A single gene encodes isopentenyl diphosphate isomerase isoformstargeted to plastids, mitochondria and peroxisomesin Catharanthus roseus

Gregory Guirimand • Anthony Guihur • Michael A. Phillips • Audrey Oudin •

Gaelle Glevarec • Celine Melin • Nicolas Papon • Marc Clastre • Benoit St-Pierre •

Manuel Rodrıguez-Concepcion • Vincent Burlat • Vincent Courdavault

Received: 17 February 2012 / Accepted: 5 May 2012 / Published online: 26 May 2012

� Springer Science+Business Media B.V. 2012

Abstract Isopentenyl diphosphate isomerases (IDI) cat-

alyze the interconversion of the two isoprenoid universal

C5 units, isopentenyl diphosphate and dimethylally

diphosphate, to allow the biosynthesis of the large variety

of isoprenoids including both primary and specialized

metabolites. This isomerisation is usually performed by

two distinct IDI isoforms located either in plastids/

peroxisomes or mitochondria/peroxisomes as recently

established in Arabidopsis thaliana mainly accumulating

primary isoprenoids. By contrast, almost nothing is known

in plants accumulating specialized isoprenoids. Here we

report the cloning and functional validation of an IDI

encoding cDNA (CrIDI1) from Catharanthus roseus that

produces high amount of monoterpenoid indole alkaloids.

The corresponding gene is expressed in all organs

including roots, flowers and young leaves where transcripts

have been detected in internal phloem parenchyma and

epidermis. The CrIDI1 gene also produces long and short

transcripts giving rise to corresponding proteins with and

without a N-terminal transit peptide (TP), respectively.

Expression of green fluorescent protein fusions revealed

that the long isoform is targeted to both plastids and

mitochondria with an apparent similar efficiency. Deletion/

fusion experiments established that the first 18-residues of

the N-terminal TP are solely responsible of the mitochon-

dria targeting while the entire 77-residue long TP is needed

for an additional plastid localization. The short isoform is

targeted to peroxisomes in agreement with the presence of

peroxisome targeting sequence at its C-terminal end. This

complex plastid/mitochondria/peroxisomes triple targeting

occurring in C. roseus producing specialized isoprenoid

secondary metabolites is somehow different from the sit-

uation observed in A. thaliana mainly producing house-

keeping isoprenoid metabolites.

Keywords Catharanthus roseus � Dual targeting �Isoprenoid � Isopentenyl diphosphate isomerase �Methylerythritol phosphate pathway � Mevalonate pathway

Introduction

Isoprenoids encompass an extraordinary variety of essen-

tial (primary) and specialized (previously named second-

ary) metabolites in bacteria, animals, and fungi and also in

plants in which more than 30,000 different molecules have

been identified to date. These molecules originate from

the head-to-tail condensation of the basic five-carbon units

isopentenyl diphosphate (IPP) and its allylic isomer

dimethylallyl diphosphate (DMAPP) (Sacchettini and

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11103-012-9923-0) contains supplementarymaterial, which is available to authorized users.

G. Guirimand � A. Guihur � A. Oudin � G. Glevarec �C. Melin � N. Papon � M. Clastre � B. St-Pierre �V. Courdavault (&)

EA2106 ‘‘Biomolecules et Biotechnologies Vegetales’’,

Universite Francois Rabelais de Tours, 37200 Tours, France

e-mail: vincent.courdavault@univ-tours.fr

M. A. Phillips � M. Rodrıguez-Concepcion

Department of Molecular Genetics, Centre for Research

in Agricultural Genomics (CRAG), CSIC-IRTA-UAB,

08034 Barcelona, Spain

V. Burlat

Laboratoire de Recherche en Sciences Vegetales, Universite de

Toulouse, UPS, UMR 5546, BP 42617, 31326 Castanet-Tolosan,

France

V. Burlat

CNRS, UMR 5546, BP 42617, 31326 Castanet-Tolosan, France

123

Plant Mol Biol (2012) 79:443–459

DOI 10.1007/s11103-012-9923-0

Poulter 1997). Sequential condensations of IPP to DMAPP

lead to the synthesis of polyprenyl diphosphates of

increasing size such as geranyl diphosphate (C10), farnesyl

diphosphate (C15) or geranylgeranyl diphosphate (C20),

which could be further metabolized to monoterpenes, ses-

quiterpenes and diterpenes, respectively. While fungi and

animals synthesize both IPP and DMAPP via the mevalo-

nate (MVA) pathway, most of bacteria produce these pre-

cursors through the 2-C-methyl-D-erythritol 4-phosphate

(MEP) pathway. Interestingly, plants harbor both pathways

giving rise to specific end-products including phytosterols,

sesquiterpenes, triterpenes and the side-chain of ubiqui-

none for the MVA pathway (Bouvier et al. 2005) and

monoterpenes, diterpenes, carotenoids as well as the side-

chain of chlorophylls and tocopherols for the MEP pathway

(Rodrıguez-Concepcion and Boronat 2002). These specific

biosyntheses result from a complex subcellular compart-

mentalization within plant cells. Indeed, the MEP pathway

enzymes have been exclusively localized in plastids (Hsieh

et al. 2008) while the MVA pathway has been considered

until recently to occur mainly in the cytosol (Tholl and Lee

2011). However, the specific localization of the final steps

of the MVA pathway within peroxisomes has revealed a

more complex organization than previously supposed

(Sapir-Mir et al. 2008; Simkin et al. 2011). Both pathways

also differ by their terminal products since the MEP

pathway directly synthesizes both IPP and DMAPP (6:1

mixture) while the MVA pathway produces only IPP

(Bloch et al. 1959; Hoeffler et al. 2002; Rohdich et al.

2003). Due to the molar ratio required to synthesize the

different polyprenyl diphosphates, isomerization of IPP to

DMAPP is needed to allow an efficient isoprenoid bio-

synthesis, especially for the MVA pathway. The enzyme

IPP isomerase (IDI; EC 5.3.3.2) catalyzes the reversible

conversion of IPP to DMAPP and is found in all living

organisms (Ramos-Valdivia et al. 1997). Depending on the

cofactor required for activity, IDIs have been classified into

two types. Type I IDIs are zinc metalloproteins also

needing Mg2? for their catalytic activity (Muehlbacher and

Poulter 1988; Anderson et al. 1989; Carrigan and Poulter

2003; Lee and Poulter 2006). Type II IDIs are flavoproteins

requiring the reduced form of flavin mononucleotide and

Mg2? for activity and they have been identified in archaea

and some bacteria (Kaneda et al. 2001; Sharma et al. 2010).

Therefore, plants only display type I IDIs that are usually

encoded by two distinct genes (Campbell et al. 1998;

Cunningham and Gantt 2000; Nakamura et al. 2001). In

Arabidopsis thaliana, both genes (AtIDI1, At5g16440 and

AtIDI2, At3g02780) are expressed as long and short tran-

scripts encoding proteins that differ in length at their

N-terminal end (Phillips et al. 2008). Such mechanism

engenders a complex subcellular localization of the cor-

responding isoenzymes since AtIDI1 and AtIDI2 long

isoforms are targeted to plastids and mitochondria,

respectively (Phillips et al. 2008; Okada et al. 2008), while

both short isoforms are directed to peroxisomes (Sapir-Mir

et al. 2008) reflecting the compartmentalization of the MEP

and MVA pathways and the central role of IDI in iso-

prenoid biosynthesis. These combined studies have largely

contributed to the re-evaluation of the first model of type I

IDIs subcellular distribution in plant cells proposing a dual

localization within plastids and cytosol (Nakamura et al.

