arabidopsis Peroxisomal ATP Carrier Candidate Genes, At3g05290, At5g27520, and

At2g39970, with Rice, Yeast, and Human Homologs.

Black shading indicates identical amino acid residues in all six sequences, whereas gray shading indicates identical amino acid residues in at least four

sequences. The mitochondrial energy transfer signature domains are boxed, and the six predicted transmembrane-spanning domains are underlined.

The arrows flank the region that was used for the RNAi approach to silence expression of PNC1 and PNC2 simultaneously.

Arabidopsis Peroxisomal ATP Transporters 3243

were assigned the acronyms PNC1 and PNC2, for Arabidopsis

peroxisomal adenine nucleotide carrier 1 and 2, respectively.

Although the third candidate, At2g39970, is targeted to peroxi-

somes in yeast, it does not complement the phenotype of the

ant1Dmutant. It is thus unlikely that it functions as a peroxisomal

ATP transporter.

PNC1 and PNC2 Are Targeted to Peroxisomes in Tobacco

Epidermal Cells

To assesswhether PNC1 and PNC2 are targeted to peroxisomes

in plants as well, young tobacco (Nicotiana benthamiana) leaves

were infiltrated with Agrobacterium tumefaciens that were co-

transformed with the corresponding PNC1/2-EYFP construct

and a plasmid carrying the peroxisomal-targeted marker CFP-

PTS1. Three days after infiltration, tobacco epidermal cells were

analyzed in vivo by fluorescence microscopy (Figure 3B). The

peroxisomes were visualized as blue fluorescent dots by the

peroxisomal CFP-PTS1 marker construct. The EYFP and CFP

signals colocalized, indicating that PNC1-EYFP andPNC2-EYFP

are targeted to peroxisomes not only in yeast, but also in plant

cells.

PNC1 and PNC2 Catalyze an ATP/AMP Exchange

While genetic complementation of a yeast knockout mutant

deficient in its peroxisomal ATP carrier by PNC1 and PNC2

strongly indicated that the plant proteins encode peroxisomal

ATP importers, this experiment does not provide direct proof of

protein function. To substantiate their transport properties, we

analyzed whether recombinant PNC1 and PNC2 reconstituted

into liposomes were able to transport ATP in vitro (Flugge and

Weber, 1994;Weber et al., 1995). The peroxisomal ATP transport

proteins fromArabidopsis and the yeastANT1were expressed in

the yeast knockout mutant ant1D (Palmieri et al., 2001). Mem-

brane proteins from the corresponding transgenic yeast cells

were extracted, directly reconstituted into liposomes, and as-

sayed for ATP import activity. The previously characterized ANT1

catalyzes the transport of ATP, ADP, or AMP in a strict counter-

exchange mode (Palmieri et al., 2001). Thus, the uptake of

radioactively labeled [a-32P]ATP into proteoliposomes wasmea-

sured in the presence or absence of a suitable counterexchange

substrate. Given that the mitochondrial ATP/ADP carriers from

yeast are highly specific for ATP and ADP, respectively (Heimpel

et al., 2001; Haferkamp et al., 2002), AMP was selected as the

counterexchange substrate to avoid possible background from

mitochondrial ATP carrier activity. Using this experimental setup,

no significant ATP uptake into liposomes reconstituted with

membrane proteins from ant1D control cells containing the

empty vector was measured (Figure 4A). By contrast, liposomes

reconstituted withmembranes from the yeastmutant expressing

either PNC1 or PNC2 showed a substantial ATP/AMP counter-

exchange activity (Figures 4B and 4C, closed symbols). In the

absence of AMP, no ATP uptake was observed (Figures 4B and

4C, open symbols). The transport rates obtained by both

Arabidopsis proteins were comparable to those observed for

Figure 2. Complementation of a Yeast Mutant Deficient in Peroxisomal

ATP Uptake (ant1D).

The ant1D yeast mutant transformed with either empty vector (pDR195)

or with the ORFs of At2g39970, At3g05290, At5g27520, and Sc ANT1

cloned into pDR195 was tested for growth on agar plates containing

medium with lauric acid as the sole carbon source. As an additional

control, the wild-type strain transformed with the pDR195 vector was

also used for the growth assays. The plates were photographed after

incubation at 308C for 6 d.

Figure 3. Colocalization Studies of EYFP Fusion Proteins and CFP-

PTS1 in the ant1D Yeast Mutant and in Tobacco Epidermal Cells.

(A) The ORFs of PNC1 and PNC2 were fused at their C termini to EYFP,

whereas the coding sequence of At2g39970 was fused at its N terminus

to EYFP. The EYFP fusion proteins were coexpressed with the perox-

isomal marker CFP-PTS1 under the control of a constitutive promoter.

The subcellular localization was analyzed in the ant1D knockout yeast

mutant by fluorescence microscopy. The merged images of CFP and

EYFP fluorescence are also shown.

(B) PNC1 and PNC2 fused to EYFP were transiently coexpressed with

CFP-PTS1 in tobacco epidermal cells. Images were taken 3 d after

Agrobacterium infiltration. The colocalization is demonstrated in the

merged images of the PNC1/2-EYFP fusions with those of CFP-PTS1.

3244 The Plant Cell

ANT1 (Figure 4D), further emphasizing that both PNC1 andPNC2

are able to catalyze an ATP/AMP exchange, like their putative

yeast ortholog.

Since we could not directly test whether yeast-expressed

Arabidopsis PNC1 and PNC2 are able to catalyze the exchange

of ADP with ATP, due to the background activity of the yeast

mitochondrial ATP/ADP carrier, we also expressed PNC1 and

PNC2 in Escherichia coli, which does not show endogenous ATP

carrier activities (see Supplemental Figure 2A online). Uptake of

radiolabeled [a-32P]ADP into proteoliposomes preloaded with

either ATP, ADP, or AMP was measured for reconstituted re-

combinant proteins purified from E. coli. ADP was efficiently

taken up in the presence of the internal substrates ATP, ADP, or

AMP, but not in their absence (see Supplemental Figure 2B

online). Thus, PNC1 and PNC2 represent nucleotide carriers with

specificity for ATP, ADP, and AMP in a strict counterexchange

mode, like the yeast ANT1 transporter.

PNC1 and PNC2 Are Highly Specific for ATP, ADP, and AMP

A detailed knowledge of substrate affinity and specificity is

required to understand the function of a transport protein in vivo.

To determine the apparent Km values of PNC1 and PNC2 for

ATP, the initial rate of [a-32P]ATP uptake into AMP-preloaded

proteoliposomes was measured at different external ATP con-

centrations. PNC1 and PNC2 exhibit an apparent Km value for

ATP of 83 mM (6 15 SE, n = 3) and 150 mM (6 36 SE, n = 3),

respectively, whereas ANT1 has an apparent Km value of 54 mM

(6 13 SE, n = 3) for ATP (Table 1). To assess the substrate

specificity of the ATP carriers, the inhibition constant (Ki) was

determined. For this, the import of radiolabeled ATP into pro-

teoliposomes preloaded with AMP was measured in the pres-

ence of nonlabeled ADP, AMP, and inorganic ortho-phosphate

(Pi). For PNC1, PNC2, and ANT1, both ADP and AMP compet-

itively inhibited the uptake of ATP, whereas Pi exerted no

significant inhibitory effect. In addition, the obtained apparent

Ki values shown in Table 1 exhibit similar Ki/Km ratios for all three

recombinant proteins. The estimated Ki values of ADP are

twofold to threefold higher than their corresponding Km values

for ATP. In the case of the apparent Ki value for AMP, the half-

maximal inhibition of ATP transport was observed at even higher

concentrations.

