The Arabidopsis thaliana Type I Isopentenyl DiphosphateIsomerases Are Targeted to Multiple SubcellularCompartments and Have Overlapping Functions inIsoprenoid Biosynthesis W

Michael A. Phillips,a,1 John C. D’Auria,a Jonathan Gershenzon,a,2 and Eran Picherskyb

a Max Planck Institute for Chemical Ecology, D-07745 Jena, Germanyb Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048

To form the building blocks of isoprenoids, isopentenyl diphosphate (IPP) isomerase activity, which converts IPP to dimethylallyl

diphosphate (DMAPP), appears to be necessary in cytosol, plastids, and mitochondria. Arabidopsis thaliana contains only two

IPP isomerases (Isopentenyl Diphosphate Isomerase1 [IDI1] and IDI2). Both encode proteins with N-terminal extensions similar

to transit peptides and are expressed in all organs, with IDI1 less abundant than IDI2. Examination of enhanced green fluorescent

protein fusions established that IDI1 is mainly in the plastid, whereas IDI2 is mainly in the mitochondria. Both proteins are also in

the cytosol as a result of their translation from naturally occurring shorter transcripts lacking transit peptides, as demonstrated

by59 rapid amplificationofcDNAendscloning. IPP isomeraseactivity in the cytosol wasconfirmedbyuniform labelingof IPP-and

DMAPP-derived units of the cytoplasmic isoprenoid product, sitosterol, when labeled mevalonate was administered. Analysis of

mutant lines showed that double mutants were nonviable, while homozygous single mutants had no major morphological or

chemical differences from the wild type except for flowers with fused sepals and underdeveloped petals on idi2 mutants. Thus,

each of the two Arabidopsis IPP isomerases is found in multiple but partially overlapping subcellular locations, and each can

compensate for the loss of the other through partial redundancy in the cytosol.

INTRODUCTION

Isoprenoids are essential compounds in all organisms, but their

largest structural variety occurs in plants. There, they serve not

only universal roles as photosynthetic pigments, redox cofac-

tors, and hormones but also specialized ecological roles as

feeding deterrents, pollination vector attractants, and phyto-

alexins. Animals and fungi produce the precursors for isoprenoid

biosynthesis via the mevalonic acid pathway, and many bacteria

synthesize the same building blocks through the methylerythritol

phosphate (MEP) pathway. Plants, by contrast, possess both

pathways, with the mevalonic acid pathway operating in the

cytosol (McGarvey and Croteau, 1995) and the MEP pathway

operating in plastids (Rodrıguez-Concepcion and Boronat,

2002). Although proceeding via chemically distinct routes, the

two pathways converge at the formation of isopentenyl diphos-

phate (IPP). Isoprenoid biosynthesis also requires the formation

of dimethylallyl diphosphate (DMAPP), a double bond isomer of

IPP, as both of these C5 diphosphate compounds are used by

prenyltransferases to form the C10, C15, C20, and larger prenyl

diphosphates that serve as precursors in the monoterpene,

sesquiterpene, and diterpene biosynthetic pathways, respec-

tively. In each reaction series, DMAPP is the initial allylic diphos-

phate to which successive units of IPP are added via head-to-tail

condensations (Burke et al., 1999).

While the plastidic MEP pathway results in the synthesis of

both IPP and DMAPP (Hoeffler et al., 2002), the cytosol-localized

mevalonate pathway produces only IPP. IPP can be isomerized

to DMAPP by isopentenyl diphosphate isomerase (IDI), a divalent

metal ion–requiring enzyme found in all living organisms

(Gershenzon and Kreis, 1999). IDI can also catalyze the reverse

reaction from DMAPP to IPP, but the equilibrium favors the forward

reaction. Animals, fungi, and some bacteria contain an enzyme

designated type I IPP isomerase (Muehlbacher and Poulter, 1988;

Anderson et al., 1989; Carrigan and Poulter, 2003); other bacteria

have a nonhomologous type II IPP isomerase (Kaneda et al., 2001).

In plants, only DNA sequences encoding proteins homologous

with type I IPP isomerase have been identified to date (Blanc and

Pichersky, 1995; Campbell et al., 1997; Cunningham and Gantt,

2000; Nakamura et al., 2001; Page et al., 2004).

Based on the pathways of isoprenoid biosynthesis found in

plants, the molar ratio of IPP to DMAPP needed to make various

terpenoid classes varies from 1:1 for monoterpenes to 2:1 for

sesquiterpenes and sterols, 3:1 for diterpenes, carotenoids, and

phytol, and much higher for long-chain polyprenols and polyter-

penes (Gershenzon and Kreis, 1999) (Figure 1). Thus, by con-

trolling the conversion of IPP to DMAPP, IPP isomerase may

have an important role in regulating the biosynthesis of the major

1 Current address: Laboratori de Genetica Molecular Vegetal, ConsejoSuperior de Investigaciones Cientificas, Institut de Recerca i TecnologiaAgroalimentaries, Barcelona 08034, Spain.2 Address correspondence to gershenzon@ice.mpg.de.The authors 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) are: MichaelA. Phillips (mapgmy@ibmb.csic.es) and John C. D’Auria (dauria@ice.mpg.de).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.107.053926

The Plant Cell, Vol. 20: 677–696, March 2008, www.plantcell.org ª 2008 American Society of Plant Biologists

types of isoprenoid products formed. In principle, DMAPP for

isoprenoid formation in nonplastidic compartments could arise

not only from IPP isomerase but also directly from the plastidial

MEP pathway via transport from plastids to other sites of

isoprenoid biosynthesis. However, since the IPP:DMAPP ratio

produced in vitro by 1-hydroxy-2-methylbutenyl 4-diphosphate

reductase, the last step in the MEP pathway, is 6:1 (Rohdich

et al., 2003; Eisenreich et al., 2004), IPP isomerase activity might

still be necessary to optimize the relative amounts of these two

C5 intermediates for efficient isoprenoid biosynthesis both in the

plastids and elsewhere in the cell.

With the distribution of the various branches of isoprenoid

biosynthesis in different subcellular locations, the compartmen-

tation of IPP isomerase could have a significant influence on the

flux to different end products. Intriguingly, most known plant

genes encoding type I IPP isomerases appear to encode pro-

teins with N-terminal transit peptides, suggesting organellar

localization (Figure 2). For example, Clarkia breweri IDI1 and

IDI2 have N-terminal extensions of 70 and 69 amino acids,

respectively, compared with Escherichia coli IDI (Blanc and

Pichersky, 1995), and Arabidopsis thaliana has been shown to

contain only two IPP isomerase genes (At5g16440, designated

IDI1, and At3g02780, designated IDI2), both of which encode a

protein with an N-terminal extension (compared with the E. coli

IPP isomerase) that has been shown to not be necessary for

catalytic activity (Campbell et al., 1997) (Figure 2). An exception

was found in tobacco (Nicotiana tabacum), in which two cDNAs,

designated IDI1 and IDI2, were obtained, one encoding a protein

with an apparent transit peptide, while the other did not. When

these proteins were expressed as GFP fusions in this plant, the

tobacco IDI1 protein was found to be targeted to the plastid,

while the IDI2 protein was found in the cytosol (Nakamura et al.,

2001). However, it was not conclusively demonstrated that the

tobacco IDI2 cDNA was in fact a copy of a full-length transcript

(e.g., by comparison with a genomic sequence or by primer

extension experiments).

The presence of N-terminal extensions on the IPP isomerases

found in Arabidopsis and other species suggests that they might

be directed to plastids or mitochondria, both sites of isoprenoid

formation in plants. Plastids, in addition to the MEP pathway,

also contain biosynthetic machinery for monoterpene, diterpene,

gibberellin, carotenoid, and phytol production. Mitochondria, on

the other hand, are sites of ubiquinone biosynthesis (Lutke-

Brinkhaus et al., 1984; Ohara et al., 2006) and form other iso-

prenoids in organisms outside the plant kingdom (Felkai et al.,

1999; Marbois et al., 2005). Curiously, in both C. breweri and

Arabidopsis, none of the IDI proteins appear to be targeted to the

cytosol (since they all appear to have transit peptides), even

though this compartment produces sesquiterpenes, sterols, and

dolichols and the cytosolic mevalonate pathway supplies only

IPP and not DMAPP. Conceivably, alternative splicing or alter-

native transcription start sites of the IDI genes could lead to

cytosolic protein targeting, as is known for another Arabidopsis

protein involved in isoprenoid biosynthesis, farnesyl diphosphate

synthase1, which can be located in both the cytosol and the

mitochondria (Cunillera et al., 1997). Thus, it is not clear at pres-

ent which plant compartments possess IPP isomerase activity

and whether such activity is essential for the synthesis of iso-

prenoid compounds in that compartment.

Here, we present evidence that in Arabidopsis, one IPP isom-

erase (IDI1) is targeted to plastids, the other (IDI2) is targeted to

mitochondria, and additional IPP isomerase activity encoded by

both genes is present in the cytoplasm. While even one func-

tional IDI gene is sufficient to support growth and development,

Figure 1. Overview of Isoprenoid Biosynthesis by Compartment in a Higher Plant Cell.

Indicated are the IPP and DMAPP requirements for various classes of isoprenoids. AcCoA, acetyl-CoA; FPP, farnesyl diphosphate; GPP, geranyl

diphosphate; GGPP, geranylgeranyl diphosphate; G3P, glyceraldehyde 3-phosphate; MEP, methylerythritol phosphate pathway; MVA, mevalonate

pathway; OPP, diphosphate moiety. The block arrows denote multiple steps, and the asymmetric arrows reflect the expected equilibrium of the

reversible IPP isomerase (IDI) reaction.

678 The Plant Cell

we demonstrate that IDI1 and IDI2 are not completely redundant

and play distinct roles in isoprenoid metabolism.

RESULTS

Transcripts of Arabidopsis IDI1 and IDI2 Encode Proteins

with an N-Terminal Extension

The present annotation in The Arabidopsis Information Resource

(TAIR; http://www.plantgdb.org/AtGDB/) shows that the IDI1

gene contains six exons and five introns, as does IDI2 (Figure 3).