2001). In addition, by contrast with type II IDIs organized

in tetramers, elucidation of the crystal structure of the

Escherichia coli type I IDI indicates that they mainly

operate as monomers in agreement with plant IDI purifi-

cation (Ramos-Valdivia et al. 1997; Durbecq et al. 2001;

Kaneda et al. 2001). However, an elegant and sensitive

analysis of the yeast proteome complexes has revealed the

existence of type I IDIs self interactions in multi-protein

structures suggesting the existence of higher levels of

association (Gavin et al. 2002).

The current knowledge concerning the architecture of

the isoprenoid biosynthetic pathways and the IPP/DMAPP

isomerization in plant cells mainly relies on results

obtained in A. thaliana. However, a growing set of results

has been also produced during the last 10 years in plants

such as Catharanthus roseus displaying a particularly

active production of specialized isoprenoids (Hemmerlin

et al. 2012). C. roseus, also called Madagascar periwinkle,

is known to produce valuable monoterpenoid indole

alkaloids (MIA; Guirimand et al. 2010a), which contain an

isoprenoid moiety synthesized by the MEP pathway. In

C. roseus, we previously demonstrated that MEP pathway

genes and geraniol 10-hydroxylase gene (CrG10H) were

mainly expressed in a specialized tissue of aerial organs,

the internal phloem associated parenchyma (IPAP; Burlat

et al. 2004; Courdavault et al. 2005; Oudin et al. 2007)

and that a MEP pathway enzyme and geranylgeranyl

diphosphate synthase accumulated in the stroma of plas-

tids and in long stroma-filled chlorophyll-free protrusion

budding from plastids named stromules (Oudin et al.

2007; Guirimand et al. 2009; Thabet et al. 2012). In

addition, we have also recently established the pivotal role

of peroxisomes in isoprenoid production by the localiza-

tion of the enzymes catalyzing the final steps of the MVA

pathway in this subcellular compartment as well as those

of a farnesyl diphosphate synthase involved in sesquit-

erpenoid biogenesis (Clastre et al. 2011; Simkin et al.

2011; Thabet et al. 2011; Guirimand et al. 2012). To gain

further insight into the role of the enzyme compartmen-

talization during isoprenoid biosynthesis in plants dis-

playing an active production of specialized isoprenoids,

we describe here the characterization of C. roseus type I

IDI (CrIDI1), including the cloning and functional vali-

dation of the enzyme, the CrIDI1 transcript distribution

444 Plant Mol Biol (2012) 79:443–459

123

studied by real-time PCR and RNA in situ hybridization,

the IDI subcellular localization analyzed by fluorescent

protein fusion approaches and its propensity to self

interact in vivo using bimolecular complementation

assays. These analyses indicate that CrIDI1 displays a

triple subcellular localization in plastids/mitochondria and

peroxisomes suggesting that this single protein allows the

biosynthesis of distinct isoprenoids originated from either

the MVA or MEP pathways in three distinct subcellular

compartments.

Methods

Plant material and cell culture conditions

Mature C. roseus (L.) G. Don, cultivar Pacifica Pink

(Apocynaceae) plants grown from seeds (Ball Ducrettet,

Thonon, France) in the botanical garden of Tours were

harvested in summer for microscopy fixation (RNA in situ

hybridization experiments) and RNA extraction (cloning

experiments and gene expression measurements). C. roseus

cell suspensions used for subcellular localization studies

(C20A chlorophyll-free cells and CR6 chlorophyll con-

taining cells) were propagated in Gamborg B5 medium

(Duchefa) at 24 �C under continuous shaking (100 rpm)

for 7 days (C20A cells) or 14 days (CR6 cells) as previ-

ously described (Merillon et al. 1989; Kodja et al. 1989;

Guirimand et al. 2009).

RNA extraction and cDNA synthesis

After grounding of young leaves in liquid nitrogen, total

RNA was extracted using the NucleoSpin RNA Plant kit

(Macherey-Nalgel, France) according to the manufacturer’s

instructions. First-strand cDNA was synthesized from 5 lg

of total RNA using 500 ng of oligo(dT)18 primers and 15

units of Thermoscript reverse transcriptase (Invitrogen)

according to the manufacturer’s instructions. Following

retro-transcription, RNA complementary to cDNA was

removed by treatment with E. coli RNase H (Invitrogen)

during 20 min at 37 �C. For 50 RACE analysis, similar

reactions were carried out except that total RNA was sub-

sequently removed by treatment with RNase A (Sigma) and

the reaction product was purified using the NucleoSpin

Extract II kit. For real-time PCR, total RNA was extracted

with RNAeasy Plant mini kit (Qiagen) and treated (1.5 lg)

with RQ1 RNase-free DNase (Promega) before being used

for first-strand cDNA synthesis by priming with oligo (dT)17

(0.5 lM). Reverse transcription was carried out using

superscript III RT (Invitrogen) at 50 �C according to the

manufacturer’s instructions.

Cloning of the C. roseus cDNA encoding isopentyl

diphosphate isomerase 1

On the basis of the alignment of known plant IDI amino

acid sequences, a conserved region was identified and used

to design the degenerated primer IDIrev 1 (50-YTTRTGRA

TGSTYTTCATRCT-30). IDIrev1 was used in combination

with the M13 reverse universal primer to carry out PCR

amplifications on a kZAPII-oriented C. roseus cDNA

library using GoTaq DNA polymerase (Promega). A partial

cDNA encompassing the 50 end of a putative IDI coding

sequence was obtained and cloned into pGEM-T easy

vector (Promega) prior to sequencing. This sequence

allowed designing the ISOfor2 primer (50-TTGCTCTAA

TTCTACTACGCTTGC-30) used in combination with the

T7 universal primer to amplify the 30 end of the corre-

sponding cDNA using the C. roseus cDNA library as a

matrix. After sequencing of the resulting PCR product, the

two specific primers IPPfull-for (50-CCAACTCACTCATT

ACTACTCAAAAGCT-30) and IPPfull-rev (50-CTTAAAC

AGTGCACATCTAGAATACAGT-30) were then designed

to amplify a full-length open reading frame using Pfu DNA

polymerase (Promega). The resulting amplicon was cloned

into pSC-A vector (Stratagene) following A-tailing and

sequenced before deposition at NCBI under Genbank

accession number (EU135981).

Functional characterization of CrIDI1 and purification

of the recombinant enzyme

Based on ChloroP prediction (Table 1), a truncated cDNA

of CrIDI1 was amplified using primers pQE-IDI-Bam

(50-GCGGATCCATGGGTGCTGCCGTTACTGATTCC-30)and pQE-IDI-Hind (50-GCAAGCTTTTAAATCTTCTTGT

GAATGGTTTTCAT-30) to remove the 77 amino acid-

predicted transit peptide and to introduce BamHI and

HindIII restriction sites at the 50 and 30 end, respectively.

Amplification was performed using Pfu DNA polymerase

(Promega) and the corresponding PCR product was

cloned into pQE-30 (Qiagen, Courtaboeuf, France). After

sequencing, the plasmid expressing the pseudomature form

of CrIDI was used to carry out the functional character-

ization of IDI according to Phillips et al. (2008). The

recombinant protein was expressed in E. coli BL21 cells

cultivated at 37 �C in Luria–Bertani medium supplemented

with ampicillin (75 lg ml-1) under vigorous agitation

(250 rpm) until absorbance at 600 nm reached 0.6. After

addition of 1 mM isopropyl-b-D-thiogalactoside (IPTG),

cells were allowed to grow for 4 h under the same condi-

tions prior to protein purification with a Co2? column

according to the manufacturer’s protocol (Talon Resin

metal ion, Clontech). The quaternary structure of the pro-

tein was analyzed by migration in a native polyacrylamide

Plant Mol Biol (2012) 79:443–459 445

123

gel (PAGE) (12 %) following incubation with 10 mM

dithiothreitol (DTT) or water as previously described

(Takahashi et al. 1998).