Figure 4. Time Kinetics of ATP Uptake into Proteoliposomes Harboring Recombinant PNC1, PNC2, and ANT1 Proteins.

Liposomes were reconstituted with the membrane fraction from ant1Dmutant yeast containing the corresponding expression constructs for PNC1 (B),

PNC2 (C), and ANT1 (D) or with the empty vector as a control (A). The uptake of radioactively labeled [a-32P]ATP (0.2 mM) was measured into

proteoliposomes preloaded internally with 30 mM AMP (closed symbols) or in the absence of AMP as a counterexchange substrate (open symbols).

ATP transport was terminated by loading the proteoliposomes onto anion-exchange columns. Radioactivity of the column flow-through in the sample

was quantified by liquid scintillation counting. The means of a representative experiment with three technical replicates 6 SE are shown.

Table 1. Apparent Km (ATP) and Ki Values of Recombinant PNC1,

PNC2, and ANT1 for Various Metabolites

Substrate PNC1 PNC2 ANT1

Km (mM) Km (mM) Km (mM)

ATP 83 (615) 150 (636) 54 (613)

Ki (mM) Ki (mM) Ki (mM)

ADP 211 (652) 254 (665) 171 (651)

AMP 266 (666) 427 (6136) 199 (663)

Pi >100,000 >100,000 >100,000

All experiments were performed with liposomes preloaded with 30 mM

AMP. The data represent the mean values 6 SE from three experiments.

Arabidopsis Peroxisomal ATP Transporters 3245

Expression Pattern of PNC1 and PNC2

As a first step to elucidate the role of PNC1 and PNC2 in planta,

their expression patterns were investigated by searching the

publicly available microarray databases using the Arabidopsis

eFP Browser (Winter et al., 2007). Both genes are ubiquitously

expressed in Arabidopsis throughout all developmental stages

(see Supplemental Figure 3 online). Notably, PNC2 is highly

expressed in tissues where b-oxidation plays an important role

(Baker et al., 2006), such as flowers, stamens, pollen, developing

seeds, and senescent leaves. In addition, the Arabidopsis

coresponse database CSB.DB was searched for genes that

are coexpressed withPNC1 and PNC2 (Steinhauser et al., 2004).

Most of the genes coexpressed with PNC1 and PNC2 are part of

the glyoxylate cycle and gluconeogenesis, processes known to

be primarily associated with b-oxidation (see Supplemental

Table 1 online). Importantly, one of the highest-ranking coex-

pressed genes (Spearman rank correlation coefficient > 0.7) for

both PNC1 and PNC2 is the long-chain acyl-CoA synthetase

LACS7 that plays an important role in the activation of fatty acids

during seedling establishment (Fulda et al., 2004).

Generation of Transgenic Arabidopsis Plants Displaying

Impaired Peroxisomal ATP Import

To elucidate the impact of peroxisomal ATP transporters in vivo,

T-DNA insertion lines for PNC1 (SAIL_303H02, referred to as

pnc1-1) and PNC2 (SALK_014579, referred to as pnc2-1) were

isolated from the publicly available collections (see Supplemen-

tal Figure 4A online). No abberant phenotype was observed

under normal growth conditions in homozygous pnc1-1 and

pnc1-2 plants. Importantly, compared with the wild type, seed-

ling development was not delayed in either of the homozygous

insertion lines, indicating that storage oil mobilization was not

compromised (see Supplemental Figure 4B online). However, in

the pnc1-1 mutant, the T-DNA is located in the last exon of the

PNC1 gene, leading to the expression of a truncated transcript

that encodes a protein lacking the last 19 amino acids of the C

terminus. We expressed recombinant PNC1D19C protein in the

yeast knockout ant1D as described above and found that the

truncated transport protein retains modest protein-mediated

ATP uptake activity when reconstituted into liposomes (see

Supplemental Figure 5 online). Thus, the Arabidopsis T-DNA

insertion line pnc1-1 does not represent a loss-of-function mu-

tant, although the T-DNA had inserted into an exon of the

corresponding gene.

To produce Arabidopsis lines with impaired ATP import into

peroxisomes mediated by PNC1 and PNC2, plants repressing

the expression of both transporter genes were generated using

an RNAi approach (Wesley et al., 2001) with an ethanol-inducible

promoter AlcA (Roslan et al., 2001; Sweetman et al., 2002). A

480-bp cDNA fragment of PNC1 was introduced into an intron-

spliced hairpin RNA construct (see Supplemental Figure 6 on-

line). The selected cDNA fragment of PNC1 is highly similar (86%

identity) to the corresponding region of PNC2, thus enabling

simultaneous silencing of both genes from a single construct.

The ethanol-inducible RNAi construct (AlcApro:PNC1RNAi) was

introduced into Arabidopsis plants by Agrobacterium-mediated

transformation. Several independent transgenic lines (T1 gener-

ation) were obtained after kanamycin selection and integration of

the T-DNA was confirmed by genomic PCR. After selfing, plants

of the T2 generation were screened for homozygous iPNC1

iPNC2 lines by segregation analysis of the nptII resistance gene,

further propagated, and named iPNC12.

To determine the degree of simultaneous repression of PNC1

and PNC2 mRNA in these transgenic lines, we assessed PNC1

and PNC2 steady state transcript levels using RT-PCR. RNA

from independent iPNC1/2 lines grown in the presence or

absence of ethanol was isolated from 5-d-old seedlings. A

strong simultaneous suppression of PNC1 and PNC2 was ob-

served in two independent RNAi lines after ethanol induction,

consistent with their high nucleotide sequence identity, whereas

the expression of the third gene, At2g39970, was not affected

(Figure 5A). The results obtained by RT-PCR for iPNC1/2 lines

1 and 2 were independently verified by quantitative RT-PCR

(qRT-PCR) using two biological replicates (Figure 5B). PNC1 and

PNC2 are;15- and 11-fold downregulated after ethanol induc-

tion in iPNC1/2 line 1 when compared with empty vector control

plants. An even stronger reduction of the relative PNC mRNA

amounts was observed in the second iPNC1/2 line. The relative

transcript levels of both PNC genes showed a slight repression

(twofold and fourfold reduction in lines 1 and 2, respectively) prior

to induction when compared with control plants. Roslan et al.

(2001) described previously that growth on Murashige and

Skoog (MS) agar plates might trigger endogenous ethanol pro-

duction by anaerobic fermentation due to lowoxygen conditions.

This could explain the slight decrease of target genes in iPNC1/2

lines prior to ethanol application relative to the control. Impor-

tantly, the transgenic lines showed growth defects only after

ethanol induction (Figure 6), in parallel with the strong simulta-

neous repression of both PNC1 and PNC2 under these condi-

tions (Figure 5B).

The PNC1/2 RNAi Lines Are Compromised in

Seedling Establishment

The role of PNC1 and PNC2 in b-oxidation during postgermina-

tive growth was investigated by examining seedling establish-

ment of homozygous iPNC1/2 plants. Mutants defective in

import (cts-1), activation (lacs6/7), and degradation of fatty acids

(b-oxidation mutants, such as 3-ketoacyl-CoA thiolase mutant

[kat2-1]/peroxisome defective 1 [ped1]) are unable to mobilize

seed storage oil and seedling growth is arrested (Hayashi et al.,

1998, 2002; Germain et al., 2001; Footitt et al., 2002; Fulda et al.,

2004). However, the presence of an alternative carbon source,

such as sucrose, can rescue the phenotype.