These two genes encode proteins of 291 and 284 amino acids,

respectively, which contain N-terminal extensions of 75 and 68

residues, respectively, compared with the sequence of E. coli IDI

(Figure 2). This extension, present in most plant IDI sequences

(Cunningham and Gantt, 2000), is quite variable, with the excep-

tion of the 16 residues just upstream of the Met residue in the

equivalent position to the starting Met in the E. coli sequence

(Figure 2). This 16-residue sequence starts with a Met that is

conserved in almost all known plant IDI sequences and contains

Figure 2. Amino Acid Sequence Alignment of Plant, Yeast, and E. coli Type I IPP Isomerase Proteins.

At, Arabidopsis thaliana IDI1 (At5g16440) and IDI2 (At3g02780); Cb, Clarkia breweri IPP isomerase 1 (X82627) and IPP isomerase 2 (U48963); Ec,

Escherichia coli IPP isomerase (NP_417365); Sc, Saccharomyces cerevisiae IPP isomerase (P15496). Residues in black are absolutely conserved in all

sequences shown, while those in dark gray and light gray are conserved among 70 and 50%, respectively. Alignments were performed using Blosum62

(AlignX, VectorNTI 10) with a gap-opening penalty of 10 and a gap-extension penalty of 0.1. Asterisks indicate Met residues conserved in nearly all

known plant species. The horizontal bar represents the conserved 16-residue region of the N-terminal extension (relative to E. coli IDI).

Figure 3. Genomic Structures of Arabidopsis IDI1 (At5g16440) and IDI2 (At3g02780).

Green arrows indicate exons, while the thin yellow lines in between indicate introns. The red boxes show the 59 untranslated regions. The 39 untranslated

regions are shown as yellow boxes. qRT-PCR forward and reverse primers are represented as black arrowheads. The insertion sites of the T-DNA

mutants used in this study are indicated with black vertical lines. They are idi1-1 (GABI insertion line 217HO4, exon 3, 799 nucleotides downstream of

the predicted start codon), idi1-2 (SALK insertion line 006330, intron 3, 841 nucleotides downstream of the predicted start codon), and idi2-1 (SALK

insertion line 014625, exon 4, 1651 nucleotides downstream of the predicted start codon).

The Role of IPP Isomerase in Arabidopsis 679

a second conserved Met residue halfway through (Cunningham

and Gantt, 2000) (Figure 2). Interestingly, yeast IDI also contains

a large N-terminal extension relative to E. coli IDI, but this

sequence as a whole shows little similarity to the corresponding

plant sequences, with the exception of the above-mentioned

16-residue region. The yeast sequence does not contain any Met

residues in this region (Figure 2), but since some enzymes of the

mevalonate pathway have been associated with mitochondria in

yeast (Shimizu et al., 1971), it may indeed serve some non-

catalytic function. When the N-terminal extension upstream of

this 16-residue region, which is also highly variable between At

IDI1 and At IDI2, is discounted, At IDI1 and At IDI2 are 91%

identical to each other, 84 and 83% identical, respectively, to C.

breweri IDI1, 45 and 46% identical, respectively, to yeast IDI, and

27 and 26% identical, respectively, to E. coli IDI. Interestingly, the

first exon of At IDI1 and At IDI2 (236 and 215 nucleotides,

respectively, starting from the initiating ATG codon) encodes the

entire N-terminal part of the protein that has no equivalent in the

E. coli IDI protein plus a few additional amino acids.

The prediction of N-terminal extensions for both IDI1 and IDI2,

as outlined above, is consistent with other available sequence

information for these genes. For example, the starting codon of

IDI1 is likely to be as indicated in Figure 2 due to the presence of

an in-frame stop codon in the 59 untranslated region (UTR) of IDI1

at position �9 relative to the predicted initiating ATG in several

available cDNAs. For IDI2, available cDNAs include a maximum

of 26 nucleotides of 59 UTR sequence upstream of the predicted

ATG initiating codon, and this region does not include any

additional start codons or stop codons in-frame with the pro-

posed start codon. However, an in-frame stop codon is present

at �57 with no additional start codons in between, making it

extremely likely that the proposed start codon for IDI2 is also the

correct one. Examination of 36 cDNAs for IDI1 and 41 cDNAs for

IDI2 in the databases also supported the identification of all

introns as annotated in TAIR (except when the cDNAs were

incomplete). The sequences of RT-PCR products that we ob-

tained for IDI1 and IDI2 using oligonucleotides based on the

beginning and end of the open reading frames as annotated in

TAIR (idi1-attB1 and idi1-attB2 [for IDI1] or idi2-attB1 and idi2-

attB2 [for IDI2] [see Supplemental Table 1 online]) also confirmed

the intron–exon demarcation.

In order to gain insight into the possible subcellular localiza-

tions of the two IDI enzymes as directed by the N-terminal ex-

tensions, the full-length deduced amino acid sequences for the

two proteins were used to query several public servers, including

ChloroP (http://www.cbs.dtu.dk/services/ChloroP/) (Emanuelsson

et al., 1999), TargetP (http://www.cbs.dtu.dk/services/TargetP/)

(Emanuelsson et al., 2000), Predotar (http://urgi.versailles.inra.fr/

predotar/predotar.html), MitoProt (http://mips.gsf.de/cgi-bin/

proj/medgen/mitofilter) (Claros and Vincens, 1996), and PSORT

(http://wolfpsort.org/) (Nakai and Horton, 1999). Most algorithms

suggested plastid localization with a high level of certainty for

IDI1, although MitoProt also suggested mitochondrial localiza-

tion for this protein. There was less agreement regarding IDI2,

with targeting predictions roughly split between the mitochondria

and plastid. Interestingly, ChloroP predicted plastid localization

for both proteins, while MitoProt predicted mitochondrial local-

ization for both.

IDI1 Is Directed to the Plastid and IDI2 Is Directed to

the Mitochondrion

To determine the subcellular localization of both IDI proteins, we

transformed Arabidopsis with constructs in which the genomic

sequence for either IDI1 or IDI2 had been fused to enhanced

green fluorescent protein (eGFP) under the control of the cauli-

flower mosaic virus 35S promoter and visualized targeting by con-

focal and fluorescence microscopy. Two other transformed lines

were used as controls. First, we cloned the eGFP gene into the

sameeGFP-readyGatewayplant transformationvector (pB7FWG2)

(Karimi et al., 2002), producing an eGFP dimer lacking any transit

peptide, which served as a control for cytosolic localization.

Second, we used Arabidopsis plants transformed with a signal

peptide fused to eGFP, for which plastid localization of the eGFP

fusion protein had already been confirmed (Marques et al., 2004).

Comparisons of confocal micrographs of the plastid and

cytosolic controls (Figures 4B and 4C, respectively) and non-

transformed controls (Figure 4A) viewed under similar instrument

settings indicated that virtually no green fluorescence was de-

tectable in nontransformed controls, whereas red autofluores-

cence derived from chlorophyll was readily visible (Figure 4A). In

the plastid control, intense green fluorescence colocalized with

chloroplast autofluorescence (Figure 4B). However, in the cyto-

solic localization control, green fluorescence was visible in the

cytosol, the nucleus, and at the cell wall but not in the red

autofluorescent plastids (Figure 4C). Thus, under these experi-

mental conditions, plastid targeting could readily be distin-

guished from cytosolic localization.

Plant lines transformed with IDI1 were visualized in a similar

way and revealed eGFP fluorescence that colocalized with

plastids in roots, stems, and leaves (Figure 4D; localization in

stems and roots not shown). However, plants transformed with

IDI2 exhibited green fluorescence in discrete subcellular organ-

elles that did not colocalize with plastid autofluorescence (Figure

4E). When seedling roots from these same transformed lines

were stained with a mitochondria-specific orange fluorescent

dye, eGFP fluorescence was observed to colocalize with the

MitoTracker Orange–derived fluorescence (Figure 4F), confirm-

ing targeting to mitochondria.

The same transformation vectors containing the cDNAs in-

stead of the full-length genomic sequences were also tested in

this way. Cloning and sequencing of the transcripts using trans-

gene-specific primers showed that plants transformed with the

genomic sequence and cDNAs produced identical transcripts.

Although the cDNAs were found to have generally the same

localization patterns as the genomic sequences, some expres-

sion of the IDI1 cDNA was occasionally detected in both the

cytosol and plastid simultaneously (Figure 5A). A third set of

eGFP fusion constructs for IDI1 and IDI2 bearing the native

promoter was analyzed, but expression levels were too low to

visualize eGFP fluorescence.

At Least One IPP Isomerase Gene Is Required for

Arabidopsis Viability, and IDI2 Is Necessary for Normal

Flower Development

Lines of Arabidopsis with mutations at each IPP isomerase locus

were investigated to learn more about the roles of these genes in

680 The Plant Cell

Figure 4. Subcellular Localization of IDI-eGFP Fusion Proteins in Transformed or Control Arabidopsis Leaf Tissue.

The first column shows green fluorescence from eGFP, the second shows red autofluorescence from chlorophyll, and the third shows an overlay of the

The Role of IPP Isomerase in Arabidopsis 681

planta. Using publicly available T-DNA insertion line collections,

we obtained two lines with insertions in the IDI1 gene (idi1-1,

GABI 217_ H04, and idi1-2, SALK_006330, N506330) and one

line with an insertion in the IDI2 gene (idi2-1, SALK_014625,

N514625). The insertion in idi1-1 is in exon 3, the insertion in idi1-2

is after the fourth nucleotide in intron 3, and the insertion in idi2-1

is in exon 5 (Figure 3). RT-PCR revealed that none of these

mutant lines produced a functional full-length transcript from the

affected gene (Figure 6). All single idi mutants (Figure 3) were

confirmed as homozygous for their respective insertions in the T3

generation by DNA gel blot analysis, T-DNA–specific PCR, and

left border sequencing of the resulting PCR products. The

segregation of T-DNA insertions and associated herbicide re-

sistance markers was Mendelian in all cases.

Plants homozygous for idi1-1 or idi1-2 alleles showed no

obvious phenotype. However, plants homozygous for the idi2-1

allele formed flowers in which the sepals were fused and the

petals were highly underdeveloped (Figure 7A). The pollen from

these mutants was viable, and backcrossing of this mutant for

three generations to ecotype Columbia (Col-0) wild-type plants

followed by self-fertilization confirmed that the phenotype was

linked to the idi2-1 mutant allele.