Bioinformatic sequence analysis

The predictions of protein subcellular localization were

performed using PSORT (http://psort.ims.u-tokyo.ac.jp/),

Predotar (http://urgi.versailles.inra.fr/predotar/predotar.html),

ChloroP 1.1 (http://www.cbs.dtu.dk/services/ChloroP/), Mi-

toProt (http://ihg.gsf.de/ihg/mitoprot.html), TargetP 1.1 (http://

www.cbs.dtu.dk/services/TargetP/) and Target Signal Pre-

dictor (Target SP)—PTS1 binding sites (http://www.peroxiso

medb.org/diy_PTS1.html) softwares.

50 RACE analysis

A homopolymeric tail of cytosine was added at the 30 end

of the synthesized cDNA using terminal deoxinucleotidyl

transferase (Roche) according to the manufacturer’s

instructions. Unincorporated nucleotides and enzyme were

removed by purification using the NucleoSpin Extract II kit

and the purified cDNAs used as a matrix for nested PCR

using oligo(dG)18 primers and nested gene-specific primers

50 RACE-IDI2 (50-TGAACTCATCTACTGGGACATCTT

CAG-0) and 50 RACE-IDI3 (50-GATGGCTGCAGCATGT

GTTTGTC-30). 50 RACE PCR products were separated in a

2.5 % agarose gel, excised, purified and cloned into the

pGEM-T easy vector (Promega) prior to sequencing.

Gene expression measurements (real-time PCR

analysis)

The expression of the CrIDI1 (GenBank EU135981) and

RPS9 (GenBank AJ749993) genes was analyzed by real-

time PCR using primers qIDI-up (50-TGCTGCCGTTACT

GATTCCG-30), qIDI-down (50-ATACGCTGAAAGCCCT

GTGC-30), qRPS9-for (50-TTACAAGTCCCTTCGGTGG

T-30) and qRSP9-rev (50-TGCTTATTCTTCATCCTCTTC

ATC-30) on retro-transcribed RNA extracted from C.

roseus organs including roots, first internodes, young and

mature leaves, flower buds and flowers. Real-time PCR

was run on an ABI Prism 7000 SDS light cycler (Applied

Biosystems, Foster City, CA, USA) using the SYBR�

Green I technology. Each reaction was performed in a total

reaction volume of 25 ll containing 1 ll of a 1/3 dilution

of the first cDNA strand synthesis reaction, 0.2 lM for-

ward and reverse primers and 19 MESA GREEN qPCR

Master mix Plus buffer (Eurogentec, Angers, France). The

reaction was initiated by a denaturation step at 95 �C for

10 min, followed by 40 cycles at 95 �C for 15 s and 60 �C

for 1 min. CrRPS9 was used as a control gene to allow the

normalization of the gene copy numbers in each sample.

Each assay was performed in triplicate. Data correspond

to average values (n = 3) ± standard deviation (SD).

Relative transcripts levels in each sample are expressed as

a ratio of the abundance of the CrRPS9 transcripts and

normalized to the transcript level of young leaves that was

set to one for each gene separately.

In situ hybridization of C. roseus leaves

Full-length CrIDI1 cDNA cloned in pSC-A vector (Strat-

agene) was used for the synthesis of sense and anti-sense

RNA-probes as previously described (Mahroug et al.

2006). For CrG10H, a previously described plasmid was

used for the riboprobe transcription (Burlat et al. 2004).

Paraffin-embedded serial longitudinal sections of young

developing leaves were hybridized with digoxigenin-

labeled transcripts and localized with anti-digoxigenin-

alkaline phosphatase conjugates according to Mahroug

et al. (2006).

Constructs for subcellular localization studies

and bimolecular fluorescent complementation assays

The subcellular localization of CrIDI1 has been studied

by creating fluorescent fusion proteins using the pSCA-

Table 1 Predictions of CrIDI1 subcellular localization

Enzyme PSORT Predotar ChloroP MitoProt TargetP Target SP

CrIDI1 P (stroma) 0.928 P 0.89 Y (0.582)

77 residues

Y (0.922)

18 residues

cTP (0.779)

mTP (0.062)

0.8

HKLM (matrix) 0.595 M 0.11

P (thy mb) 0.595 E 0.10

For PSORT and Predotar predictions, the three most favorable localizations are reported with the corresponding score. For ChloroP, MitoProt

and TargetP predictions, the score of transit peptide presence and transit peptide length are given. For target signal predictor (Target SP)

prediction, the score and sequence of the peroxisome targeting sequence (PTS1) is given

cTP chloroplast transit peptide, E elsewhere, M mitochondria, mTP mitochondria transit peptide, P plastid, thy mb thylakoid membrane

446 Plant Mol Biol (2012) 79:443–459

123

cassette GFPi and pSCA-cassette YFPi plasmids (Guiri-

mand et al. 2009). Full-length ORF of CrIDI1 as well as

its truncated variants were amplified with Pfu DNA poly-

merase using the specific primers described in supple-

mental Table 1, designed to introduce BglII and/or SpeI

restriction sites at both cDNA extremities. After amplifi-

cation, these cDNA were sequenced and cloned in frame

with the 50 end of the GFP coding sequence to generate

CrIDI–GFP fusion protein for example. Details on primers

and constructs are given in supplemental Table 1. The

subcellular localization of AtIDI1 (At5g16440) and AtIDI2

(At3g02780) has been studied by a similar approach: the

AtIDI1 coding sequence has been amplified with primers

AtIDI1-Bgl (50-GTAGATCTGATGTCTACTGCTTCACT

ATTTAGCTTC-30) and AtIDI1-Spe (50-GTACTAGTGAG

CTTGTGAATGGTTTTCATGTCT-30) and cloned in the

BglII and SpeI restriction sites of the pSCA-cassette GFPi

plasmid; the AtIDI2 coding sequence has been amplified

with primers AtIDI2-for-Spe (50-GTACTAGTATGTCTG

CTTCTTCTTTATTTAATCTCCCATTG-30) and AtIDI2-

rev-Spe (50-GTACTAGTGAGTTTGTGGATGGTTTTCA

TGTCTATAGC-30) and cloned in the BglII restriction site

of the pSCA-cassette GFPi plasmid. For testing the effect

of the presence of an additional isoleucine residue located

downstream of the SKL PTS1 on the peroxisome import

efficiency, the YFP coding sequence of the pSCA-cassette

YFPi plasmid has been removed and replaced by an YFP

coding sequence coding for either YFP-SKL, amplified

with primers YFPfor2 (50-GAAGATCTGACTAGTATGG

TGAGCAAGGGCGAGGAGCTGTTCACC-30) and YF-

Prev-SKL-stop (50-GCGCTAGCTTACAGCTTCGAGCC

ACCGCCGCCGCCTCTAGATCCGGTGGATCTGAGTC

C-30) or YFP–SKLI, amplified with primers YFPfor2 (50-GAAGATCTGACTAGTATGGTGAGCAAGGGCGAGG