In our study, seeds of several independent iPNC1/2 lines were

grown on agar plates containing 0.2% (v/v) ethanol under con-

trolled growth conditions. To assess whether sucrose was re-

quired for seedling establishment, the seeds were placed on

media either with or without sucrose. A defect in seedling growth

upon ethanol-induction of RNAi expression was reproducibly

observed in the absence of sucrose (Figure 6A). Growth and

development were arrested in iPNC1/2 seedlings compared with

control plants carrying the empty vector. In contrast with other

3246 The Plant Cell

known mutants that exhibit the sucrose-dependent phenotype

described above, iPNC1/2 treated with ethanol did develop

seedlings on plates with sucrose but grew significantly slower

than the control plants (delayed seedling growth). In the absence

of the inducer ethanol, the growth rates and phenotypes of

iPNC1/2 were indistinguishable from the wild type (Figure 6A).

Similarly, dark-grown hypocotyls of PNC1/2 RNAi seedlings

were much shorter than those of wild-type or empty vector

controls when grown in the absence of sucrose, indicative of

their inability to mobilize storage oil for hypocotyl elongation

(Penfield et al., 2004). Sucrose increased the elongation of RNAi

hypocotyls but did not restore them to the same length as the

wild type, indicating other functions for PNC1 and 2, in addition

to storage oil mobilization (Figure 6B).

Although some mutants in which b-oxidation is severely im-

paired exhibit a defect in germination (Pinfield-Wells et al., 2005;

Pracharoenwattana et al., 2005; Footitt et al., 2006), germination

efficiency was not affected in the iPNC1/2 repression lines as

shown for the kat2-1 and cts-1mutant (see Supplemental Figure

7 online). Two independent iPNC1/2 lines, line 1 and line 2, that

showed a severe ethanol-inducible phenotype were selected for

further studies.

Transgenic Lines with an Impaired ATP Import into

Peroxisomes Accumulate Fatty Acids and Fatty Acyl-CoA

and Retain Oil Bodies

In wild-type plants, TAG stored in oil bodies is degraded via

lipolysis and peroxisomal b-oxidation and converted to sucrose

to fuel postgerminative seedling establishment (Cooper and

Beevers, 1969). To examine whether the arrested seedling

growth of the PNC1/2 RNAi lines is linked to impaired lipid

Figure 5. Analysis of the PNC1 and PNC2 Transcripts in Arabidopsis Seedlings of PNC1/2 RNAi and Control Plants.

Seedlings of two representative PNC1/2 RNAi lines and seedlings transformed with the empty vector (control) were grown for 5 d on half-strength MS

agar plates in the presence (+) of 0.2% (v/v) ethanol or in the absence (�) of ethanol.

(A) For the RT-PCR analysis, gene-specific primers were used to amplify PNC1, PNC2, At2g39970, and ACT7 from total RNA. RNA gel electrophoresis

was performed to verify that equal amounts of total RNA were used for the cDNA synthesis by staining with ethidium bromide.

(B) Relative transcript levels of PNC1 and PNC2 were determined by qRT-PCR using gene-specific primers binding at the exon-exon junction and the 39

untranslated region. The reference gene, TIP41-like gene (At4g34270), was used for normalization of gene transcript levels among all samples. The data

represent the mean normalized expression (MNE) values 6 SE of three technical replicates from two independent experiments.

Arabidopsis Peroxisomal ATP Transporters 3247

breakdown, levels of fatty acids and acyl-CoA esters were

measured in 5-d-old seedlings of two independent iPNC1/2

lines treated with ethanol, and the presence of oil bodies in

seedling tissues was investigated by microscopy. The total

content of fatty acids was increased eightfold in 5-d-old

iPNC1/2 seedlings compared with seedlings transformed with

the empty vector (control). As shown in Figure 7A, C16:0, C18:0,

C18:1. C18:2, C18:3, C20:1, C20:2, and C20:3 fatty acids accu-

mulated in the repression lines. In particular, the content of

eicosenoic acid (C20:1), which is considered to be diagnostic of

TAG (Lemieux et al., 1990), was 10 to 20 times higher in iPNC1/2

seedlings than in control plants. Consistent with the accumula-

tion of fatty acids, a strong increase in the quantity of long/very-

long-chain acyl-CoA esters (C20:0, C20:1, and C22:1) was

observed in iPNC1/2 seedlings in comparison with the controls

(Figure 7B). In the absence of ethanol, little difference in either the

level of fatty acids or acyl-CoAs was observed in iPNC1/2

seedlings compared with the empty vector control (see Supple-

mental Figure 8 online), indicating that ethanol-induced silencing

of PNC1 and PNC2 gene expression causes the accumulation of

fatty acids and acyl-CoA esters.

Examination of seedlings stained with the lipophilic dye, Nile

Red, revealed that, while oil bodies were largely absent in wild-

type hypocotyls 5 d after germination, the RNAi seedlings

exhibited a marked retention of oil bodies (Figure 7C). Taken

together, the retention of oil bodies and the accumulation of fatty

acids and acyl CoAs indicate that both lipolysis and b-oxidation

are impaired in iPNC1/2 seedlings.

The PNC1/2 RNAi Lines Are Resistant to Root Growth

Inhibition in the Presence of IBA and 2,4-DB

The requirement of peroxisomal ATP import for b-oxidation of

compounds other than fatty acids was assessed by screening

the response of pnc1/2 RNAi lines to IBA and 2,4-DB. In wild-

type plants, both compounds are activated with CoA in an

ATP-dependent manner and then converted by one cycle of

b-oxidation to the active auxin indole-3-acetic acid and the

herbicide 2,4-D, respectively, which severely inhibit root and

hypocotyl growth (Hayashi et al., 1998; Zolman et al., 2000;

Rashotte et al., 2003). Primary root elongation was analyzed in

PNC1/2 RNAi seedlings grown on agar plates supplemented

with 0.5% (w/v) sucrose, 0.2% (v/v) ethanol, and either 30 mM

IBA or 0.8 mM 2,4-DB. As shown in Figure 8A, the PNC1/2 RNAi

seedlings exhibited a moderate level of resistance to IBA and

2,4-DB compared with wild-type seedlings and empty vector

controls. We also investigated auxin responses of dark-grown

hypocotyls from seedlings of two PNC1/2 RNAi lines, empty

vector controls, and the wild type. Although hypocotyls are

somewhat less sensitive to the application of exogenous auxins

than roots (Zolman et al., 2000; Rashotte et al., 2003), both IBA

and 2,4-DB inhibited elongation of wild-type and control hypo-

cotyls. By contrast, the hypocotyls of PNC1/2 RNAi seedlings

exhibited resistance to these compounds, consistent with a

block in their b-oxidation (Figure 8B). These results indicate that

the activation of IBA and 2,4-DB is dependent on peroxisomal

ATP import catalyzed by PNC1/2.

Figure 6. Postgerminative Seedling Growth of Arabidopsis PNC1/2

RNAi Plants.

(A) Seedlings of two representative PNC1/2 RNAi lines and seedlings

transformed with the empty vector (control) were grown for 15 d vertically

on half-strength MS agar plates supplemented with 1% (w/v) sucrose

(+Suc) or lacking sucrose (�Suc) in the presence of 0.2% (v/v) ethanol

(+EtOH) or in the absence of ethanol (�EtOH). Seedlings were rear-

ranged on fresh plates and photographed. Bars = 1 cm.

(B) Effect of exogenous sucrose on hypocotyl growth in the dark. Seed-

lings of two representative PNC1/2 RNAi lines, wild type, and plants

transformed with empty vector (control) were plated on half-strength

MS agar plates supplemented with 0.2% (v/v) ethanol in the absence

(�Suc) or presence of 0.8% (w/v) sucrose (+Suc). Following 24 h of ex-

posure to light to permit germination, plates were incubated for 5 d in the

dark. Values are means6 SE of measurements from 20 to 30 hypocotyls.