We were unsuccessful in obtaining double homozygous mu-

tants by crossing either idi1 mutant with idi2-1. Following

screening of >400 progeny from each of these crosses (>800

plants total), no double homozygous plant was observed (1 in 16

was expected). In addition, we were unable to detect any plants

that were homozygous at one locus and heterozygous at an-

other. The expected frequency of this genotype, assuming

strictly Mendelian segregation, would be one in four plants.

In an attempt to rescue the double homozygous mutant

containing both the idi1-1 and idi2-1 alleles, we transformed

double heterozygous lines with a truncated version of the IDI1

cDNA lacking the putative signal peptide but fused to eGFP in an

effort to provide IPP isomerase activity to the cytosol (see kinetic

characterization for a description of the active, truncated forms

of IDI1 and IDI2). The retention of the truncated fusion protein

(cytIDI-eGFP) in the cytoplasm was confirmed by confocal

microscopy (Figure 5B). Transformed plants were screened by

PCR for the presence of the idi1-1 or idi2-1 allele. We determined

the genotypes of 40 plants by PCR and further monitored the

expression of the transgene in these plants by measuring GFP

fluorescence in crude protein extracts of seedlings (data not

shown). Based on this screening strategy, we selected six

Figure 4. (continued).

two channels. All confocal images were scanned using similar laser gain and offset settings. Confocal images are as follows: nontransformed

Arabidopsis (A), plastid localization–positive control ferredoxin NADP(H) oxidoreductase (Marques et al., 2004) (B), cytosolic eGFP expression (empty

vector control) (C), genomic IDI1-eGFP fusion (D), and genomic IDI2-eGFP fusion (E). (F) shows an epifluorescence micrograph of MitoTracker Orange–

stained root hairs of Arabidopsis stably transformed with the IDI2-eGFP construct. eGFP fluorescence is shown in the left column, MitoTracker Orange

fluorescence in the central column, and the overlay of the two in the right column. Bars ¼ 20 mm.

Figure 5. Confocal Micrographs Showing the Simultaneous Plastid and Cytosolic Localization of IDI1c-eGFP in the Stem of a Wild-Type Plant and of

the Truncated cytIDI-eGFP in the Cytoplasm of Leaf Tissue in the idi2-1 Mutant Background.

(A) IDI1c-eGFP expression in the stem (wild type).

(B) cytIDI-eGFP expression in leaf (idi2-1).

The first column shows green fluorescence from eGFP, the second shows red autofluorescence from chlorophyll, and the third shows an overlay of the

two channels. All confocal images were scanned using similar laser gain and offset settings. Cytosolic expression of IDI did not restore the fused-sepal

phenotype of the mutant plant but made possible the isolation of plants homozygous for the idi2 allele and heterozygous for the idi1 allele. Confocal

conditions were the same as those described for Figure 4. Bars ¼ 20 mm.

682 The Plant Cell

independent lines from the T0 generation that were heterozy-

gous for both the idi1-1 and idi2-1 mutant alleles and had high

levels of expression of the truncated IDI1 (as determined by GFP

fluorescence) and allowed them to self-fertilize. Screening of 240

individuals in the T1 generation again failed to yield a double

homozygous mutant. However, we were able to find plants that

were heterozygous at the IDI1 locus and homozygous for idi2-1.

Of 240 plants, 36 had such a genotype. In nearly every case,

these plants displayed the same flower morphology defect as the

idi2-1 single mutant, although two lines (cytIDI-11 and cytIDI-14)

displayed partial restoration to the wild type (Figure 7B). No

plants homozygous for the idi1-1 mutant allele and heterozygous

at the IDI2 locus were isolated in this screen.

Transformation of the idi2-1 Mutant with the Full-Length

IDI2 cDNA but Not the Full-Length IDI1 cDNA Restores

the Wild-Type Floral Phenotype

Since the fused sepal phenotype was observed to segregate

with the idi2-1 allele, we attempted to restore the wild-type

phenotype in this mutant by transformation with vectors encod-

ing either the IDI1 or IDI2 full-length cDNA. T1 glufosinate

ammonium–resistant plants were allowed to self-fertilize, and

seeds were collected from individual transformed lines. T2 plants

were grown on sterile Murashige and Skoog (MS)–glufosinate

ammonium plates, and transformed lines with apparent single

inserts (as judged by a 3:1 resistant-to-susceptible ratio) were

transferred to soil and allowed to flower under long-day condi-

tions. Plants transformed with the IDI1 cDNA retained the mutant

phenotype (Figure 7C), while the flowers of those transformed

with the IDI2 cDNA were completely restored to the wild type

(Figure 7D). The subcellular localization of IDI2 in the rescued

mutant background was confirmed with confocal microscopy

and determined to be identical to that shown in Figure 4E.

LossofEither IDI1or IDI2 IndividuallyHasaMinimalEffecton

Isoprenoid Content in Leaf Tissue or in Flowers

The single homozygous mutants were subjected to chemical

analyses to quantify the major isoprenoid metabolites produced

in the chloroplasts and mitochondria. Chlorophyll and caroten-

oids are probably the most abundant isoprenoid products of the

chloroplasts, while ubiquinone is the major isoprenoid produced

in mitochondria. Given the role of carotenoids as photoprotec-

tants of the photosynthetic apparatus, we anticipated that if

either IDI protein played a limiting role in carotenoid production,

their absence would be most clearly noted under high light

conditions. Therefore, we compared carotenoid and chlorophyll

levels in acetone extracts of leaves from wild-type and mutant

plants grown under high light levels (600 mmol�m�2�s�1) as

measured by HPLC. A simple ion-trap liquid chromatography–

mass spectrometry (LC-MS) method was implemented to se-

lectively detect ubiquinone levels in the same extracts. We

detected no differences between wild-type plants and idi1-1

and idi1-2 mutant lines for any of the isoprenoids analyzed,

except that a slight decrease (20%) in both the carotenoids and

chlorophylls was noted for idi2-1 (Figure 8). A sample of the

plants used for HPLC analysis was also used to again confirm the

absence of functional transcripts in the mutant lines.

To examine whether the idi2-1 mutant flower phenotype was

due to a deficiency in a specific class of essential isoprenoids,

the above chemical analysis was repeated using wild-type and

idi2-1 flowers grown under normal conditions. No significant

differences were noted between the levels of carotenoids, chlo-

rophylls, phytosterols, or ubiquinone in flowers from the idi2-1

mutant versus those of wild-type flowers (Figure 9).

IDI2 Transcripts Are More Abundant Than Those of IDI1

Overall, but Both Genes Have Similar Organ-Specific

Expression Profiles

The organ-specific expression of IDI1 and IDI2 was monitored by

quantitative real-time PCR (qRT-PCR) using primers designed to

recognize a portion of the 39 UTR of each gene. Steady state

transcript levels of each gene in four organs (roots, stems,

leaves, and flowers) were measured and normalized to those of

leaves. Within a given organ, overall expression between IDI1

and IDI2 was also compared based on the Ct values of the

common normalizer gene. Qualitatively, expression of both

genes was detectable in all organs surveyed. Quantitatively,

expression of IDI1 was 4.1-fold higher in flowers than in leaves,

1.5-fold higher in roots than leaves, and about the same in stems

as in leaves (Figure 10A). Expression of IDI2 was about the same

in flowers and stems as in leaves but ;2.5-fold higher in roots, in

agreement with Campbell et al. (1997) (Figure 10A). When the

expression of the two IDI genes was compared within a given

organ, IDI2 was always more abundant, with IDI2 transcripts

outnumbering IDI1 by ;10-fold in leaves, 22-fold in roots, 4.9-

fold in flowers, and 4.6-fold in stems.

Figure 6. RT-PCR Analysis of Wild-Type and idi Mutant Lines.

Total RNA was converted to cDNA using a poly(dT) primer and then used in

a PCR either with primers that amplify the full-length transcript of IDI1

(columns A to C) or IDI2 (columns D and E) (top panel) or a normalizer gene

(RP2LS; bottom panel) to confirm the conversion of RNA into cDNA

template. cDNA templates are as follows: wild type (A), idi1-1 (B), idi1-2 (C),

wild type (D), and idi2-1 (E). IDI1 was amplified with primers flanking the

start and stop codons and is predicted to produce a product of 930 bp,

whereas IDI2 was amplified with primers that included a small portion of the

39 UTR and is thus predicted to amplify a slightly larger product of 988 bp.

The Role of IPP Isomerase in Arabidopsis 683

The steady state transcript level of the remaining functional IDI

gene in each mutant line was similarly measured. In this case,

whole mutant seedlings were used and compared with samples

obtained from wild-type seedlings. No mutant showed a signif-

icant increase in the remaining IDI gene relative to the wild type

(#1.5 fold increase; Figure 10B).

The IDI1 and IDI2 Proteins Have Similar Kinetic Properties

To look for further differences in the roles of the two genes, we

compared the catalytic properties of the encoded proteins

expressed in vitro. It was previously shown that the N-terminal

extensions of IDI1 and IDI2 were not required for catalytic activity

(Campbell et al., 1997). Therefore, we expressed both proteins in

truncated form based on their alignment to the start codon of

E. coli IDI (Figure 2). The purification of the recombinant proteins

was aided by expression with an N-terminal His tag. However,

due to the catalytic role of divalent metal ions in the IPP isom-

erase reaction (Carrigan and Poulter, 2003), the presence of

the His tag posed problems for accurately measuring the affinity

of each enzyme for IPP. Therefore, after introducing a thrombin

cleavage site, the His tags were removed by thrombolytic cleav-

age following purification on a charged Ni affinity column. The Km

values of IDI1 and IDI2 for IPP were determined to be 20 6 3.8

and 16 6 2.6 mM, respectively, while their kcat values were 2.24 6

0.1 and 0.89 6 0.04 min�1, consistent with previous literature

reports for this class of IDI enzymes (Muehlbacher and Poulter,

1988; Anderson et al., 1989; Street and Poulter, 1990; Carrigan

and Poulter, 2003). A pH optimum curve of each protein revealed

that both had a similar preference for a neutral pH (Figure 11).