AGCTGTTCACC-30) and YFPrev-SKLI-stop (50-GCGCT

AGCTTAAATCAGCTTCGAGCCACCGCCGCCGCCTC

TAGATCCGGTGGATCTGAGTCC-30).The CrIDI1 self interactions have been characterized by

bimolecular fluorescence complementation (BiFC) assays

using the pSCA-SPYNE173 and pSCA-SPYCE(M) plas-

mids (Guirimand et al. 2010b) modified from original BiFC

plasmids (Waadt et al. 2008), which allow expressing

proteins fused to the N-terminal extremity of the two split-

YFP fragments (YFPN, residues 1–173 and YFPC, residues

156–239). Details on primers and cloning procedures are

given in supplemental Table 2. Negative control constructs

allowing the targeting of the split-YFP fragments to plas-

tids were generated by cloning the targeting peptide of IDI

(amplified with primers IDI–GFP–AS and pepATG4-rev)

at the 50 end of the coding sequence of the N-terminal

(YFPN, amino acids 1–173) and C-terminal (YFPC, amino

acids 156–239) fragments of YFP, in the BglII and SpeI

restriction sites of pSCA-SPYNE173 and pSCA-SPY-

CE(M). Interactions of the CrIDI1 long isoform have been

studied by the cloning of full length cDNA amplified with

primers IDI–GFP–S and IDI–GFP–AS, in the BglII and SpeI

restriction sites of pSCA-SPYNE173 and pSCA-SPY-

CE(M), to express tpIDI–YFPN and tpIDI–YFPC. Interac-

tions of the CrIDI short isoform have been studied by

creating internal split-YFP fusions within the CrIDI

sequence. For this purpose, the split-YFP coding sequence of

both pSCA-SPYNE173 and pSCA-SPYCE(M) has been

removed and replaced respectively by a split-YFPN and split-

YFPC coding sequence exhibiting the coding sequence of the

11 last residues of CrIDI1 at their 30 end. The coding

sequence of the CrIDI1 short isoform (devoid of the 11 last

residues) amplified with primers IDI-del3 and IDI-rev-del

was subsequently cloned in the BglII and SpeI restriction

sites of the resulting plasmids.

Biolistic transformation of C. roseus cells

and epifluorescence microscopy

Transient transformation of C. roseus cells by particle

bombardment and fluorescence imaging were performed

following the procedures described by Guirimand et al.

(2009, 2010b). Briefly, C. roseus plated cells were bom-

barded with DNA-coated gold particles (1 lm) and 1,100

psi rupture disc at a stopping-screen-to-target distance of

6 cm, using the Bio-Rad PDS1000/He system. Cells were

cultivated for 14 h to 38 h prior to being harvested, treated

and observed. The protein subcellular localization was

determined using an Olympus BX-51 epifluorescence

microscope equipped with an Olympus DP-71 digital

camera and a combination of YFP and CFP filters. The

pattern of localization presented in this work is represen-

tative of circa 50 observed cells.

Organelle markers and cell co-transformation

A set of organelle markers was used in co-transformation

experiments with the CrIDI–GFP constructs for identifi-

cation of the subcellular compartments accumulating

fusion proteins. The ‘‘plastid’’-mcherry (CD3–1,000) and

‘‘plastid’’-CFP (CD3–994) markers, ‘‘mitochondria’’-

mcherry (CD3–992) and ‘‘mitochondria’’-CFP (CD3–986)

markers, and the ‘‘peroxisome’’-CFP marker, described

by Nelson et al. (2007) were obtained from the ABRC

(http://www.arabidopsis.org). The mcherry-GUS cytosolic

marker and the mcherry nucleocytosolic marker were

described previously (Guirimand et al. 2011a). Plasmid

co-transformations were performed according to Guirimand

et al. (2009, 2011b) using 400 ng of each plasmid or 100 ng

for BiFC assays.

Plant Mol Biol (2012) 79:443–459 447

123

Results

Isolation of a C. roseus IDI (CrIDI1) full-length cDNA

A full-length cDNA sequence of a putative C. roseus IDI

(CrIDI1) was successively cloned from an oriented C.

roseus cells cDNA library. This amplified full-length

cDNA is 1,271-bp long and has been deposited at NCBI

under Genbank accession number (EU135981). Interest-

ingly, albeit degenerated primers were used for the first

rounds of amplification, no other sequences homologous to

CrIDI1 were amplified. In addition, Genbank EST database

screening only retrieved EST strictly homologous to CrIDI1

(supplemental Table 3) as well as in the 653,830 EST

sequences of the MAGPIE database (V. De Luca, personal

communication). However, contigs of a putative second

isoform (named CrIDI2) were identified in the Medicinal

Plant Genomics Resource (http://medicinalplantgenomics.

msu.edu/) but the associated transcriptomic data reveal

that CrIDI2 mRNAs are barely detectable as compared to

those of CrIDI1 (supplemental Table 4). Such EST/mRNA

distributions strongly suggest that CrIDI1 is the mainly

expressed isoform in C. roseus plants. As a consequence,

this work has been focused on the sole characterization of

this isoform.

The 936-bp open reading frame of CrIDI1 encodes a

311-residues long protein with an estimated mass of

35 kDa. Amino acid comparison highlights the high degree

of identity of CrIDI1 with plant orthologs (89 and 88 %

with AtIDI1 and AtIDI21, respectively) as well as with

bacterial and yeast ones (21 and 32 %, respectively)

(Fig. 1). In addition, this protein displays the fourteen

residues composing the conserved active site of IDIs as

well as a Nudix (Nucleoside Diphosphate linked to X)