3248 The Plant Cell

DISCUSSION

b-Oxidation plays an essential role in seedling establishment to

support growth and development (Baker et al., 2006; Graham,

2008). Fatty acids released from TAG are degraded via perox-

isomal b-oxidation and converted to sucrose during postgermi-

native growth to supply metabolic energy and carbon skeletons.

However, before entering b-oxidation, fatty acids are imported

into peroxisomes byCTS (Zolman et al., 2001; Footitt et al., 2002;

Hayashi et al., 2002) and further activated with CoA by LACS6

and LACS7 (Fulda et al., 2004). Both steps are required for

postgerminative storage oil breakdown to enable normal seed-

ling establishment (Footitt et al., 2002; Hayashi et al., 2002; Fulda

et al., 2004).

In this study, we investigated whether the initial ATP-depen-

dent activation reaction of b-oxidation depends on the import of

Figure 7. Fatty Acid and Acyl-CoA Profile and Visualization of Hypocotyl Oil

Bodies in 5-d-OldArabidopsisSeedlings ofPNC1/2RNAi andControl Plants.

(A) and (B) Seedlings of PNC1/2 RNAi line 1 (gray bars) and line 2 (black

bars) and seedlings transformed with the empty vector control (white

bars) were grown for 5 d on half-strength MS agar plates supplemented

with 0.5% (w/v) sucrose in the presence of 0.2% (v/v) ethanol. The values

are means6 SE of the mean ([A] three replicates of 50 seedlings; [B] five

replicates of 10 to 25 seedlings).

(C) Seedlings of a representative PNC1/2 RNAi line (line 1; A and B) and

the wild type (Col-0; C and D) were grown for 5 d on half-strength MS

agar plates supplemented with 0.5% (w/v) sucrose in the presence of

0.2% (v/v) ethanol. Hypocotyl cells were visualized by microscopy. A and

C, bright-field images; B and D, Nile Red fluorescence. Bars = 35 mm.

Figure 8. Root and Hypocotyl Growth Response to IBA and 2,4-DB.

(A) Effect of IBA and 2,4-DB on primary root elongation. Seedlings of two

representative PNC1/2 RNAi lines, wild-type seedlings, and control

plants carrying the empty vector (control) were grown for 5 d on half-

strength MS agar plates supplemented with 0.5% (w/v) sucrose and

0.2% (v/v) ethanol in the presence of 0.8 mM 2,4-DB (black bars) or 30

mM IBA (gray bars). Relative growth is expressed as a percentage of the

length of the primary root in the absence of 2,4-DB and IBA. The values

are means6 SE of the mean of measurements from three replicates of 15

to 20 seedlings.

(B) Effect of IBA and 2,4-DB on elongation of hypocotyls in the dark.

Seedlings of two representative PNC1/2 RNAi lines and of the wild type

were allowed to germinate in the light for 24 h and then grown for 5 d in

the dark on half-strength MS agar plates supplemented with 0.8% (w/v)

sucrose and 0.2% (v/v) ethanol in the presence of 0.8 mM 2,4-DB (black

bars) or 30 mM IBA (gray bars). Relative growth is expressed as a

percentage of the hypocotyl length in the absence of 2,4-DB and IBA.

The values are means 6 SE of the mean (three replicates of 20 to 30

seedlings).

Arabidopsis Peroxisomal ATP Transporters 3249

external ATP into peroxisomes by specific transport proteins

during postgerminative growth. We identified two novel perox-

isomal ATP transporters from Arabidopsis using a candidate

gene approach. Based on sequence similarity to previously

characterized ATP transporters from yeast peroxisomes

(Palmieri et al., 2001; van Roermund et al., 2001), three members

of the Arabidopsis mitochondrial carrier family encoded by the

genes At2g39970, At3g05290, and At5g27520 were identified.

Complementation studies of the yeast mutant deficient in per-

oxisomal ATP uptake revealed that At3g05290 and At5g27520

could be involved in ATP import into yeast peroxisomes. Such a

transport function is required to metabolize medium-chain fatty

acids and restore the yeast growth phenotype (Figure 2). Thus,

both proteins (PNC1 and PNC2) are peroxisomal nucleotide

carriers in Arabidopsis.

Interestingly, the third candidate, which is encoded by

At2g39970 and annotated as peroxisomal membrane protein

PMP38, did not complement the yeast growth phenotype, al-

though colocalization experiments using fluorescence micros-

copy confirmed the peroxisomal localization in yeast (Figure 3).

The peroxisomal localization of Arabidopsis PMP38 in pumpkin

(Cucurbita sp) has already been demonstrated by cell fraction-

ation and immunocytochemical analysis in an earlier study

(Fukao et al., 2001). Because the abundance of the PMP38

protein was coregulated with that of the fatty acid b-oxidation

enzymes during postgerminative growth, this transporter was

proposed to be involved in fatty acid degradation as a putative

peroxisomal ATP carrier (Fukao et al., 2001). However, PMP38

most likely has a different substrate specificity, and our results

strongly indicate that PMP38 does not function in transport of

ATP across the peroxisomal membrane. Further analysis to

determine the transport function or in vivo role of mutants

deficient in this protein has yet to be done.

Our in vitro ATP uptake studies showed that recombinant

reconstituted PNC1 and PNC2 catalyze the strict counterex-

change of ATP with AMP (Figure 4). This is consistent with the

physiological function of the peroxisomal carrier. ATP is imported

into peroxisomes, where it is converted to AMP by fatty acid

activation, and AMP is then subsequently exported to the cytosol

(Rottensteiner and Theodoulou, 2006). Like their putative yeast

ortholog ANT1, both Arabidopsis transport proteins accept ADP

as an additional substrate. Among the diverse group of eukary-

otic adenylate transporters, only the Brittle1 homolog from

potato (Solanum tuberosum) (Leroch et al., 2005) shares the

unique substrate specificity for AMP with the peroxisomal ATP

carriers. All other ATP transporters from mitochondria, plastids,

and ER are highly specific for ATP and ADP and do not transport

AMP (Heimpel et al., 2001; Haferkamp et al., 2002; Leroch et al.,

2008). In the physiological context, counterexchange of ATPwith

AMP across the peroxisomal membrane is appropriate because

during b-oxidation LACS proteins consume ATP and produce

AMP (Fulda et al., 2004). Since several protein kinases and ATP-

dependent chaperones have recently been identified by proteo-

mics in the peroxisomal matrix (Reumann et al., 2004, 2007),

which requires ATP and generates ADP, an ATP/ADP counter-

exchange activity is needed. The determination of inhibition

constants (Ki) revealed that peroxisomal ATP transporters from

Arabidopsis and S. cerevisiae exhibit a two to three times higher

apparent Ki for AMP than for ATP (Table 1), indicating that AMP

only inhibits the import of ATP into peroxisomes at high concen-

trations of AMP. Especially in sink tissues, such as cotyledons

during postgerminative growth, the cellular ATP level is low and

the adenosine monophosphate kinase (adenylate kinase) pro-

vides additional ATP by converting two ADP molecules into ATP

and AMP, resulting in a low ATP/AMP ratio (Stitt et al., 1982;

Geigenberger and Stitt, 2000; Igamberdiev and Kleczkowski,

2006). Under these conditions, the import of ATP into peroxi-

somes is favored due to the kinetic properties of the PNC

proteins.

Since PNC1 and PNC2 exhibit similar biochemical transport

properties and tissue expression profiles (see Supplemental

Figure 3 online), it is likely that these peroxisomal transporters are

functionally redundant. To investigate the in vivo role of ATP

import into peroxisomes, we initiated a strategy to construct a

double knockout for both PNC genes. As the pnc1-1 T-DNA

insertion line did not lead to a dysfunctional PNC1 protein and a

second allele is not available in any of the publicly accessible

collections, we generated ethanol-inducible pnc1/2 RNAi plants

to suppress the peroxisomal ATP import activity in Arabidopsis.