Incorporation of [2-13C]Mevalonate into Sitosterol

Demonstrates IPP Isomerase Activity in the Cytosol

Except for the floral phenotype of the idi2 mutant, there were no

significant morphological or chemical alterations in the individual

idi mutants, making it hard to draw any conclusions about the

specific roles of these genes in isoprenoid metabolism. The nega-

tive impact of a single idi mutation may be ameliorated by DMAPP

supplied from the isomerase encoded by the other IDI gene. To

investigate this possibility, we compared the biosynthesis of

sitosterol, the most abundant plant sterol, in the wild type ver-

sus all three idi mutant lines. Sitosterol is known to be biosyn-

thesized in the cytosol. Thus, to assess the source of the IPP and

DMAPP for sitosterol biosynthesis, we labeled the cytosolic

mevalonate pathway by supplying [2-13C]mevalonate to plants

Figure 7. Phenotype of idi2-1 Mutant Flowers and Genetic Comple-

mentation.

Flowers from the idi2-1 mutant ([A], left) demonstrate a fused-sepal

phenotype with highly underdeveloped petals but produce fertile pollen

and can self-fertilize. A Col-0 wild-type flower ([A], right) is shown for

comparison. Two homozygous idi2 lines (out of 36) expressing cytosolic

IDI showed partial reversion to the wild-type flower morphology (B). The

black arrow in (B) indicates a mutant flower, whereas the white arrows

show wild-type flowers. The fused-sepal phenotype of the idi2-1 mutant

was not affected by transformation with the IDI1 cDNA (C), while this

phenotype was fully restored to that of the wild type by transformation of

this mutant with a cDNA encoding IDI2 (D). Shown are three independent

lines transformed with the IDI1 cDNA (C) and three lines transformed with

the IDI2 cDNA (D); a Col-0 wild-type flower is pictured at left in (D).

684 The Plant Cell

in a MS growth medium while simultaneously administering

mevastatin, an inhibitor of an essential step in the mevalonate

pathway, HMG-CoA reductase, to block the endogenous for-

mation of unlabeled mevalonate (Rodrıguez-Concepcion et al.,

2004). The biosynthesis of a sitosterol molecule requires four IPP

units and two DMAPP units (see Supplemental Figure 1 online). If

there were a cytosolic IPP isomerase, all IPP and DMAPP units

should be labeled by [2-13C]mevalonate, resulting in an incorpo-

ration of five 13C atoms (one 13C atom is lost during demethyl-

ation of the cycloartenol intermediate) (Schaeffer et al., 2001).

However, if there is no IDI activity in the cytosol, only the IPP units

would be labeled directly from [2-13C]mevalonate, resulting in the

incorporation of four 13C atoms. The DMAPP would have to

come either from IPP generated in the cytosol and isomerized to

DMAPP in the plastids or mitochondria or from IPP generated in

the plastid that was isomerized there. In either case, the incor-

poration of label would be expected to be lower than for a

cytosol-generated DMAPP unit.

We detected high levels of [2-13C]mevalonate incorporation

into sitosterol in these experiments using GC-MS and 13C NMR.

The mass spectra for the trisilyl-sitosterol derivatives from the

wild type and idi mutant lines were identical (Figure 12). In

extracts from labeled plants (Figure 12B), a [M þ 5]þ� molecular

peak dominated the molecular ion cluster in the wild type and in

all three mutant lines, indicating the existence of a cytosolic IPP

isomerase activity in all of these plants. When spectra from plants

supplemented with [2-13C]mevalonate were compared with

those derived from unlabeled plants (Figures 12B versus 12A),

many differences in the fragmentation patterns were evident.

Aside from the dominant mass ions (m/z 491 versus 486),

sitosterol fragments from labeled plants bearing one (m/z 130

versus 129), two (m/z 161 versus 159), three (m/z 258 versus

255), four (m/z 361 versus 357), and five (m/z 401 vs. 396) 13C

labels were detected, corresponding to sitosterol fragments

containing one to five 13C-labeled isoprene units. The strongest

mass spectral evidence for a cytosolic isomerase comes from

the detection of 13C labeling in the aliphatic side chain of the

sitosterol skeleton, most of whose carbon atoms originate from

DMAPP. Specific fragments corresponding to this side chain

showed a significant enrichment of 1 mass unit (m/z 44 versus 43

and m/z 86 versus 85) in all samples supplemented with

[2-13C]mevalonate (Figure 12B) but not in the unlabeled controls

(Figure 12A), consistent with 13C labeling of the C26 position of

the aliphatic chain of sitosterol via a DMAPP unit derived from

[2-13C]mevalonic acid (see Supplemental Figure 1 online). The

absolute levels of sitosterol in the plants used in these experi-

ments were similar in mutant and wild-type lines grown on MS

medium alone (132 to 141 ng/mg fresh weight, based on com-

parison with the internal stigmastanol standard). Meanwhile,

plants grown in the presence of [2-13C]mevalonate and meva-

statin displayed a decrease of ;30% in the levels of sitosterol

(99 to 109 ng/mg fresh weight).

To confirm that the labeling of sitosterol mass spectral frag-

ments was due to DMAPP originating from an isomerase in the

cytosol, labeled sitosterol from wild-type and mutant plants was

purified by preparative HPLC and then measured by 13C NMR.

The incorporation of [2-13C]mevalonic acid would be expected to

label five specific carbon atoms in the sitosterol skeleton (see

Supplemental Figure 1 online). For sitosterol isolated from both

Figure 9. Analysis of Isoprenoid Levels in idi2-1 Mutant Flowers.

Carotenoid and chlorophyll levels were determined by HPLC analysis

(monitoring at 445 and 650 nm). An aliquot of the same sample was used

for the analysis of ubiquinone-9 levels by ion-trap LC-MS as described in

Methods. Quantification was based on external standard curves. Quan-

tification of sitosterol was based on the relative peak area of stigmas-

tanol added to flower tissue prior to extraction as an internal standard

(10 mg), while identification was based on comparison with a derivatized

authentic sitosterol standard. Error bars indicate SD from seven repli-

cates. Ubiquinone-9 levels are shown on the right vertical axis.

Figure 8. Carotenoid, Chlorophyll, and Ubiquinone Contents of Wild-

Type and idi Mutant Leaf Tissue Exposed to Continuous High Light (600

mmol�m�2�s�1).

Frozen, ground leaf tissue (500 mg) from six pooled individual plants was

extracted with 2.5 mL of acetone:water (9:1) and analyzed by HPLC (for

carotenoids and chlorophyll) or ion-trap LC-MS (for ubiquinone). Values

shown are averages 6 SD of eight replicates. Quantification was based

on external standard curves. Ubiquinone-9 levels are shown on the right

vertical axis.

The Role of IPP Isomerase in Arabidopsis 685

mutant and wild-type plants, each of these five atoms was

indeed found to be enriched (Figure 13). Most notable was the

strong enrichment of a signal at 19.82 ppm corresponding to

C26, a carbon atom in the side chain derived from DMAPP. This

furnished strong evidence that the DMAPP used in the formation

of sterols is derived from the cytosolic mevalonate pathway via a

cytosolic IPP isomerase.

Shortened Transcripts of Both IDI1 and IDI2 Are

Expressed That Lack the N-Terminal Transit Peptide for

Organellar Targeting

The presence of the IDI protein in the cytosol might be due to the

translation of a shorter transcript lacking the N-terminal signaling

peptide. To determine whether such alternative transcription

occurred at a significant rate, 59 rapid amplification of cDNA ends

(RACE) was employed to assess the lengths of the IDI1 and IDI2

transcripts. Using reverse primers for each gene optimized for 59

RACE (see Supplemental Table 1 online), we found two distinct

size classes of 59 RACE products for IDI1 and IDI2. Because the

reverse primers were designed to be complementary to exon 2

(IDI1) and exon 4 (IDI2), we were able to rule out genomic DNA

contamination as a possible source for these clones. Sequence

analysis of these fragments indicated that the longer 59 RACE

products from transcripts of both genes from both leaf and flower

tissues came from full-length transcripts that included the pro-

posed start codons (Figure 14; fragments 1 and 3 representing

IDI1 and IDI2, respectively). For IDI1, even longer transcripts than

those represented by fragment 1 were observed in both tissues.

Importantly, 59 RACE analysis and sequencing detected shorter

transcripts for both IDI1 and IDI2 (represented by fragments 2 and

5 for IDI1 in leaf and flowers, respectively, and by fragment 4 for

IDI2 in both tissues). Fragment 2 represents an IDI1 transcript that

begins 180 bp downstream from the predicted start codon, while

fragment 5 represents an IDI1 transcript beginning 48 bp down-

stream from the same position. Fragment 4 represents an IDI2

transcript that begins 52 bp from its start codon. The shorter

transcripts include the region encoding the Met-rich, conserved

portion of the N-terminal extension corresponding to Met codons

at positions�16 and�8 (IDI2 fragment 4 and IDI1 fragment 5) or

only at position�8 (IDI1 fragment 4) relative to the E. coli IDI start

codon (Figure 2). A highly truncated IDI1 RACE product was also

observed from leaf templates, which is unlikely to produce

functional proteins.

Figure 10. Steady State Transcript Levels of IDI1 and IDI2 in Arabidopsis

as Measured by qRT-PCR.

Relative quantification was performed according to the Pfaffl method

(Pfaffl, 2001). Error bars indicate SD.

(A) Organ-specific expression. Fold change values compare expression

levels of each gene with that in the leaf. When IDI1 and IDI2 were

compared in the same organ, IDI2 was found to be more abundant in all

organs surveyed (described in text). cDNA template loading was nor-

malized to the Ct value of the Arabidopsis RP2LS gene (At4g35800).

(B) Expression of the remaining IDI gene in idi mutant lines. Total RNA

was extracted from whole seedlings, converted to cDNA, and used in

relative quantification experiments with wild-type seedling extracts.

Figure 11. pH Optima of the Two Mature IDI Proteins from Arabidopsis.

Activity is based on the conversion of [1-14C]IPP to its acid-labile isomer,

DMAPP, and scintillation counting of an ether extract of the reaction

following acid hydrolysis. Error bars indicate SD of four replicates at each

pH. Linear conditions were established previously by assaying a range of

enzyme concentrations and incubation times. IDI1 and IDI2 are repre-

sented by open and closed circles, respectively.