motif of the Nudix hydrolase family, classically found in

phosphohydrolases and other IDIs (Fig. 1). CrIDI1 also

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ CrIDI 1 MSSTLTTSYFQTILKRISVCSLPSSTSSSNSLYPILLKHKFSSFPSVSFSSSASSPC-------SNSTTLAAFSSIAAPS AtIDI1 1 --------------------------MSTASLFSFPSFHLRSLLPSLSSSSSSSSSRFAPPRLSPIRSPAPRTQLSVRAF AtIDI2 1 --------------------------MSASSLFNLPLIRLRSLALSSSFSSFRFAHR-------PLSSISPRKLPNFRAF EcIDI 1 -------------------------------------------------------------------------------- ScIDI 1 ------------------------MTADNNSMPHGAVSSYAKLVQNQTPEDILEEFP-------EIIPLQQRPNTRSSET

~~~ # # # # # #CrIDI 74 FSSMGAAVTDSAMDAVQRRLMFEDECILVDENDHVVGHDTKYNCHLMEKIESENLLHRAFSVFLFNSKFELLLQQRSATKAtIDI1 55 SAVTMTDSNDAGMDAVQRRLMFEDECILVDENDRVVGHDTKYNCHLMEKIEAENLLHRAFSVFLFNSKYELLLQQRSKTKAtIDI2 48 SGTAMTDTKDAGMDAVQRRLMFEDECILVDETDRVVGHDSKYNCHLMENIEAKNLLHRAFSVFLFNSKYELLLQQRSNTKEcIDI 1 --------------------MQTEHVILLNAQGVPTGTLEKYAAHT-----ADTRLHLAFSSWLFNAKGQLLVTRRALSKScIDI 50 SNDESGETCFSGHDEEQIKLMNEN-CIVLDWDDNAIGAGTKKVCHLMENIE-KGLLHRAFSVFIFNEQGELLLQQRATEK

# # # # # # # CrIDI 154 VTFPLVWTNTCCSHPLYRESELIE-----ENVLGVRNAAQRKLLDELGIVA-EDVPVDEFMPLGRMLYKAPSDGIWGEHEAtIDI1 135 VTFPLVWTNTCCSHPLYRESELIE-----ENVLGVRNAAQRKLFDELGIVA-EDVPVDEFTPLGRMLYKAPSDGKWGEHEAtIDI2 128 VTFPLVWTNTCCSHPLYRESELIQ-----DNALGVRNAAQRKLLDELGIVA-EDVPVDEFTPLGRMLYKAPSDGKWGEHEEcIDI 56 KAWPGVWTNSVCGHPQLGESNEDAVIRRC------RYELGVEITPPESIYP-DFRYRATDPSGIVENEVCPVFAARTTSA ScIDI 128 ITFPDLWTNTCCSHPLCIDDELGLKGKLDDKIKGAITAAVRKLDHELGIPEDETKTRGKFHFLNRIHYMAPSNEPWGEHE

# CrIDI 228 LDYLLFIVRDVKVN----PNPDEVADVKYMTRDDLKELLRKADAGEEGLKLSPWFRLVVDNFLFKWWDHVEKGTLLEAADAtIDI1 209 VDYLLFIVRDVKLQ----PNPDEVAEIKYVSREELKELVKKADAGDEAVKLSPWFRLVVDNFLMKWWDHVEKGTITEAADAtIDI2 202 LDYLLFIVRDVKVQ----PNPDEVAEIKYVSREELKELVKKADAGEEGLKLSPWFRLVVDNFLMKWWDHVEKGTLVEAIDEcIDI 129 LQINDDEVMDYQWC----DLADVLHGIDATPWAFSPWMVMQATNREARKRLSAFTQLK---------------------- ScIDI 208 IDYILFYKINAKENLTVNPNVNEVRDFKWVSPNDL-----KTMFADPSYKFTPWFKIICENYLFNWWEQLDDLSEVENDR

CrIDI 304 MKTIHKLI AtIDI1 285 MKTIHKL- AtIDI2 278 MKTIHKL- EcIDI -------- ScIDI 283 QIHRML--

* * *

mTP cTP

PTS1

Fig. 1 Multiple sequence alignment of type I IDIs. Amino acid

comparison of IDI from C. roseus (CrIDI1; GenPept ABW98669) with

A. thaliana long isoform 1 (AtIDI1, AGI accession number

At5g16440), A. thaliana long isoform 2 (AtIDI2, AGI accession

number At3g02780), E. coli (EcIDI, GenPept NP_417365) and

Saccharomyces cerevisiae (ScIDI; GenPept NP_015208). Identical

and similar residues are highlighted by dark and gray shading,

respectively. Asterisks indicate methionine residues conserved in IDI

from plant species and the horizontal bar represents the conserved

region of the N-terminal extension. #Designates the conserved catalytic

residues of IDI according to the NCBI conserved domain search.

Arrowheads delimitate the Nudix hydrolase conserved sequence

(PFAM00293). Putative type 1 peroxisome targeting sequence

(PTS1), mitochondrial (mTP) and chloroplast (cTP) target peptides

are shown

448 Plant Mol Biol (2012) 79:443–459

123

contains an N-terminal extension of 93 residues as com-

pared to E. coli IDI, which is moreover 18- and 25-residues

longer than the AtIDI1 and AtIDI2 extension, respectively

(Fig. 1). Such extension ends with a conserved region

containing three methionine residues conserved in plant

IDI that could be used for alternative initiation of transla-

tion (Cunningham and Gantt 2000; Phillips et al. 2008).

Since plant IDIs display a complex localization within

cells, the subcellular targeting features of CrIDI1 were

investigated with protein subcellular localization prediction

servers. As detailed in Table 1, most of the algorithms

predicted a plastidial localization based on the identifica-

tion of a putative plastid targeting peptide (TP) of 77 res-

idues (ChloroP prediction) located in the N-terminal

extension of CrIDI1. Interestingly, both PSORT and Mi-

toprot predicted a putative mitochondrial localization with

a high confidence for Mitoprot based on the identification

of a mitochondrial TP corresponding to the first 18-residues

of CrIDI1 (Fig. 1). Finally, the target signal predictor of the

peroxisome database also detected a potential type I per-

oxisome targeting sequence (PTS1) at the C-terminal end

of the protein (HKL, residues 308–310) as previously

observed in both Arabidopsis IDIs (Sapir-Mir et al. 2008)

albeit an additional isoleucine residue was present down-

stream of the PTS1 in CrIDI1 (Fig. 1).

Functional characterization of CrIDI1

To confirm that the cloned cDNA encodes a functional

IDI, the activity of the recombinant protein has been

evaluated by the measurement of the catalytic conversion

of IPP into DMAPP (Fig. 2). Since the N-terminal exten-

sions of plant IDIs are not required for enzyme activity

(Campbell et al. 1998), the recombinant protein was

expressed as a truncated protein (Fig. 2a), devoid of the

first 76 residues, after cloning the deleted coding sequence

in the pQE-30 plasmid (IDI–pQE). The amount of isom-

erized IPP was measured in protein extracts from IDI–pQE

transformed BL21 E. coli cells in comparison with protein

extracts from untransformed BL21 cells and boiled protein

extracts from IDI to pQE transformed BL21 cells (control

conditions). Extracts were obtained from cultures adjusted

to nearly identical cell densities. A significant IPP isom-

erization ([1.50 nmol h-1) was measured in the induced

protein extracts from IDI–pQE transformed cells with no

significant background in control conditions (Fig. 2b). IPP

isomerization (0.44 nmol h-1) was also obtained in the

uninduced protein extract from these cells, resulting

probably from uncontrolled expression from the plasmid

promoter as observed in Fig. 2a. On the basis of these

results and the high identity of CrIDI1 to previously

characterized plant IDIs, it could be assumed that CrIDI1

encodes a functional IDI.

Spatial distribution of CrIDI1-encoding transcripts

The distribution of the CrIDI1 transcripts was first ana-

lyzed by real-time PCR (qPCR) within distinct organs of

fully developed C. roseus plants. CrIDI1 mRNAs were

detectable in all the tested organs with two patterns of

expression (Fig. 3a). In roots, young leaves and flowers of

C. roseus, CrIDI1 transcript levels are circa twofold higher

than those measured in internodes, mature leaves and

flower buds in agreement with transcriptomic data (sup-

plemental Table 4). This represents an intermediate situa-

tion as compared to A. thaliana where the highest amount

of AtIDI1 and AtIDI2 gene products are mainly restricted to

flowers and roots, respectively (Okada et al. 2008; Phillips

et al. 2008).

The tissue-specific distribution of CrIDI1 transcripts was

subsequently investigated by RNA in situ hybridization

performed on serial sections of young developing C. roseus

leaves since these organs constitute the main site of CrIDI1

expression as revealed by qPCR analysis. As depicted on

Fig. 3b, labeling with the antisense probe allowed detecting

CrIDI1 transcripts in the adaxial part of the leaf vascular

Fig. 2 Functional characterization of CrIDI1. a SDS–PAGE of

protein crude extracts from E. coli cells harboring the pQE-30 empty

vector or the pQE-30-IDI vector after 0, 2 or 4 h following the

induction of protein expression. b The functional activity of

recombinant CrIDI1 was evaluated by the measurement of the IPP

to DMAPP catalytic conversion as described by Phillips et al. (2008)

Plant Mol Biol (2012) 79:443–459 449

123

region, corresponding to the internal phloem associated

parenchyma (IPAP) cells as confirmed by the colocalization