The repression of both PNC genes after ethanol induction was

confirmed at the transcript level in these RNAi lines. The phys-

iological and biochemical analyses of iPNC1/2 plants strongly

indicate that the storage oil breakdown in peroxisomes during

postgerminative growth requires an external supply of ATP,

suggesting that PNC1/2 activity is critical for mobilizing fatty

acids through b-oxidation.

The iPNC1/2 plants are defective in seedling growth and

development when induced with ethanol (Figure 6). Supply of

sucrose restored seedling growth (Figure 6), which is also the

case for several other known mutants affected in the import of

fatty acids into peroxisomes (cts-1), activation of fatty acids

(lacs6/7), and in fatty acid b-oxidation, such as the kat2/ped1

mutant, which is deficient in thiolase activity (Hayashi et al., 1998,

2002; Germain et al., 2001; Footitt et al., 2002; Fulda et al., 2004).

Intriguingly, however, the observation that sucrose could not com-

pletely rescue the arrested growth phenotype of the iPNC1/2

seedlings in the presence of ethanol (Figure 6) indicates a

pleiotropic effect of impaired peroxisomal ATP import beyond

activation of fatty acids. We assume that hitherto unknown

ATP-dependent processes within peroxisomes, besides the

activation of fatty acids, require ATP and depend on external

ATP import into peroxisomes mediated by PNC1 and PNC2

(Reumann et al., 2004, 2007).

A common feature of b-oxidation mutants, including the cts-1

mutant and the lacs6 lacs7 double mutant, is impaired fatty acid

breakdown, associated with retention of storage oil–specific

fatty acids in the cotyledons and hypocotyls (Baker et al., 2006;

Graham, 2008). After ethanol induction, iPNC1/2 seedlings also

accumulated fatty acids, such as C18:2, C18:3, and C20:1

(Lemieux et al., 1990; Millar and Kunst, 1999). This indicates

that ATP import is required for complete TAG breakdown to

support seedling establishment. A similar fatty acid profile as

observed in the iPNC1/2 lines was reported for seedlings defec-

tive in the peroxisomal ABC transporter CTS and in fatty acid

activating enzymes LACS6/7 (Footitt et al., 2002; Fulda et al.,

2004). Together with the results from the cts-1 and lacs6 lacs7

3250 The Plant Cell

mutants, it can be concluded that storage oil–derived fatty acid

breakdown is compromised in the PNC1/2 RNAi lines, causing

the defect in seedling establishment. Interestingly, the marked

increase in corresponding acyl-CoA esters in the iPNC1/2 seed-

ling is also observed in the cts-1 and lacs6 lacs7 mutants. It is

likely that the CoA pool serves as a sink for nonesterified fatty

acids released by lipolysis of TAG in these mutants. Because

an accumulation of free fatty acids in the cytosol is potentially

deleterious, they have to be diverted to other pools. While long-

chain fatty acids can be incorporated into membrane lipids,

very-long-chain fatty acids are converted to CoA esters by an

unknown cytosolic or microsomal acyl-CoA synthetase (Larson

et al., 2002). The Arabidopsis genome encodes a large family of

acyl-activating enzymes with diverse substrate specificity and

subcellular localization (Shockey et al., 2003; Kienow et al.,

2008). Although not supported by experimental evidence to date,

it is likely that one of the currently uncharacterized AAEmembers

is involved in the CoA transfer.

At first sight, the retention of oil bodies and accumulation of

TAG-derived fatty acyl-CoAs appears counterintuitive. However,

this phenotype, shared by other b-oxidation mutants, is consis-

tent with a combination of limited lipolysis and a defect in

b-oxidation. It has been proposed that accumulation of acyl-

CoAs in mutant seedlings leads to a block in storage oil mobili-

zation from the oil body, perhaps by inhibition of lipolysis (Germain

et al., 2001;Grahamet al., 2002;Graham, 2008). In agreementwith

this, activity of neutral TAG lipase from castor bean (Ricinus

communis) is inhibited by oleoyl-CoA (Hills et al., 1989).

Mutants in which b-oxidation is severely impaired, such as

kat2/ped1, cts-1, and the peroxisomal citrate synthase double

mutant, csy2 csy3, exhibit a defect not only in postgerminative

seedling establishment, but also in germination (Hayashi et al.,

1998; Footitt et al., 2002, 2006; Pinfield-Wells et al., 2005;

Pracharoenwattana et al., 2005). Interestingly, the germination

rate of PNC1/2 RNAi seeds was not compromised (see Supple-

mental Figure 7 online). This indicates that the unknown function

of b-oxidation, which is required for germination, is not signifi-

cantly affected in PNC1/2 RNAi seeds, possibly reflecting the

incomplete suppression of these genes in the transgenic lines. In

this context, it should be noted that the lacs6 lacs7 double

mutant also germinates, albeit more slowly than the wild type

(Fulda et al., 2004; Footitt et al., 2006).

The iPNC1/2 plants are partially resistant to the inhibitory

effect of IBA and 2,4-DB on elongation of primary roots and dark-

grown hypocotyls, suggesting that the activation of the corre-

sponding precursors and hence their subsequent b-oxidation is

dependent on peroxisomal ATP import. However, the degree of

resistance is lower than that observed in known b-oxidation

mutants, including cts-1, kat2/ped1, and acx3 (Hayashi et al.,

1998; Zolman et al., 2000, 2001; Footitt et al., 2002; Rylott et al.,

2003). A residual ATP import activity in the iPNC1/2 lines

might be sufficient to channel low levels of IBA and 2,4-DB into

b-oxidation for conversion to indole-3-acetic acid and 2,4-D,

resulting in partial inhibition of root elongation. Root growth of the

lacs6 lacs7 double mutant is inhibited in the presence of IBA and

2,4-DB, indicating that LACS6 and LACS7 are only specific for

long-chain fatty acids (Fulda et al., 2004). Thus, an unknown

acyl-activating enzyme must catalyze the activation of the IBA

and 2,4-DB precursor. To date, it was not known whether the

protein(s) catalyzing IBA/2,4-DB activation is (are) localized

inside the peroxisomes or whether CTS imports the activated

precursor into peroxisomes. Our results suggest that the acti-

vation depends on peroxisomal ATP import, implying that the

activation reaction takes place within peroxisomes and not in the

surrounding cytosol.

Taken together, the data presented here are consistent with

the hypothesis that the diverse substrates of the b-oxidation,

such as fatty acids, IBA, and 2,4-DB, are transported across the

peroxisomalmembrane by CTS as free acids and then activated

by ATP-dependent reactions inside peroxisomes, which are

supplied with external ATP by PNC1/2. PNC1 and PNC2 rep-

resent the exclusive route for peroxisomal ATP supply because

Arabidopsis plants defective in peroxisomal ATP import are

compromised in storage oil mobilization and resistance to root

growth inhibitors as a consequence of a limiting supply of ATP

within peroxisomes. However, it remains a formal possibility

that at least some b-oxidation substrates are imported by

CTS as CoA esters and require an intraperoxisomal ATP supply

because they are deesterified at some stage during the

b-oxidation spiral. This is consistent with the identification of

three peroxisomal acyl-activating enzymes of the AAE family,

which are involved in JA biosynthesis (Schneider et al., 2005;

Theodoulou et al., 2005; Koo et al., 2006; Kienow et al., 2008).