686 The Plant Cell

DISCUSSION

The Arabidopsis IDI1 and IDI2 Genes Encode Similar

Proteins with Divergent N Termini That Are Predicted to

Serve as Transit Peptides

Our studies of the two Arabidopsis IPP isomerase genes, IDI1 and

IDI2, indicated no great differences in organ expression patterns

between them, and the catalytic properties of the encoded pro-

teins were also similar. However, there are some distinctions in

their metabolic roles caused by differences in subcellular localiza-

tion resulting from divergence in their N-terminal sequences.

An analysis of the gene structure of IDI1 and IDI2, based on

available cDNAs and our own RT-PCR products, indicated that

the annotated versions available in public databases correctly

show the intron and exon structure of these genes and that

alternative splicing of primary transcripts, if it occurs, must be

rare. Furthermore, the presence of upstream in-frame stop

codons in the 59 UTR (or promoter region) of both genes strongly

suggested that the start codons shown in Figure 2 are the true

translational start signals used in planta for the complete tran-

script. The predicted amino acid sequences include the pres-

ence of a 65- to 75-residue N-terminal extension in both proteins

compared with the E. coli homolog. These extensions are highly

divergent from one another (and from the corresponding se-

quences in other plant IDI proteins) with the exception of the last

16 residues. This observation, together with the results of tar-

geting prediction software and the observation that both proteins

are catalytically active when expressed as truncations without

the extension (Campbell et al., 1997; this study), all indicated

likely organellar targeting of IDI1 and IDI2 when translated from

full-length complete transcripts.

The IDI1 Protein Participates in Plastid Isoprenoid

Formation, but Knocking Out the IDI1 Gene Has No

Morphological or Chemical Effects

Expression of the IDI1 gene fused to the eGFP open reading

frame in plants resulted in clear plastid localization of this protein

(Figure 4). While the terminal step of the MEP pathway produces

Figure 12. Sitosterol Mass Spectra from Arabidopsis Plants Grown in the Absence or Presence of [2-13C]Mevalonate and Mevastatin.

(A) Plants grown on MS medium.

(B) Plants grown on MS medium containing [2-13C]mevalonate mevastatin.

In each case, no difference was detected between spectra from wild-type or idi mutant plants (spectra from a wild-type plant are shown; see also

Supplemental Figure 2 online). The inset in (A) shows the mass spectrum of authentic sitosterol standard; the inset in (B) shows the incorporation of

[2-13C]mevalonate into sitosterol in wild-type and idi lines grown in the presence of mevastatin, as determined by the relative proportions of isotopomers

in the mass ion cluster representing [M]þ (m/z 486) through [Mþ5]þ (m/z 491) species after subtraction of the natural abundance of 13C in the control

samples. Error bars indicate SD of four replicates. Sterols were extracted from 2-week-old seedlings with methanol:CH2Cl2 (2:1, v/v) using stigmastanol

as an internal standard, derivatized with N-methyl-N- trimethylsilyltrifluoroacetamide, and analyzed by GC-MS.

The Role of IPP Isomerase in Arabidopsis 687

both IPP and DMAPP (Eisenreich et al., 2004), previous work has

suggested that IDI activity in the plastid is necessary for maximal

rates of synthesis of isoprenoids such as carotenoids (Albrecht

and Sandmann, 1994; Page et al., 2004). Furthermore, it has also

been shown that in E. coli cells that were engineered to synthe-

size carotenoids from DMAPP and IPP produced via the MEP

pathway, IDI activity was a limiting factor in carotenoid biosyn-

thesis (Kajiwara et al., 1997; Cunningham and Gantt, 2000). That

exogenously supplied mevalonate can complement a block in

1-deoxyxylulose 5-phosphate synthase activity and chlorophyll

biosynthesis (presumably following the transport of IPP to the

plastid) also suggests the presence of IDI activity in this com-

partment (Estevez et al., 2000; Nagata et al., 2002). Thus, plastid

localization for a plant IDI is not surprising. However, plants

homozygous for the idi1 null alleles are viable and contain wild-

type levels of carotenoids and chlorophylls even under high light

conditions (Figure 8), suggesting that IDI2 may somehow com-

pensate for the lack of IDI1.

The IDI2 Protein Participates in Mitochondrial Isoprenoid

Formation, and idi2 Mutants Have Abnormal

Floral Morphology

The expression in plants of IDI2 fused to eGFP unambiguously

demonstrated localization to a nonplastid organelle (Figure 4E).

Using a mitochondria-specific fluorescent dye, we were able to

show the site of IDI2 targeting as the mitochondria (Figure 4F). A

mitochondria-specific isoform of IPP isomerase is not surpris-

ing given the previously reported role of mitochondria in the bio-

synthesis of some essential isoprenoids, such as ubiquinone

(Lutke-Brinkhaus et al., 1984), and the targeting of a specific

isoform of FPP synthase to this compartment (Cunillera et al.,

1997). While IPP has been shown to be taken up by mitochondria

(Lutke-Brinkhaus et al., 1984), if DMAPP cannot similarly be

absorbed, then there would be an absolute requirement for

an IPP isomerase in the mitochondrion to provide DMAPP

for the construction of isoprenoids. Direct import of FPP into

Figure 13. Upfield Portion of the 13C NMR Spectra of Purified Sitosterol Extracted from Wild-Type and idi Mutant Arabidopsis Seedlings Cultivated in

the Presence of [2-13C]Mevalonate and Mevastatin.

Sitosterol from wild-type and mutant plants gave virtually identical spectra (see Supplemental Figure 3 online). The wild-type spectrum is shown above.

These signals were identified by previously published chemical shift values for sitosterol (Goad and Akihisa, 1997). These allowed unambiguous

assignment of all carbon atoms in the molecule. Labeling was found to occur at the predicted positions based on the scheme for sitosterol biosynthesis

from IPP and DMAPP shown in Supplemental Figure 1 online. The peaks at 24.31 (C15), 31.93 (C7), 33.96 (C22), and 37.27 (C1) ppm were enriched by

labeling via IPP. The peak at 19.82 ppm (C26) was enriched by labeling via DMAPP. Since the extent of IPP and DMAPP labeling of sitosterol, a cytosolic

isoprenoid product, is comparable, both substrates must come from the cytosol, indicating the presence of a cytosolic IPP isomerase. The structure

and carbon atom numbering of sitosterol are shown above the spectrum, with enriched carbons shown in boldface; numbers in parentheses indicate

the reported chemical shifts in parts per million.

688 The Plant Cell

mitochondria, inferred from the observation that the administra-

tion of [3H]farnesol to cultured tobacco BY-2 cells resulted in

labeled ubiquinone (Hartmann and Bach, 2001), would obviate

the need for an IPP isomerase there. But it is possible that the

label from farnesol in this study was imported in a form other

than FPP, such as ubiquinone itself, which has been shown to be

imported into mitochondria in tobacco after assembly in the

endoplasmic reticulum (Swiezewska et al., 1993; Ohara et al.,

2004).

Compared with IDI1, IDI2 is more highly expressed overall. At

the organ level, we observed that IDI2 expression is highest in

roots but that IDI1 expression is highest in flowers. These results

partially agree with those of Campbell et al. (1997), who found

that IDI1 and IDI2 were both most highly expressed in roots. This

discrepancy may be due to cross-hybridization of the cDNA

probes used with RNA gel blots in the previous study, since the

portions of IDI1 and IDI2 that encode the mature protein share

89% identity at the nucleotide level. In our case, the design of

primers in the 39 UTR, where IDI1 and IDI2 share no similarity, and

the sequencing of multiple qRT-PCR products ensured the

specificity of IDI transcript measurements.

The mitochondrial localization of IDI2 and the fused-sepal

phenotype of the idi2-1 mutant (Figure 7) led us to speculate that

specific isoprenoid metabolites derived from the mitochondria

Figure 14. The 59 RACE Products of IDI1 and IDI2 in Leaves and Flowers of Wild-Type Arabidopsis Plants.

Nested RACE products were separated on a 4% agarose gel, excised, cloned, and sequenced. A nucleotide alignment of five representative RACE

products is shown for bands representing the IDI1 full-length transcript (1), the IDI1 short transcript (2 and 5), the IDI2 full-length transcript (3), and the

IDI2 short transcript (4). Alignments were performed using Blosum62 (VectorNTI 10) with a gap-opening penalty of 10 and a gap-extension penalty of

0.1. The presumed start codons for the full-length IDI1 and IDI2 proteins are boxed, while the dotted lines represent possible alternative start codons for

shortened transcripts, including that corresponding to the E. coli start codon (defined as Metþ1) and conserved Met codons at amino acid positions�8

and �16 relative to the E. coli start position. The sizes of representative marker bands are indicated in base pairs.

The Role of IPP Isomerase in Arabidopsis 689

are necessary for normal floral development. In addition to the

synthesis of ubiquinone, some protein prenylation is thought to

take place in the mitochondria of spinach (Spinacia oleracea)

(Shipton et al., 1995). However, in Arabidopsis, no protein

prenyltransferase activity is known to occur in this organelle

(Crowell, 2000). While mitochondria have previously been shown

to be involved in floral organ development in Nicotiana spp

(Bonnett et al., 1991), it is not currently clear why a lack of IDI2

activity in Arabidopsis would produce the observed floral phe-

notype.

Representative isoprenoids biosynthesized in the plastid, cy-

tosol, and mitochondrion were analyzed in idi2-1 flowers in an

attempt to identify the cause of the mutant phenotype. However,

these analyses failed to show any specific isoprenoid deficiency

in mutant flowers. Furthermore, transformation experiments in

which a truncated isomerase expressed in the idi2-1 mutant

background failed to fully restore the wild-type phenotype sug-

gest that the basis of the idi2-1 phenotype is not related to any of

the compounds we have examined here or to the lack of

cytosolic isomerase activity.