of CrG10H mRNA in this tissue (Fig. 3d; Burlat et al. 2004).

Transcripts were also detected in both the adaxial and

abaxial epidermis of leaves albeit the mRNA level was

dramatically lower than those measured in IPAP cells

(Fig. 3b). No significant background was observed using the

sense probes (Fig. 3c, e). On the basis of these results, it

could be hypothesized that the distribution of CrIDI1 tran-

scripts is restricted to the major sites of isoprenoid biosyn-

thesis within C. roseus leaves.

CrIDI1 is expressed as short and long transcripts

In A. thaliana, AtIDI1 and AtIDI2 are both expressed as

long and short isoforms that differ by the presence of either

a plastid (AtIDI1) or a mitochondrion (AtIDI2) TP at the

N-terminal end of the long isoform. This mechanism

directly influences the subcellular localization of the cor-

responding proteins since AtIDI1 long isoform is mostly

targeted to plastids and the AtIDI2 long isoform is mostly

targeted to mitochondria while both short isoforms are

sorted to peroxisomes via a type 1 PTS1 located at the

C-terminal end of the proteins (Okada et al. 2008; Phillips

et al. 2008; Sapir-Mir et al. 2008). To determine whether

this kind of alternative transcription also occurs for CrIDI1,

50 RACE was performed on RNA extracted from young C.

roseus leaves. Separation of amplification products by

electrophoresis allowed identifying cDNAs of two different

sizes (Fig. 4a). Sequence analysis revealed that the longer

50 RACE products originate from full-length transcripts

including the initiation codon of the putative plastid/

mitochondrion TP (Fig. 4b; Table 1). The shorter 50 RACE

products lack 145 bp at the 50 end and begin 37 bp

downstream of this initiation codon but contain three

internal Met codons that could initiate translation of a

shorter CrIDI1 isoform lacking the putative plastid/mito-

chondrion TP. These results demonstrate that similar pro-

cesses of alternative transcription exist for IDI genes in A.

thaliana and C. roseus yielding to the formation of long

and short isoforms of CrIDI1, bearing or not the N-terminal

transit TP.

CrIDI1 long isoform is dually targeted to plastids

and mitochondria

The presence of both long and short isoforms of CrIDI1

prompted us to study the localization of both types of

protein within C. roseus cells. The subcellular localization

of the long isoform was first investigated by GFP/YFP

tagging. The full length coding sequence of CrIDI1 was

fused to the N-terminal end of GFP and the corresponding

construct was transiently expressed in C. roseus cells

(Fig. 5a). The IDI–GFP fusion protein displayed a dual

pattern of fluorescence signal located in both plastids

including stromules, and mitochondria as confirmed by co-

localisation with specific markers of each compartment

(Fig. 5b–m). This dual targeting appears to occur at an

apparent similar efficiency as suggested by the high fluo-

rescence observed in plastids or mitochondria. Interest-

ingly, this differs from the situation described in A.

Fig. 3 Analysis of CrIDI1 transcript distribution in C. roseus.

a Analysis of CrIDI1 transcript levels as determined by real-time

RT–PCR analyses from different C. roseus organs. Total RNA was

extracted from organs and subjected to reverse transcription. Tran-

script levels for CrIDI1 and CrRPS9 were determined by real-time

PCR using gene-specific primers. CrIDI1 expression level was

normalized using CrRPS9. The transcript level of young leaves was

set to ‘‘1’’. b–e Cellular distribution of CrIDI1 transcripts in young

leaves analyzed by in situ RNA hybridization. Paraffin-embedded

serial sections of young leaves were hybridized with digoxigenin-

labelled transcripts, which were subsequently localized with anti-

digoxigenin-alkaline phosphatase conjugates followed by nitro blue

tetrazolium chloride (NBT)/5-bromo 4-chloro-3-indolyl phosphate

(BCIP) color development. Hybridization of the geraniol 10-hydrox-

ylase (CrG10H) transcripts was used as a positive control. The anti-

sense (AS) probes used for RNA labeling (b, d) and control

hybridization with sense (S) RNA probes (c, e) are mentioned on

the figure. Arrows and arrowheads show labeling within the internal

parenchyma associated phloem (IPAP) and both the adaxial and

abaxial epidermis, respectively. Bars 100 lm

450 Plant Mol Biol (2012) 79:443–459

123

thaliana in which a preferential localization to either

plastids or mitochondria has been described for AtIDI1 and

AtIDI2, respectively (Phillips et al. 2008) and confirmed in

C. roseus cells (Supplemental Fig. 1). To gain insight into

this dual localization, fusion/deletion experiments of the

predicted TP of CrIDI1 were also conducted. First, pre-

dicted TP of various lengths (from the first residue to

residues surrounding one of the three internal methionine

residues) were fused to YFP and transiently expressed in

C. roseus cells (Fig. 6a). Irrespective to the length of the

TP, a dual localization in plastids and mitochondria was

observed as exemplified for the shorter (pepATG1) and

longer (pepATG4) peptide, respectively (Fig. 6b–i). The

same dual localization was also observed for the two

additional constructs pepATG2 and pepATG3 (data not

shown). Similarly, gradually TP deleted versions of CrIDI1

were expressed as YFP-fused proteins initiated from each

of the three internal methionine residues (Fig. 6j). For the

three constructs, a similar nucleocytosolic localization was

observed as illustrated by the perfect overlapping of fluo-

rescence with the mcherry nucleocytosolic marker as

exemplified for the deletion of the first 76-residues (IDI-

del1, Fig. 6k–n). Taken together, these results show that

the first 76 residues of CrIDI1 encompass the minimal TP

of this enzyme and are solely responsible for the dual

mitochondria/plastids targeting. In addition, since the

extreme N-terminus of CrIDI1 has been predicted to allow

a mitochondrial targeting (Table 1), the first 18-residues

of the CrIDI1 TP were also fused to GFP (Fig. 7a). In C.

roseus transiently transformed cells, the corresponding

fusion protein displayed a specific localization in mito-

chondria as revealed by the merge of fluorescence with the

mito-mcherry marker (Fig. 7b–e). However, the remaining

part of the TP (including residues 19–76, Fig. 7f) was

unable to address the fusion protein to plastids or mito-

chondria (Fig. 7g–j). This suggests that the first 18 residues

of CrIDI1 TP allow the mitochondrial targeting but are also

required for the plastidial targeting in association with the

remaining part of the TP.

CrIDI1 short isoform is targeted to peroxisomes

Since short isoforms of AtIDI1 and AtIDI2 are addressed to

peroxisomes (Sapir-Mir et al. 2008), the potential targeting

of the CrIDI1 short isoform to this subcellular compart-

ment has been also investigated. For this purpose, YFP

internal fusions were constructed to maintain the accessi-

bility of the putative PTS1 (HKL) located at the C-terminal

extremity of CrIDI (Fig. 8). As previously mentioned by

Sapir-Mir et al. (2008), the sole addition of the predicted

PTS1 or the entire CrIDI1 short isoform to the C-terminal

end of YFP failed to target the fusion protein to peroxi-

somes. A similar result was also obtained for the YFP

internal fusion proteins engaging the last 11 residues of

CrIDI1 at the C-terminus of YFP and the remaining part of

the short isoform at the N-terminus of YFP (data not

shown). As a consequence, internal fusions within long and

short versions of CrIDI1 were performed with a modified