The JA precursor 12-oxophytodienoic acid is imported into

the peroxisome by two routes, one of which requires CTS

(Theodoulou et al., 2005), where it is reduced and further

converted to JA by three consecutive rounds of b-oxidation

(Schaller et al., 2004; Baker et al., 2006; Delker et al., 2007).

The observation that the peroxisomal AAE enzymes exhibit

specificity for different JA biosynthetic intermediates (12-

oxophytodienoic acid, OPC:8, OPC:6, and OPCC:4) indicates

that the CoA moiety is removed at several alternate steps in the

pathway. Accordingly, peroxisomes of all organisms contain a

range of thioesterases, one of which has been shown to play an

essential role in the breakdown of short straight-chain and

branched-chain fatty acids in yeast (Maeda et al., 2006). Two

peroxisomal thioesterases have been reported in Arabidopsis,

but their physiological function is not yet clear (Tilton et al.,

2004). It has been suggested that removal of the CoA moiety

may play a role in the regulation of b-oxidation and prevent

sequestration of free CoA due to buildup of unusual CoA-

esterified species that are inefficiently metabolized (Hunt and

Alexson, 2008). Thus, the requirement of several physiological

processes for peroxisomal ATP import could also reflect the

need to reactivate intermediates to enter b-oxidation.

Knowledge of metabolite transport across the peroxisomal

membrane is scarce. Multiple peroxisomal pathways are con-

nected with metabolic pathways in other cellular compartments,

implying that a large variety of small molecules must be trans-

ported across the peroxisomal membrane. The characterization

of PNC1 and PNC2 reported here is a step toward obtaining a

mechanistic and molecular understanding of peroxisomal trans-

porters in plants. The PNC1/2 RNAi lines established in this work

provide an opportunity to further investigate whether, in addition

to the initial activation of b-oxidation substrates, yet to be

defined reactions demand ATP in plant peroxisomes.

Arabidopsis Peroxisomal ATP Transporters 3251

METHODS

Materials

Chemicals were purchased from Sigma-Aldrich, and anion exchange

resin Dowex AG1-X8 Resin was supplied by Bio-Rad Laboratories.

Radiochemical [a-32P]ATP was provided by GE Healthcare. Reagents

and enzymes for recombinant DNA techniques were obtained from

Invitrogen, New England Biolabs, and Promega.

Plant Material and Growth Conditions

Wild-type Arabidopsis thaliana (ecotype Col-0) and the Arabidopsis

T-DNA insertion lines, SAIL_303H02 (pnc1-1) and SALK_014579 (pnc2-1),

were obtained from the ABRC at the Ohio State University. Seeds of

Arabidopsis plants were surface-sterilized, stratified for 4 d at 48C, and

germinated on 0.8% (w/v) agar-solidified half-strength MS medium

supplemented with 1% (w/v) sucrose. Unless stated otherwise, plants

were grown at 228C (70% humidity) in a 16-h-light/8-h-dark cycle in

growth chambers (100 mmol m22 s21 light intensity). Selection of the

transgenic Arabidopsis plants was performed with kanamycin at a

concentration of 50 mg/mL. Selected plants were transferred to soil for

further growth in a chamber under the same conditions. For the analysis

of postgerminative seedling growth, plants were grown on half-strength

MS agar plates supplemented or not with 1% (w/v) sucrose in the

presence or absence of 0.2% (v/v) ethanol at 16-h-light/8-h-dark cycles

and 100 mmol m22 s21 light intensity. To study the response to 2,4-DB

and IBA and for lipid analysis, seedswere plated on half-strengthMSagar

plates supplementedwith 0.5% (w/v) sucrose6 30mM IBAor 0.8mM2,4-

DB (added from stocks dissolved in ethanol) in the presence of 0.2% (v/v)

ethanol at constant illumination (150 mmol m22 s21). After 5 d, seedlings

were photographed and roots measured using MetaMorph software

(Molecular Devices). For measuring the hypocotyl growth in the dark,

seeds were plated on half-strength MS agar supplemented with 0.2% (v/

v) ethanol. Sucrose (0.8% [w/v]), 2,4-DB (final concentration 0.8mM), and/

or IBA (final concentration 30 mM) were added as indicated. Following 2 d

of stratification and 24 h of exposure to light to induce germination, plates

were kept in the dark for 5 d. Hypocotyls were photographed and

measured using MetaMorph software (Molecular Devices). For subcellu-

lar colocalization studies, Nicotiana benthamiana was grown on soil in a

greenhouse under a light regime of 8 h of darkness and 16 h of light.

Phylogenetic Analyses

The sequences were retrieved from the GenBank database of the

National Center for Biotechnology Information (http://www.ncbi.nlm.nih.

gov). The BLAST program (Altschul et al., 1990) was used to search for

putative peroxisomal ATP transport proteins in Arabidopsis based on

sequence similarity to the previously identified ANT1 from Saccharomy-

ces cerevisiae (Palmieri et al., 2001). The amino acid sequences were

aligned by the CLUSTALW program (http://www.ebi.ac.uk/Tools/

clustalw) using default settings. The alignment layout was performed with

the GeneDoc program (http://www.nrbsc.org/gfx/genedoc/index.html).

DNA Sequencing and Cloning Procedures

The clones U13126 and U18363 containing the full-length ORF cDNA of

At3g05290 and At5g27520 were obtained from the ABRC. The corre-

sponding clone for At2g39970 (pda.00682) was provided by Riken

BioResource Center.

For general cloning, DNA sequences were amplified by PCR using

proofreading polymerase (PlatinumPfx polymerase; Invitrogen). The PCR

primers designed for this study are listed in Supplemental Table 2 online.

The PCRproducts were subcloned into pGEM-T Easy (Promega), and the

sequences were verified by sequencing (Research Technology Support

Facility, Michigan State University). Subsequent cloning steps were

performed using standard molecular techniques (Sambrook et al., 1989).

Yeast Complementation Studies

The ant1D knockout mutant deficient in the peroxisomal ATP transporter

ANT1 was purchased from Open Biosystems. For complementation

studies, the ORFs of At2g39970, At3g05290, and At5g27520 were

amplified from the cDNA clones by PCR as described above. The

following gene-specific primers were used: NL16/NL17 for At2g39970,

NL64/NL65 for At3g05290, and NL97/NL106 for At5g27520. The PCR

fragments were cloned via XhoI and BamHI into yeast expression vector

pDR195 under the control of a constitutive promoter of the plasma

membrane H+-ATPase (PMA1pro) from S. cerevisiae (Rentsch et al.,

1995). In addition, the coding sequence of ANT1was PCR-amplified from

genomic DNA of the lab yeast strain INVSc1 (Invitrogen) using the primers

NL55 and NL25, and a yeast expression construct was obtained

according to the cloning strategy described above.

The ant1D strain was cultivated using standard YPD media according

to Sambrook et al. (1989) and transformedwith the expression constructs

using the lithium chloride method (Schiestl and Gietz, 1989). The trans-

formants were selected on synthetic complete minimal medium lacking

uracil (SC-U). For yeast complementation studies, the transformed

knockout mutants were analyzed on SC-U agar plates using lauric acid

as the sole carbon source (Palmieri et al., 2001).

Subcellular Localization of YFP Fusions in Yeast

For localization studies in yeast, a pDR195-based expression vector was

generated containing EYFP for C-terminal fusion of the gene of interest.