Single Mutants in IDI1 or IDI2 Are Rescued by the

Remaining IDI

Characterization of mutant lines in which the IDI genes had been

disrupted showed that the simultaneous loss of both genes

results in a lethal phenotype. However, there were no major

morphological or chemical abnormalities associated with the

loss of either gene singly, with the exception of the loss of IDI2 in

flowers. What is the source of DMAPP in these single idi mu-

tants? We can rule out the MEP pathway as a significant source

of DMAPP in the single mutant lines, because then the double idi

mutant (which should have a functioning MEP pathway) would

also be viable. By the same reasoning, we can exclude the

existence of a heretofore unrecognized type II IPP isomerase in

Arabidopsis or the presence of any other protein catalyzing the

conversion of IPP to DMAPP in planta. Other explanations for the

viability of the single idi mutant lines require the remaining IDI

protein to be the source of the DMAPP. Although it was impos-

sible to obtain viable plants with three idi null alleles in crosses

between the mutants, overexpressing IDI1 as a cytosolic protein

gave viable plants that were homozygous for the idi2 null allele

and heterozygous for an idi1 null allele. To be sure, there are

limits to the ability of one IDI activity to complement another, in

that expression of IDI1 as a plastidic protein or a cytosolic protein

completely failed to restore the idi2-1 flower phenotype to that of

the wild type (Figure 7). Nevertheless, it seems clear that a single

IDI gene is sufficient for plant viability, whether it encodes IDI1,

which we observed in the plastids, or IDI2, which was found in the

mitochondria. In both cases, the transport of DMAPP or later

intermediates between organelles would seem essential for

normal isoprenoid formation.

Scattered evidence suggests that isoprenoid biosynthetic in-

termediates can be transported among subcellular components.

For example, IPP import into mitochondria (Lutke-Brinkhaus

et al., 1984) and exchange of IPP between the cytosol and plastid

have been documented by inhibitor studies in Arabidopsis (Laule

et al., 2003) and in vivo feeding studies in several plant systems,

including tobacco BY-2 cultures (Hemmerlin et al., 2003), snap-

dragon (Antirrhinum majus) (Dudareva et al., 2005), chamomile

(Chamaemelum nobile) (Adam et al., 1999), and lima bean (Pha-

seolus lunatus) (Piel et al., 1998). In vitro studies with isolated

membranes also support the transport of IPP from plastids to the

cytosol (and possibly DMAPP, albeit at very low levels) (Bick and

Lange, 2003) and from the cytosol to plastids (Kreuz and Kleinig,

1984; Soler et al., 1993; Flugge and Gao, 2005). Among earlier

isoprenoid intermediates, exogenously supplied mevalonate, an

intermediate in the cytosolic IPP pathway, has been shown to par-

tially rescue a block in the plastidic MEP pathway (Estevez et al.,

2000; Nagata et al., 2002; Gutierrez-Nava et al., 2004; Rodrıguez-

Concepcion et al., 2004). The transport of bona fide MEP pathway

intermediates such as 1-deoxy D-xylulose 5-phosphate (DXP)

from the cytosol into plastids has been shown to proceed via an

inorganic phosphate antiport transporter (Flugge and Gao, 2005).

In fact, a cytosolic xylulose kinase was recently identified in

Arabidopsis that converts 1-deoxy D-xylulose to DXP (Hemmerlin

et al., 2006), although a role for DXP in the cytosol is unclear at

present.

To find the source of DMAPP in cytosolic isoprenoid formation,

we investigated the origin of the C5 units of a major cytosol-

produced isoprenoid, sitosterol, in both wild-type and single idi

mutant Arabidopsis plants. When [2-13C]mevalonate, an inter-

mediate of the cytosolic pathway, was supplied, all C5 units in all

idi1 and idi2 mutant lines were uniformly labeled whether derived

from IPP or DMAPP. These findings point to the existence of a

cytosolic IPP isomerase activity arising from both IDI1 and IDI2

and provide no support for the incorporation of DMAPP units

originating in another compartment (which would be unlabeled)

or for the transport of cytosolic IPP to the plastids or mitochon-

dria for the isomerization to DMAPP followed by incorporation

upon return to the cytosol (which would give lower specific

labeling).

Arabidopsis Has IPP Isomerase Activity in the Cytosol

Arising from Shorter Transcripts of IDI1 and IDI2

While the GFP studies indicated the localization of IPP isomerase

protein only in plastids (IDI1) or mitochondria (IDI2), inspection of

the gene sequences indicated additional downstream Met co-

dons of IDI1 and IDI2 that could serve as initiators of translation of

shortened proteins without signal peptides that might be local-

ized to the cytosol. Using 59 RACE, shorter transcripts were

isolated for both genes; these lacked the 59 UTR and 48 bp or

more of the coding sequence of the full-length transcripts. These

short transcripts can be expected to give shortened proteins

without transit peptides, providing further evidence of cytosol-

localized IPP isomerase. The fact that the in vitro activity of both

proteins has an optimum at pH 7.0 is also consistent with a

cytosolic location. However, in relation to mitochondrial and

plastidial IPP isomerase forms, such truncated cytosolic proteins

probably represent only a small proportion of total IPP isomerase

proteins. While 59 RACE is not a quantitative technique, it is

expected to favor the amplification of shorter fragments. Hence,

the observation that RACE products corresponding to full-length

transcripts are more abundant than RACE products representing

shorter ones (Figure 14) suggests that full-length transcripts

690 The Plant Cell

predominate in planta. The greater proportion of longer plastid-

targeted (IDI1) or mitochondria-targeted (IDI2) transcripts versus

cytosolic transcripts (from both IDI1 and IDI2) may also explain

the general lack of cytosolic eGFP expression observed in IDI-

eGFP–transformed lines. Fluorescence from a small amount of

cytosolically localized fusion protein dispersed over the large

space of the cytosol is likely much less visible by confocal

microscopy than large amounts of plastidial and mitochondrial

fusion proteins accumulated in the smaller volumes of these

organelles. However, since our IDI-eGFP constructs did not

include the native 59 UTR sequences, the analysis of 59 RACE

products is probably a more physiologically relevant indicator of

protein targeting.

The presence of IPP isomerase activity in the cytosol is not

only consistent with the sitosterol feeding studies and the exis-

tence of shorter transcripts but also rationalizes the lack of gross

phenotypes in the idi mutants. It also provides a logical way to

support the formation of sesquiterpenes, sterols, dolichols, and a

range of other isoprenoids in this compartment, since the basic

isoprenoid pathway in the cytosol produces only IPP and not

DMAPP, the C5 starter unit for terpenoid construction.

The Properties and Localization of IDI May Vary among

Plant Species

While the IPP isomerase of E. coli is dispensable for this bacte-

rium (Hahn et al., 1999), plants require this activity to supply the

C5 starter units for isoprenoid chain elongation; indeed, most

plant species studied, like Arabidopsis, have at least two differ-

ent IDI genes (Cunningham and Gantt, 2000; Nakamura et al.,

2001). The products of multiple IDI genes may be targeted to

different subcellular locations, as shown here for Arabidopsis,

because isoprenoid formation in plants is typically distributed

among different subcellular compartments. Previous studies

with tobacco showed that two IDI cDNAs encode proteins that

are localized to the cytoplasm and the plastid, respectively

(Nakamura et al., 2001), although it remains to be determined

whether these cDNAs in fact represent actual transcripts of IDI

forms present in this species.

Interestingly, sequence analyses of IDI genes have demon-

strated that proteins from the same species are more similar to

each other than to IDI sequences of other species (Cunningham

and Gantt, 2000). This is also true in Arabidopsis, in which IDI1

and IDI2 share 92% identity when the N-terminal region is ex-

cluded from comparison but are only 84 to 90% identical to other

known plant IPP isomerase sequences. Hence, IDI gene dupli-

cation is relatively recent on the scale of higher plant evolution,

and there may be differences in IPP isomerase properties or

localization among different plant lineages (Ramos-Valdivia et al.,

1997a, 1998). For example, in Cinchona robusta cell cultures that

produce anthraquinones requiring a DMAPP unit, two IDI pro-

teins are known with different kinetic characteristics and patterns

of occurrence (Ramos-Valdivia et al., 1997b). These results are in

contrast with this study, in which it was shown that the two

Arabidopsis proteins have no significant differences in kinetic

properties but have different patterns of subcellular localization.

There are vast differences among plant species in the amounts

and types of terpenoid secondary metabolites they produce.

Given that particular terpene types differ in their relative require-

ments for IPP and DMAPP, and in the subcellular compartments

in which they are produced, various species may require distinct

types of IPP isomerases operating in separate compartments to

efficiently regulate their IPP and DMAPP pools. Arabidopsis, with

its relatively low output of terpenoid secondary metabolites

(Chen et al., 2003; D’Auria and Gershenzon, 2005) and two

biochemically similar IPP isomerases operating in various com-

partments, may provide a basic model for IPP isomerase diver-

sification and distribution in a plant species.

METHODS

Plant Materials and Treatments

Arabidopsis thaliana (ecotype Col-0) plants were grown on soil in a climate-

controlled growth chamber (228C, 55% RH, and 100 mmol�m�2�s�1 pho-

tosynthetically active radiation) under either short days (12 h of light/12 h

of dark) or long days (16 h of light/8 h of dark) for 4 to 6 weeks. The

three independent T-DNA insertion mutants used in this study were

idi1-1 (SALK_006330, N506330), idi1-2 (GABI 217_H04), and idi2-1

(SALK_014625, N514625). Seed stocks were obtained either from the

European Arabidopsis Stock Center or from the GABI-KAT consortium

(Bielefeld University). Initial segregation analysis was performed by

seeding plants on MS medium plates supplemented with either 50 mg/

mL kanamycin (SALK insertion lines) or 7.5 mg/mL sulfadiazine (GABI

insertion line) as described previously (Alonso et al., 2003; Rosso et al.,

2003). In addition, a PCR-based assay was designed to detect the

presence of the T-DNA insertion. The primers used for this assay con-

sisted of T-DNA left border–specific primers (SALK_LBb1or GABI_LBb1)

in combination with the gene-specific primers IDI1L, IDI1R, IDI2L, and

IDI2R (see Supplemental Table 1 online). Plants segregating as homozy-

gous as confirmed by herbicide resistance and PCR were further ana-

lyzed by DNA gel blot analysis performed as described previously

(D’Auria et al., 2007). Hybridization probes included either a HindIII frag-

ment from the genomic clone of IDI1 or the XbaI–NcoI fragment obtained

from the genomic clone of IDI2.

Homozygous idi2-1 plants were backcrossed for three generations to

Col-0 plants for confirmation of phenotype segregation. In every gener-

ation, 30 progeny were screened for the presence of the T-DNA insert,

and a minimum of five positive plants based on the PCR assay were used

as the pollen donors for backcrossing. Plants positive for a T-DNA insert

after the third generation of backcrossing were allowed to self-fertilize,

and their progeny were genotyped by DNA gel blot. In order to obtain

plant lines containing T-DNA insertion alleles for both IDI1 and IDI2,

homozygous mutants of each of the four insertion lines were crossed in a

reciprocal manner and analyzed in the self-fertilized F2 for a double

mutant genotype.