version of YFP flanked by 6 or 7 residues-long spacers

(Fig. 8a, f) as previously performed by Sapir-Mir et al.

(2008). In C. roseus transiently transformed cells, the

fusion protein engaging the CrIDI1 long isoform (tp-IDI–

YFP–PTS1, Fig. 8a) displayed a dual fluorescence signal

located in plastids and mitochondria as revealed by the

Fig. 4 The CrIDI1 gene displays two transcripts of different length.

a 50 RACE analysis was conducted on RNA extracted from young

C. roseus leaves allowing the amplification of long (a) and short

(b) products. b Nucleotidic and deduced amino-acid sequences of the

two RACE products. The initiation codon of the full length coding

sequence and the alternative internal initiation codon of short

transcripts are indicated by solid lines

Plant Mol Biol (2012) 79:443–459 451

123

overlapping with the fluorescence signal emitted by the

‘‘mito’’-CFP marker and by the comparison with plastid

morphology (Fig. 8b–e). By contrast, the localization

studies of the fusion protein engaging the CrIDI1 short

isoform (IDI–YFP–PTS1, Fig. 8f) resulted in a simple

punctuated fluorescence, which entirely overlaps the fluo-

rescence signal of the peroxisome-CFP marker (Fig. 8g–j).

This demonstrates that the CrIDI1 short isoform is targeted

to peroxisomes despite the presence of an additional iso-

leucine residue located downstream of the PTS1. The

absence of effect of this residue after a PTS1 has also been

confirmed by the peroxisomal targeting of both the YFP–

SKLI and YFP–SKL fusion proteins (Supplemental Fig. 2).

Taken together, these results show that the gene products of

CrIDI1 displayed a unique triple localization within cells

including a dual targeting to plastids and mitochondria for

the long isoforms and a peroxisome targeting for the short

isoforms.

Fig. 5 CrIDI1–GFP is targeted to the plastids and mitochondria. C.roseus cells were transiently co-transformed with plasmids expressing

the CrIDI1–GFP fusion proteins (a, b, f, j) and the ‘‘plastid’’-mcherry

(c, g) or ‘‘mitochondria’’-mcherry (k) markers. Co-localization of the

two signals appeared in yellow while merging the two individual

(green/red) color images (d, h, l). Cell morphology is observed with

differential interference contrast (DIC, e, i, m). Bars 10 lm

Fig. 6 N-terminus target peptides of different length from the CrIDI1

long isoform allow the dual targeting to plastids and mitochondria. C.roseus cells were transiently co-transformed with plasmids expressing

the truncated version of the transit peptide of CrIDI1 fused to GFP

including the 76 (pepATG1), 81 (pepATG2), 91 (pepATG3) or 97

(pepATG4) first residues (a, b, f) or transit peptide deleted version of

CrIDI1 (initiated at residue 77 for IDI-del1, 86 for IDI-del2 or 94 for IDI-

del3) fused to GFP (j, k) and the ‘‘plastid’’-mcherry (c), ‘‘mitochondria’’-

mcherry (g) or ‘‘nucleocytosolic’’-mcherry (l) markers. Co-localization

of the two signals appeared in yellow while merging the two individual

(green/red) color images (d, h, m). Cell morphology is observed with

differential interference contrast (DIC, e, i, n). Bars 10 lm

c

452 Plant Mol Biol (2012) 79:443–459

123

Plant Mol Biol (2012) 79:443–459 453

123

CrIDI1 is capable of self-interactions in plastids,

mitochondria and peroxisomes

Since systematic analysis of protein complexes strongly

suggested that type I IDIs are able of self-interactions in

yeast (Gavin et al. 2002), we performed BiFC assays in C.

roseus cells. The full coding sequence of CrIDI1 was

cloned in frame with the 50 end of the coding sequence of

the two split-YFP fragments (YFPN and YFPC) to generate

the IDI–YFPN and IDI–YFPC fusion proteins bearing an

accessible N-terminal TP. As a control, the two split-YFP

fragments were also fused to the sole TP of CrIDI1. This

allowed to target the two split-YFP fragments to plastids

and mitochondria as pseudo-mature non fused protein

(tpIDI–YFPN; tpIDI–YFPC) and to evaluate the spontane-

ous association of these proteins and the resulting non

specific fluorescence in cell organelles. As shown in

Fig. 9a–d, the transient expression of the two control pro-

teins in C. roseus cells did not result in the reformation of a

BiFC complex since no fluorescence was detected, dem-

onstrating that the two split-YFP fragments were unable

to interact in the absence of a protein partner under

our experimental conditions. By contrast, the transient

co-expression of IDI–YFPN and IDI–YFPC gave a fluo-

rescent signal in both plastids and mitochondria (Fig. 9e–k)

as previously observed for IDI–GFP (Fig. 5a–m). This

suggests that CrIDI1 is able of self-interaction in these two

types of organelles. In addition, since CrIDI1 is also tar-

geted to peroxisomes, the ability of the protein to self-

interact within this compartment was studied by the creation

of internal fusions with the split-YFP fragments on the basis

of those constructed for the analysis of the subcellular

localization of the CrIDI1 short isoform (Figs. 8f, 9l). Using

these constructs, we also observed the formation of a BiFC

complex within peroxisomes as revealed by the perfect

overlapping of the YFP signals and of the CFP peroxisome

marker (Fig. 9m–o), suggesting that CrIDI1 is also able of

self interaction within this subcellular compartment. Self-

interaction capacities were further confirmed using the

purified recombinant enzyme (Fig. 9p) following migration

on native PAGE (Fig. 9q). In this condition, the recombi-

nant enzyme displayed distinct levels of organization

including complexes of low and high molecular weights as

revealed by the presence of slow and fast migrating bands

on the gel. Following the incubation of the enzyme with

DTT, the slow migrating bands tempted to disappear to the

Fig. 7 The first 18-residues of the CrIDI1 long isoform transit

peptide allow the unique mitochondrial targeting. C. roseus cells were

transiently co-transformed with plasmids expressing the first 18-res-

idues of the CrIDI1 transit peptide fused to GFP (a pepIDI-mito) or

the remaining part of the transit peptide (residues 19–76) fused to

GFP (f, pepIDI-D) with the ‘‘mitochondria’’-mcherry (c) and ‘‘nucle-

ocytosolic’’-mcherry (h) markers, respectively. Co-localization of the

two signals appeared in yellow while merging the two individual

(green/red) color images (d, i). Cell morphology is observed with

differential interference contrast (DIC, e, j). Bars 10 lm

454 Plant Mol Biol (2012) 79:443–459

123

benefit of the fastest migrating band. This confirms that

CrIDI1 is able of self-interactions and suggests that the

formation of the higher molecular weight complexes also

requires disulfide bonds. However, DTT concentration had

virtually no effect on CrIDI1 activity in our in vitro assays,

suggesting that protein–protein interactions are not strictly

required for CrIDI1 activity.

Discussion

Isomerization of IPP into DMAPP is a crucial step for the

synthesis of isoprenoids of various lengths and nature. In

plant cells, this reaction is catalyzed by type 1 IDIs that

display a complex subcellular localization well character-

ized in A. thaliana (Okada et al. 2008; Phillips et al. 2008;

Sapir-Mir et al. 2008). To pursue this characterization in

plants producing high amount of specialized isoprenoids,

we have cloned and functionally validated a cDNA

encoding a type 1 IDI from C. roseus (CrIDI1), a plant

widely studied due to its capacity to synthesize valuable

MIA (Facchini and De Luca 2008). Albeit generally

encoded by at least two distinct genes (Campbell et al.

1998; Cunningham and Gantt 2000; Nakamura et al. 2001),