The EYFP sequence was obtained from the pEYFP-C1 vector (BD

Biosciences Clontech) by PCR using primers NL260/NL261, subcloned

into pBluescript II SK (Stratagene), and then introduced into pDR195. The

resulting construct was named pNL9. The ORFs of At3g05290 and

At5g27520 were amplified without stop codons by PCR using the primer

pairs NL62/NL63 and NL93/NL94, respectively, and inserted into pNL9,

leading to proteins that are tagged at the C terminus with EYFP. In the

case of At2g39970, the ORF was cloned into the pEFYP-C1 vector via

BamHI and XhoI restriction sites, which were introduced by PCR using

primers NL7 and NL8, respectively. The EYFP-At2g39970 fusion was

inserted into pDR195 using blunt-end ligation. To generate the yeast

expression vector harboring the peroxisomal marker, a Ser-Lys-Leu

motif, known as PTS1, and a stop codon were added to the end of the

CFP sequence from the pECFP-N1 vector (BD Biosciences Clontech) by

PCR using primers NL254/NL255. The CFP-PTS1 sequence was then

cloned into yeast expression vector pACTII (BD Biosciences Clontech)

under the control of a constitutive yeast alcohol dehydrogenase promoter

(ADHpro).

The resulting EYFP fusion constructs and the peroxisomal marker were

cotransformed into the ant1D mutant using the lithium chloride method

(Schiestl and Gietz, 1989). The transformed yeast cells were selected on

SC medium in the absence of uracil and leucine. The expression of the

fusion proteins in yeast was analyzed by epifluorescence microscopy.

Transient Expression of YFP Fusions in Tobacco Epidermal

Leaf Cells

The vector pNL3derived frompUC18 (NewEnglandBiolabs)wasdesigned

by introducing the cauliflowermosaic virus 35S promoter (35Spro), theORF

of EFYP for fusion to the C terminus of ORFs At3g05290 and At5g27520,

and the polyadenylation site of the ribulose-1,5-bis-phosphate

3252 The Plant Cell

carboxylase/oxygenase small subunit (rbcster) from Pisum sativum. As

described for yeast EYFP fusion constructs, the ORFs of At3g05290 and

At5g27520 were cloned via KpnI into pNL3. The resulting expression

cassetteswere then inserted viaEcoRI and a blunt-end site into the binary

plant vector pCAMBIA3301 (CAMBIA). In the case of At2g39970, theORF

was PCR-amplified using the primer set NL7/NL8 and cloned into pEFYP-

C1 via BamHI and XhoI. The sequence encoding the EYFP-At2g39970

fusion protein was inserted into pCAMBIA3301 using the blunt-end site.

To construct the plant vector harboring the peroxisomalmarker, the EYFP

sequence of the pNL3 vector was exchanged with CFP-PTS1, which was

amplified from the pECFP-N1 vector (BD Biosciences Clontech) and

modified by PCR using NL254 and NL255 primers to introduce the PTS1

signal and a stop codon. The expression cassette of the peroxisomal-

targeted marker was subsequently cloned into the plant vector pPZP211

(Hajdukiewicz et al., 1994).

Agrobacterium tumefaciens strain GV3101 (Koncz and Schell, 1986)

containing the expression cassette of EYFP fusions was coinfiltrated with

those transformed with the peroxisomal marker construct into leaves of

N. benthamiana (Romeis et al., 2001). After 72 h, the epidermal cell layers

from the tobacco leaves were peeled and analyzed for transient coex-

pression of fusion proteins by fluorescence microscopy.

Fluorescence Microscopy

Coexpression of the EYFP fusion proteins and the peroxisomal marker

(CFP-PTS1) in yeast or tobacco epidermal cells was examined using a

Zeiss Axio Imager microscope (Carl Zeiss) with either a YFP filter

(excitation, 500 6 12 nm; emission, 542 6 13.5 nm) or a CFP filter

(excitation, 438 6 12 nm; emission, 483 6 16 nm). Digital images were

processed using the AxioVision software (Carl Zeiss).

Reconstitution of Transport Activities into Liposomes

To reconstitute recombinant proteins of PNC1, PNC2, and S. cerevisiae

ANT1, the corresponding ORFs were expressed in ant1D using the

pDR195-based constructs designed in this study for yeast complemen-

tation assays. Fifty milliliters of SC-U medium containing 2% (w/v)

glucose was inoculated with an overnight culture of ant1D harboring the

various yeast expression constructs and cultured to an OD600 of 0.4. The

yeast cells were grown for 6 h aerobically at 308C. Control cultures with an

empty vector were processed in parallel. Harvest and enrichment of yeast

membranes with heterologously expressed PNC1, PNC2, and ANT1

proteins were achieved according to Bouvier et al. (2006).

Recombinant PNC1 and PNC2 proteins were also obtained by expres-

sion in the Escherichia coli strain BL21-CodonPlus (Stratagene). The

gene-specific primers NL64/65 and NL97/106 were used to amplify and

clone the corresponding ORFs into the pET16b expression vector (Merck

Biosciences, using the XhoI and BamHI restriction sites. Protein produc-

tion was induced with isopropyl-b-D-1-thiogalactopyranoside, and trans-

formed cells were harvested after 2 h. Inclusion bodies were purified as

described by Schroers et al. (1997). The expression of the N-terminal His-

tagged proteins was confirmed by SDS gel blot analysis using a mono-

clonal anti-penta-His antibody (Qiagen).

The yeast membrane fractions or E. coli inclusion bodies were

reconstituted into liposome suspension by the freeze-thaw procedure

(Kasahara and Hinkle, 1976). The liposomes had been prepared from

acetone-washed L-a-phosphatidylcholine (PC) by sonication (5 min on

ice, Branson Sonicator 250, output 2, duty cycle 30%) to a final concen-

tration of 2% (w/v) in 100 mM Tricine-KOH, pH 7.8, 20 mM potassium

gluconate, and 30 mM AMP (unless stated otherwise). For uptake in the

absence of internal substrate, phospholipids were resuspended in 100

mM Tricine-KOH, pH 7.8, and 50 mM potassium gluconate. After

thawing, the proteoliposomes were pulsed 15 times to yield unilamellar

vesicles. Unincorporated counterexchange substrates were removed by

passing the proteoliposomes over PD10 gel filtration columns (GE

Healthcare) that were equilibrated with 100 mM sodium acetate, 40 mM

potassium gluconate, and 10 mM Tricine-KOH, pH 7.2.

The uptake assay was started by adding radiolabeled [a32P]ATP or

[a32P]ADP, which was synthesized from [a32P]ATP according to Tjaden

et al. (1998) to the proteoliposomes. To terminate the reaction, 150 mL of

proteoliposomes for each measurement point was passed over Dowex

AG1-X8 columns (Chloride form, 100 to 200 mesh, equilibrated with 150

mM sodium acetate). Radiolabeled substrate that was not taken up into

liposomes was removed by binding to the anion-exchange column. The

eluted proteoliposomes were collected in scintillation vials filled with 10

mL deionized water. The imported radiolabeled ATP or ADP was quan-

tified by a liquid scintillation counter (Tri-Carb 2100TR; Packard). Kinetic

constants were determined by measuring the initial velocity of each

experiment. The Michaelis-Menten constant (Km) has been analyzed with

seven external ATP concentrations, ranging from 0.01 to 1 mM, and

competitive compounds of ATP transport were assessed by the inhibitor

constant Ki, as described by Dixon (1953). Nonlinear regression analysis

based on the Marquardt algorithm was performed with Grafit software

version 3.0 to fit all enzyme kinetic data (Erithacus Software).

Gene Expression Data

Tissue-specific gene expression patterns of PNC1 and PNC2 in Arabi-

dopsis were retrieved from the Arabidopsis eFP Browser (Winter et al.,

2007).