Isotopically Labeled Precursor Feeding and Sterol Analysis

Seeds of the four homozygous T-DNA insertion lines, along with

Col-0 seeds, were sterilized and plated on MS medium supplemented

with a combination of 10 mM mevastatin (Sigma-Aldrich) and 4.5 mM

[2-13C]mevalonolactone (Sigma-Aldrich) or with either compound alone

as controls. Plants were grown for 2 weeks. Sterols were then extracted

as follows: 100 mg of plant material (fresh weight) was ground in liquid N2

and extracted with 1.5 mL of methanol:CH2Cl2 (2:1) containing 10 mg of

stigmastanol (Sigma-Aldrich) as an internal standard. Samples were

incubated at 408C for 30 min and centrifuged at 4200g for 10 min. The

supernatant was removed and the pellet was reextracted twice with an

additional 1.5 mL of methanol:CH2Cl2, followed by pooling of the

The Role of IPP Isomerase in Arabidopsis 691

supernatants. These were then back-extracted with 2 mL of 0.8%

aqueous NaCl, and the lower organic phase was removed with a glass

pipette and transferred to a new glass vial. The aqueous phase was

extracted two additional times with 2 mL of CH2Cl2, and phases were

separated as before. The pooled organic fractions were dried under a

nitrogen stream and resuspended in 200 mL of CH2Cl2. After transfer to a

glass GC vial insert, the samples were dried again, redissolved with 70 mL

of tetrahydrofuran, incubated with 30 mL of N-methyl-N-trimethylsilyltri-

fluoroacetamide, and immediately analyzed.

The samples were analyzed on a Hewlett-Packard 6890 gas chromato-

graph coupled to a Hewlett-Packard 5973 quadrupole mass selective

detector. Separation was performed on a DB5 column (5% phenyl-

methylpolysiloxane; J&W Scientific) measuring 30 m 3 0.25 mm i.d. with

a 0.25-mm film thickness. Helium was utilized as the carrier gas at a

constant flow rate of 2 mL/min. A 1-mL aliquot of each sample was

injected in a splitless mode at 2508C. The temperature program was as

follows: 2-min hold at 2008C, then 158C/min to 2808C, followed by a

second gradient of 28C/min to 3008C, with a final 3-min hold. The mass

detector was run in scan mode from m/z 33 to 555 at 70 eV. Sitosterol, the

dominant phytosterol in Arabidopsis, eluted at 14.21 min (data not

shown). Isotopic labeling patterns and incorporation efficiencies were

determined for sitosterol following identification by retention time and

mass spectra compared with those of an authentic standard.

For NMR analysis, the methanol:CH2Cl2 plant extracts were concen-

trated and sitosterol was purified by preparative HPLC using a Supelcosil

LC-18 DB semipreparative HPLC column (Supelco) and a gradient

beginning at 10% water (solvent A) and 90% acetone (solvent B),

reaching 100% B at 11 min, followed by a hold at 100% B for 5 min

before a return to initial conditions at 20 min. Analytes were monitored at

210 nm, and sitosterol eluted as a minor peak at 10.65 min based on

comparison with an authentic standard. Sitosterol fractions from con-

secutive runs were pooled, evaporated under a nitrogen stream, and

resuspended in CDCl3. A Bruker DRX 500 NMR spectrometer was used

for recording of 1H and 13C NMR spectra of sitosterol. Operating fre-

quencies were 500.13 MHz for the acquisition of 1H spectra and 125.75

MHz for 13C spectra. CDCl3 was used as the solvent, and chemical shifts

were referenced to trimethylsilane added as an internal standard.

Vector Construction and Plant Transformation

Gateway technology (Invitrogen) was used for the generation of all

transformation constructs, which generally consisted of a target gene

bearing a C-terminal fusion to eGFP under the control of the cauliflower

mosaic virus 35S promoter for plant transformations or a target gene

bearing an N-terminal fusion to a His tag under the control of the T7

promoter for bacterial expression. attB adaptor–bearing PCR primers

(see Supplemental Table 1 online) were designed for the generation of

attB PCR products for recombination with the donor vector pDONR207

via BP Clonase reactions (Invitrogen). Primer and template combinations

used to generate the full-length cDNA or genomic clones for IDI1 and IDI2

are shown in Supplemental Table 1 online. Primers used to subclone IDI1

and IDI2 into the pB7FWG2 transformation vector (Karimi et al., 2002)

bearing a C-terminal eGFP fusion included the presumed start codon (but

none of the 59 UTR), encompassed the entire genomic sequence,

including all introns, and up to and including the last amino acid codon

of exon 6. However, the stop codons were left out to produce a

continuous open reading frame with eGFP fused to the C terminus of

either protein. The region connecting eGFP to IDI1 or IDI2 encoded a

short tether sequence (HPAFLYKVVIS) which is predicted to form a

random coil. Fully sequenced entry clones were recombined in LR

Clonase reactions with the pB7FWG2 vector (Karimi et al., 2002). After

each 10-mL recombination reaction, 3 mL was used to transform TOP10

chemically competent cells (Invitrogen), and the resulting transformed

cells were then used to inoculate 5-mL overnight cultures grown at 378C in

the presence of antibiotic (50 mg/mL gentamycin [Duchefa] for pDONR

entry clones or 100 mg/mL spectinomycin [Duchefa] for plant transfor-

mation expression vectors). Plant transformation vectors were intro-

duced into Agrobacterium tumefaciens strain GV3101 and used in

vacuum infiltration transformations with Arabidopsis (ecotype Col-0)

(Bechtold and Pelletier, 1998).

T0 seeds were sown in soil, stratified for 3 d at 48C, and sprayed with 50

mg/mL glufosinate ammonium two to three times daily starting at day 10.

Resistant plant lines were allowed to self-pollinate. Resulting T1 seeds

were characterized as follows. Single insert lines were identified by

segregation analysis based on resistance to glufosinate ammonium when

grown on sterile MS phytagar plates. Transgene expression levels of

independent lines were estimated by measuring the levels of green

fluorescence in seedling total protein extracts with a fluorescence plate

reader and a SYBR Green fluorescence filter. For this measurement, 100

mg of seedlings from each independent line was homogenized in tripli-

cate in a cold, 2-mL glass Tenbroek homogenizer containing 500 mL of

extraction buffer (100 mM Tris, pH 7.5, 10% glycerol, and 1 mM DTT). The

homogenate was spun at 48C in a benchtop microcentrifuge at maximum

speed for 10 min, and a 50-mL aliquot of each supernatant was trans-

ferred to a plate well for fluorescence measurement and compared with

wild-type (nontransformed) extracts. Apparent single insert lines (based

on 3:1 glufosinate ammonium resistance ratios) having the highest green

fluorescence values were carried on to the next generation and used in

confocal microscopy analysis. To confirm the presence of the full-length

transgene in each independent transformed line, total RNA was extracted

from each line used for microscopy and converted to cDNA as described

below in Gene Expression Measurements. This was then used as a

template in RT-PCR with the 35SF and eGFPR primers (see Supplemental

Table 1 online). Amplicons were cloned using the TOPO-TA-pCR4 cloning

kit (Invitrogen) and sequenced with M13, M13 reverse, and internal

primers to confirm that all plants used in microscopy expressed a full-

length transcript, including a complete transit peptide, that was in-frame

with the N terminus of eGFP.

Microscopy

Stably transformed Arabidopsis seedlings from the T2 or T3 generation

representing four transformation constructs (35S:IDI1c-eGFP, 35S:IDI2c-

eGFP, 35S:IDI1g-eGFP, and 35S:IDI2g-eGFP) were grown on sterile MS

agarose plates containing 25 mg/mL glufosinate ammonium (Sigma-

Aldrich) for 10 to 15 d. To test for chloroplast localization of eGFP fusion

proteins, true leaves, stems, and roots from each group were dissected

and mounted on a microscope slide with water and used in confocal

microscopy. Scans were performed on plants from three independent

lines per construct using a Zeiss LSM 510 Meta system, lasers specific for

eGFP (488-nm excitation, detection from 500 to 530 nm) and for chloro-

plast autofluorescence (561-nm excitation, 575-nm long-pass detection),

and a 403 LD C-Apochromat objective (water-corrected). To test for

mitochondrial localization, transformed Arabidopsis seedlings were

stained overnight in PBS, pH 7.2, containing 2% DMSO and 500 nM

MitoTracker Orange CMTMRos (Invitrogen) at 48C in light-protected

vessels with gentle shaking. Stained seedlings were washed two times

with PBS at 48C for 4 h with gentle shaking. Roots and stems were

mounted in PBS and visualized using a Zeiss Axiovert 200 inverted

fluorescence microscope, eGFP (filterset 38) and orange (filterset 20)

fluorescent filter blocks, an attached MRc5 Axiocam color digital camera,

and an LD Achroplan 403 objective.

Gene Expression Measurements

The expression of IDI1 and IDI2 in wild-type plant organs and homozy-

gous mutant seedlings was monitored by qRT-PCR and SYBR Green

assays. Primers were designed for each gene using Beacon Designer

692 The Plant Cell

(version 5; PremierBiosoft) in the 39 UTR (IDI1utrF, IDI1utrR, IDI2utrF, and

IDI2utrR) (see Supplemental Table 1 online). The efficiencies of the

primers were determined by the standard curve method (Pfaffl, 2001)

and according to their individual amplification curves using the Lin-

RegPCR program (Ramakers et al., 2003). For comparative quantifica-

tion, six normalizer gene candidates were screened for similarity of overall

expression levels among different organs using purified cDNA from wild-

type Arabidopsis roots, stems, flowers, and leaves. For comparison of

organ-specific expression of IDI1 and IDI2, 2.0 mg of total RNA from each

of those tissues was reverse-transcribed using SuperScript III (Invitrogen)

in a 20-mL reaction according to the manufacturer’s instructions using

50 pmol of an anchored poly(dT) primer [d(T18V)]. For each tissue, four

independent biological replicates derived from individual soil-grown

plants were reverse-transcribed into cDNA. After 1 h at 508C, reactions

were diluted 1:10 with pure water. A 1-mL aliquot of this diluted template

containing ;0.2 ng of cDNA was analyzed in triplicate in 25-mL SYBR

Green assays in which leaves were used as a calibrator against each of

the other organs. Fold change differences for IDI1 and IDI2 expression in

each of the organs surveyed were calculated according to the efficiency-

corrected model (Pfaffl, 2001) using RP2LS (At4g35800) as a reference

gene. To measure IDI gene expression in homozygous knockout mutants,

whole plate-grown seedlings were used for total RNA extractions as

described above (four biological replicates and three technical replicates

of each), then analyzed by qRT-PCR as described above using wild-type

seedling transcript levels as a calibrator for fold change calculations.

Biochemical Characterization

For a comparison of the kinetic properties of the two IDI proteins, a

truncated version of each cDNA lacking the signal peptide but containing

a thrombin cleavage site was generated using the cytIDI1clv-attB1 and

cytIDI1stp-attB2 primers for idi1 and cytIDI2clv-attB1 and cytIDI2stp-

attB2 for idi2 (see Supplemental Table 1 online). Entry clones were

generated from these attB PCR products using pDONR207 as described

above. Fully sequenced entry clones were then used in LR Clonase

reactions with a Gateway-compatible form of the bacterial expression

vector pET28 (Yu and Liu, 2006) bearing nine N-terminal His residues to

yield cytIDI1-pH28 and cytIDI2-pH28. The truncation site was based on

the alignment of IDI1 and IDI2 to the start Met of the Escherichia coli IDI

protein (GI: 1789255). Following transformation of E. coli BL21(DE3)

competent cells with purified plasmids encoding cytIDI1-pET28 and

cytIDI2-pET28, a single colony of each was used to inoculate a 200-mL

culture of OverNight Express autoinduction medium (Novagen) supple-

mented with 1% (v/v) glycerol and 50 mg/mL kanamycin. Cultures were

grown at room temperature for 48 h (220 rpm), then centrifuged for 20 min

at 4400g and 48C. The pellet was resuspended in 10 mL of ice-cold 50 mM

Tris, pH 7.5, 10% (v/v) glycerol, 500 mM NaCl, 1 mM phenylmethylsul-

fonyl fluoride, 10 mM MgCl2, 10 mM imidazole, and 50 mg/mL lysozyme

and sonicated three times for 1 min each using a Bandelin HD2070

Sonoplus sonicator at 70% power. Lysates were centrifuged for 20 min at

31,000g and 48C. Crude extracts were purified on a Pharmacia His-

TrapHQ Ni affinity column using a fast protein liquid chromatography

protein purification system (Pharmacia-Amersham) by washing the

bound sample with resuspension buffer lacking phenylmethylsulfonyl

fluoride and lysozyme and eluting with the same buffer containing 125

mM imidazole.

Following affinity purification, the His tags were removed with a

thrombin cleavage capture kit (Novagen). The biotinylated thrombin

was removed with a strepavidin–agarose spin column, and the digested

proteins, judged to be >97% pure by SDS-PAGE, were subsequently

desalted into IDI assay buffer (50 mM Tris, pH 7.0, 10% [v/v] glycerol,

5 mM MgCl2, 200 mM KCl, 1 mM DTT, 0.1 mM MnCl2, and 0.1 mM ZnCl2)

using DG-10 desalting columns (Bio-Rad) and quantified using the

Bradford reagent (Bio-Rad). Proteins were diluted to 0.25 mg/mL and

assayed by the acid lability method (Satterwhite, 1985). For Km and kcat

measurements, reactions were performed in 100-mL volumes at six

different concentrations of [1-14C]IPP (American Radiochemicals; 50

mCi/mmol). For pH optima studies, a broad-range pH buffer consisting of

50 mM acetate, MES, and bis-Tris-propane was adjusted at whole pH

intervals from 4 to 10, and reactions were performed under linear

conditions at each pH in triplicate. Each reaction included 2.5 mg of

protein and took place at room temperature. After 15 min, reactions were

stopped by the addition of 1 volume of 1 N HCl and 1 mL of diethyl ether.

Hydrolysis of the acid-labile DMAPP isomer was performed at 378C for

30 min, followed by extraction two times with 1 mL of diethyl ether. The

organic phases were pooled, purified over a Pasteur pipette column

containing glass wool, silica gel, and anhydrous MgSO4, and finally

quantified by liquid scintillation counting. Km and kcat calculations were

determined using SigmaPlot version 7.0 and the Enzyme Kinetics module

version 1.1.

Isoprenoid Analysis

Five hundred milligrams of leaf tissue from wild-type and idi mutant plants

grown in 250-mL pots under continuous high light (600 mmol�m�2�s�1) at

258C was ground in liquid nitrogen and extracted with 2.5 mL of

acetone:water (9:1) in quadruplicate. For flower analysis, 100 mg of

wild-type or idi2-1 flowers was frozen, ground, and extracted in 1.0 mL

of CH2Cl2:acetone (1:1). A 300-mL aliquot of each floral extract was dried

under a nitrogen stream and resuspended in tetrahydrofuran. Sterols

were then derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide

and analyzed by GC-MS. Both leaf and flower extracts were shielded

from light, and extraction was performed at 48C using a lab quake

followed by centrifugation at 16,000g (48C). A 10-mL aliquot of each

supernatant was analyzed by HPLC on an apparatus fitted with a diode

array detector using a Supelcosil C18 column (7.5 cm 3 4.6 mm) flowing

at 1.5 mL/min. The gradient consisted of water (A) and acetone (B),

running at 65% B for 4 min, then reaching 90% B at 12 min, and finally

100% B at 20 min, before returning to initial conditions. Carotenoids were

monitored at 445 nm and chlorophylls at 650 nm. Both classes of

compounds were identified based on their retention times and absorption

spectra compared with authentic standards.

For ubiquinone analysis, the same samples were analyzed on an 1100

series HPLC apparatus (Agilent Technologies) coupled to an Esquire

6000 ESI ion-trap mass spectrometer (Bruker Daltonics) operated in

positive mode in the range m/z 500 to 900. Specifics were as follows:

skimmer voltage, �40 eV; capillary exit voltage, �152.4 eV; capillary

voltage, �4000 V; nebulizer pressure, 35 p.s.i.; drying gas, 8 L/min; gas

temperature, 3308C. Elution was accomplished using a YMC-Pack C30

column (25 cm 3 4.6 mm, 5 mm; YMC) with a gradient of 99% methanol:

1% water (v/v) (solvent A) and tert-butyl-methylether (solvent B) at a flow

rate of 1.0 mL/min at 258C as follows: start, 0% B; 0 to 50% (v/v) B (25

min); 50 to 0% (v/v) B (1 min); 0% B (6 min). Flow coming from the column

was diverted in a ratio of 4:1 before entering the mass spectrometer

electrospray chamber. Ubiquinone-9 was detected in positive ion mode

and quantified by the integrated area of the extracted ion trace of the sum

of m/z 795.6 þ 817.6 þ 833.6 ([MþH]þ, [MþNa]þ, [MþK]þ).

59 RACE Cloning

Total RNA isolated from flowers or leaves was used to prepare RACE-

ready cDNA using the SMART RACE cDNA amplification kit (Clontech

Laboratories). Following a round of PCR using UPM and gene-specific

primers (IDI1RACE-R1 and IDI2RACE-R1), nested PCR was performed

using NUP and nested gene-specific primers (IDI1RACE-R2 and IDI2R-

ACE-R2). 59 RACE PCR products were purified on a 4% agarose gel,

excised, and cloned using the pCR4 TOPO-TA cloning kit (Invitrogen).

Cloning reaction products were used in transformations with TOP10 cells.

The Role of IPP Isomerase in Arabidopsis 693

Resulting positive colonies were grown in 5-mL Luria-Bertani cultures

containing 50 mg/mL kanamycin for plasmid mini-preps. Purified plas-

mids were sequenced using M13 and M13R primers.

Accession Numbers

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

Initiative or GenBank/EMBL databases under the following accession

numbers: Arabidopsis thaliana IDI1 (At5g16440), IDI2 (At3g02780), and

RP2LS (At4g35800); Clarkia breweri IPP isomerase 1 (X82627) and IPP

isomerase 2 (U48963); Escherichia coli IPP isomerase (NP_417365); and

Saccharomyces cerevisiae IPP isomerase (P15496).

Supplemental Data

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

Supplemental Figure 1. Scheme for the Incorporation of [2-13C]

Mevalonate into Sitosterol.

Supplemental Figure 2. Sitosterol Mass Spectra from Mutant

Arabidopsis Plants.

Supplemental Figure 3. Upfield Portion of the 13C NMR Spectrum of

Purified Sitosterol Extracted from idi Mutant Arabidopsis Seedlings.

Supplemental Table 1. Primer Sequences Used in This Study.

ACKNOWLEDGMENTS

We thank Jenny Wrede, Katrin Luck, Claudia Wenderoth, Jana Pastuscheck,

and Martin Franke for their technical assistance and Andreas Weber and

the greenhouse staff at the Max Planck Institute for Chemical Ecology

(MPICE) for their assistance in caring for the Arabidopsis plants. Stefan

Opitz (MPICE) and Ales Svatos (MPICE) are thanked for their assistance

with phytosterol and ubiquinone analysis, respectively, and we thank

Joao Pedro Marques (Institut fur Pflanzenphysiologie, Halle, Germany)

for providing us with the ferredoxin-NADP(H) oxidoreductase-eGFP

positive control Arabidopsis line. We express our gratitude to Almut

Kießlich at the Carl Zeiss Applikationszentrum (Jena, Germany) for her

assistance with confocal microscopy. This work was carried out with

support from the Max Planck Society and a postdoctoral fellowship from

the Alexander von Humboldt Foundation (J.C.D.).

Received July 3, 2007; revised January 18, 2008; accepted February 7,

2008; published March 4, 2008.

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696 The Plant Cell

DOI 10.1105/tpc.107.053926; originally published online March 4, 2008; 2008;20;677-696Plant Cell

Michael A. Phillips, John C. D'Auria, Jonathan Gershenzon and Eran PicherskySubcellular Compartments and Have Overlapping Functions in Isoprenoid Biosynthesis

Type I Isopentenyl Diphosphate Isomerases Are Targeted to MultipleArabidopsis thalianaThe

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