Isolation of T-DNA Insertion Lines

T-DNA insertion lines for PNC1 (SAIL_303H02, referred to as pnc1-1) and

PNC2 (SALK_014579, referred to as pnc2-1) were obtained from the

ABRC. To identify homozygous T-DNA insertion lines, genomic DNA was

isolated and genotyped using gene-specific primer pairs (NL268/NL269

for pnc1-1 and NL273/NL274 for pnc2-1) and a primer pair for T-DNA/

gene junction (P48/NL268 for pnc1-1 and P46/NL274 for pnc2-1). The

position of the T-DNA in the PNC genes was verified by sequencing. As a

positive control, amplification of the actin gene ACT2 (At3g18780) was

performed with the primer pair P33/P34. Independent homozygous

pnc1-1 and pnc2-1 plants were further propagated.

Generation of Arabidopsis RNAi Lines

To construct a double-stranded RNA hairpin structure, a 480-bp cDNA

fragment of PNC1 that showed 86% similarity to PNC2 on the nucleotide

level was amplified from the ABRCcDNA cloneU13126 by PCR using two

different sets of primers (NL66/NL67 and NL68/NL69) (see Supplemental

Figure 6 online). One PCR product was inserted into the pHannibal vector

(AJ311872) (Wesley et al., 2001) in the sense orientation via EcoRI and

XhoI restriction downstream of the pyruvate orthophosphate dikinase

(PDK) intron from E. coli, whereas the other was introduced in the

antisense orientation using XbaI and BamHI sites upstream of the PDK

intron. The resulting intron-spliced hairpin RNA cassette was subcloned

into the pACN vector (AF169416) between the ethanol-inducible alcohol

dehydrogenase promoter (AlcApro) from Aspergillus nidulans and the

nopaline synthase terminator (noster) (Caddick et al., 1998). For in planta

transformation, the AlcApro:PNC1RNAi-noster construct was introduced

using the HindIII site into the pBIN19-derived plant transformation vector

(Caddick et al., 1998), which carries the cassette for the expression of the

ALCR transcription factor (35Spro:ALCR-noster) required to activate the

AlcA promoter in the presence of ethanol. Wild-type Arabidopsis plants

were transformed by anAgrobacterium-mediated procedure (Clough and

Bent, 1998) and were selected using kanamycin resistance. T-DNA

insertion was confirmed in several transgenic lines (T1 generation) by

Arabidopsis Peroxisomal ATP Transporters 3253

genomic PCR using the primer pair P13/P30. T2 plants obtained after

selfing were screened for homozygous lines by segregation analysis of

the nptII resistance gene and further propagated.

RT-PCR and qRT-PCR

Total RNA was extracted from wild-type and transgenic Arabidopsis

plants generated in this study using the guanidinium thiocyanate-phenol-

chloroform extraction method (Chomczynski and Sacchi, 1987) and

subjected after DNase treatment to cDNA synthesis using Superscript II

RNaseH2 reverse transcriptase (Invitrogen). The transcript level ofPNC1,

PNC2, and At2g39970 was analyzed by PCR using gene-specific primer

sets binding to the corresponding 59 and 39 untranslated regions of the

PNC1 (primer set NL84/NL85), PNC2 (NL115/NL116), and At2g39970

(NL48/49) cDNAs. For the amplification of the truncated PNC1 transcript

in the pnc1-1 T-DNA insertion line, the primer set NL84/NL325 was used.

As a control for the cDNA quality, a cDNA fragment of an actin gene

(ACT7, At5g09810) was amplified using the primers ML167 and ML168.

PCR conditions used were as follows: 948C for 2 min, followed by 35

cycles of 948C for 30 s, 588C for 45 s, 728C for 90 s, and a final extension of

728C for 3 min. For relative quantification of the transcript level, qRT-PCR

was performed with the DNA Engine Thermal Cycler Detection system

(Bio-Rad Laboratories) and MESA GREEN qPCR MasterMix for SYBR

Assay (Eurogentec). The following primer pairs were used for gene-

specific amplification: NL326/NL85 forPNC1andNL326/NL116 for PNC2.

For all samples, cDNA levelswere normalized against those of the Type2A

phosphatase interacting protein 41 (TIP41)-like gene (At4g34270) ampli-

fied with the primer pair P71/P72 (Czechowski et al., 2005). The qRT-PCR

primers were designed with PerlPrimer (Marshall, 2004).

Lipid Analysis

Fatty acids were analyzed by gas chromatography following conversion

to methyl esters (FAMEs). The method of Browse et al. (1986) was

employed with the following modification: FAMEs were extracted with

1 mL hexane, the solvent was removed under a stream of N2, and FAMEs

were resuspended in 50 mL hexane. The fatty acid 17:0 was used as an

internal standard to permit quantification. Fatty acyl CoAsweremeasured

as described by Larson and Graham (2001).

Visualization of Oil Bodies

Seedlingswere grown as described above and stained for 5min in a 1mg/

mL solution of Nile Red (Molecular Probes). Images were recorded with a

Zeiss Axiophot fluorescencemicroscope (Carl Zeiss) using a 450- to 490-

nm filter. Corresponding bright-field images were also recorded.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the following accession

numbers: Sc ANT1 (NP_0154531), At2g39970 (NP_181526), At3g05290

(PNC1, NP_566251), At5g27520 (PNC2, NP_198104), Os05g32630

(NP_001055452), and PMP34 (NP_006349).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Complementation of ant1D with the EYFP-

Tagged Arabidopsis Proteins.

Supplemental Figure 2. Expression of PNC1 and PNC2 in E. coli and

ADP Uptake into Liposomes Reconstituted with Recombinant PNC

Proteins.

Supplemental Figure 3. Tissue-Specific Expression Pattern of PNC1

and PNC2 in Arabidopsis.

Supplemental Figure 4. Identification of Arabidopsis T-DNA Insertion

Lines for PNC1 and PNC2.

Supplemental Figure 5. Time Kinetics of ATP Uptake into Liposomes

Reconstituted with Recombinant PNC1 and Truncated PNC1D19C

Protein.

Supplemental Figure 6. Hairpin RNA Expression Construct.

Supplemental Figure 7. Germination Phenotype of PNC1/2 RNAi

Lines.

Supplemental Figure 8. Fatty Acid and Acyl-CoA Profile.

Supplemental Table 1. Genes That Are Coexpressed with PNC1 and

PNC2.

Supplemental Table 2. Gene-Specific Primers Used for Cloning in

This Study.

ACKNOWLEDGMENTS

This work was funded by a Feodor Lynen Research Fellowship of the

Alexander von Humboldt Foundation (to N.L.), National Science Foun-

dation Arabidopsis 2010 Grant MCB-0618335 (to A.P.M.W.), and

Deutsche Forschungsgemeinshaft Grant 1781/1-1 (to N.L. and A.P.M.

W.). We thank Jean Devonshire and Kirstie Halsey from the Rothamsted

Centre for Bioimaging for their expert assistance with microscopy.

Rothamsted Research receives grant-aided support from the Biotech-

nology and Biological Science Research Council of the UK. We are

grateful to Gerald Schonknecht for help with analysis of kinetic con-

stants of PNC1 and PNC2. We also thank Daniel Schubert for advice on

qRT-PCR and David Gagneul for helpful discussions. Lisa Leson and

Kirsten Abel are acknowledged for technical assistance.

Received July 11, 2008; revised October 27, 2008; accepted November

13, 2008; published December 10, 2008.

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Arabidopsis Peroxisomal ATP Transporters 3257

DOI 10.1105/tpc.108.062042; originally published online December 10, 2008; 2008;20;3241-3257Plant Cell

Ekkehard Neuhaus and Andreas P.M. WeberNicole Linka, Frederica L. Theodoulou, Richard P. Haslam, Marc Linka, Jonathan A. Napier, H.

Arabidopsis thalianaPeroxisomal ATP Import Is Essential for Seedling Development in

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