a single sequence was amplified during this work. Inter-

estingly, a search in C. roseus EST database preferentially

identify the CrIDI1 sequence (Supplemental Table 3)

suggesting that the other IDI putative gene(s) and espe-

cially CrIDI2 are expressed at very low level and/or under

specific conditions as confirmed by the transcriptomic data

obtained from the MPGR (supplemental Table 4). The

analysis of the mRNA organ distribution of the CrIDI1 by

qPCR also revealed that the corresponding transcripts

could be detected in all the tested organs with the highest

amount measured in roots, young leaves and flowers

(Fig. 3a). The organ-specific expression profile of CrIDI1

somehow corresponds to the addition of the expression

profiles of both Arabidopsis IDIs since the highest

expression of AtIDI1 and AtIDI2 has been detected in

flowers and roots, respectively (Phillips et al. 2008). This

Fig. 8 An accessible C-terminal peroxisomal targeting sequence-1

allows the peroxisomal targeting of the short CrIDI1 isoform. C.roseus cells were transiently co-transformed with plasmids expressing

internal YFP fusion of the long (a, b) and short (f, g) isoforms of

CrIDI1 with the ‘‘mitochondria’’-CFP (c) and ‘‘peroxisome’’-CFP

(h) markers, respectively. Glycine and alanine containing spacers

were added at both extremities of YFP as mentioned in italic on the

schematic representations of the constructs. Co-localization of the two

signals appeared in yellow while merging the two individual (green/

red) color images (d, i). Cell morphology is observed with differential

interference contrast (DIC, e, j). Bars 10 lm

Plant Mol Biol (2012) 79:443–459 455

123

correlates with the fact that a single coding sequence of IDI

seems to be highly expressed in C. roseus. At the cellular

level, we have established using RNA in situ hybridization

that CrIDI1 is mainly expressed in IPAP cells of young

developing leaves as previously described for MEP path-

way genes and G10H, the enzyme catalyzing the first

committed step in the formation of the monoterpenoid

precursor of MIA (Fig. 3b–e) (Burlat et al. 2004; Cour-

davault et al. 2005). This reinforces the identification of

IPAP as a prominent tissue in isoprenoid biosynthesis in

C. roseus. In addition, low amounts of transcripts were also

detected in both the adaxial and abaxial epidermis of leaves

in agreement with the identification of CrIDI1 EST within

the C. roseus leaf epidermis database (Murata et al. 2008)

and with the immunolocalization at low levels of a MEP

pathway enzyme within this tissue (Oudin et al. 2007). C.

roseus leaf epidermis has also been described as the main

site of the MVA pathway activity on the basis of the

selective gene expression profiles in this tissue (Murata

et al. 2008). Previous works in A. thaliana using mutant

analysis, radiolabelled precursor feeding and subcellular

localizations, have shown that the two IDI genes have

partially redundant functions, rendering difficult the

assignation of each isoform to either MVA or MEP path-

ways (Okada et al. 2008; Phillips et al. 2008). However, on

the basis of the cellular distribution of the CrIDI1 tran-

scripts and according to the relative abundance of CrIDI1

mRNA as compared to CrIDI2 mRNA (supplemental

Table 4), we could hypothesize that CrIDI1 isomerizes IPP

from the MEP and/or MVA pathways in a tissue-dependant

manner. Assuming that the MVA pathway is mainly

located in the epidermis of C. roseus leaves (Murata et al.

2008) and that the epidermis also houses low level of MEP

pathway enzymes (Oudin et al. 2007; Murata et al. 2008), it

could be hypothesized that CrIDI1 isomerizes IPP from

both pathways in this tissue allowing for instance the

synthesis of epidermal triterpenes from the MVA pathway.

By contrast, in IPAP cells that constitute the main site of

expression of MEP pathway genes and CrG10H, we could

reasonably think that the strong CrIDI1 expression should

be related to the isomerization of MEP-pathway derived

IPP needed for the synthesis of the monoterpenoid pre-

cursor of MIA in this tissue.

Fig. 9 CrIDI1 is able of self-interaction within plastids, mitochondria

and peroxisomes of C. roseus cells. Analysis of the CrIDI1 self

interactions was conducted by BiFC following co-transformation of

C. roseus cells using a combination of plasmids expressing the

negative control tpIDI-YFPN and tpIDI-YFPC (a–d), the CrIDI1 long

isoform fused to the split-YFP fragments IDI-YFPN and IDI-YFPC

(e–k) and internal fusions of the split-YFP fragments within the

CrIDI1 short isoform IDI-YFPN-PTS1 and IDI-YFPC-PTS1 (l–o). C.roseus cells were transiently co-transformed with each pair of

plasmids and either ‘‘plastid’’-CFP (g), ‘‘mitochondria’’-CFP (j) and

‘‘peroxisome’’-CFP (n) marker. The morphology was observed by

differential interference contrast (DIC) microscopy and combined

with the marker fluorescence signals (c, g, j, n). Superimpositions of

these combined images and of the YFP fluorescence signal emitted by

the BiFC complexes are provided on the merged images (h, k, o).

Bars 5 lm. p Purification profile of the recombinant CrIDI1 protein

analyzed by SDS–PAGE. M protein standards. q Native PAGE of the

purified enzyme incubated 10 min at room temperature in the

presence of water or 10 mM DTT. Arrowheads and stars indicate

fast and slow migrating bands on the gel, respectively

c

456 Plant Mol Biol (2012) 79:443–459

123

At the subcellular level, we have established using YFP/

GFP imaging that CrIDI1 exhibits a complex triple sub-

cellular localization based on the formation of transcripts

of various lengths as revealed by 50 RACE analysis

(Fig. 4). This phenomenon, previously described in

A. thaliana (Phillips et al. 2008), leads to the formation of

short and long isoforms of CrIDI1 that differ by the

absence/presence of the plastid (AtIDI1) or mitochondrion

(AtIDI2) TP at their N-terminal end. Thus, we showed that

the CrIDI1 short isoform lacking the N-terminal TP is

targeted to peroxisomes in agreement both with the pres-

ence of a PST1 at the C-terminus of the protein (Fig. 8f–j)

and with the results obtained for AtIDI1 and AtIDI2 (Sapir-

Mir et al. 2008). As mentioned by these authors, we noticed

that the peroxisomal targeting of IDI seems to be mediated

by particular mechanisms since both the N-terminal part

and the C-terminal end of pseudo-mature short protein

have to be accessible for proper peroxisome targeting

(Fig. 8f). The C-terminal end of CrIDI1 and its orthologs

from A. thaliana (AtIDI1 and AtIDI2) display the same

PTS1 (HKL) but differ by the presence of an additional

isoleucine residue located just after the PTS1 in CrIDI1

(Fig. 1). However, we showed that this supplemental resi-

due did not alter the efficiency of the CrIDI1 PTS1 as also

confirmed for the YFP-SKLI fusion protein (Supplemental

Fig. 2). At the physiological level, the peroxisomal locali-

zation of CrIDI1 is in direct correlation with the presence of

other isoprenoid biosynthetic enzymes acting upstream or

downstream of CrIDI1 within this compartment. These

include the 5-phosphomevalonate kinase (PMK) and the

mevalonate 5-diphosphate decarboxylase (MVD) catalyz-

ing the two last steps of the MVA pathway as well as a

farnesyl diphosphate synthase (Simkin et al. 2011; Thabet

et al. 2011; Guirimand et al. 2012). Therefore, the peroxi-

somal localization clearly suggests the involvement of short

CrIDI1 in the biosynthesis of isoprenoids derivated from the

MVA pathway and reinforces the description of peroxi-

somes as major organelles in isoprenoid biosynthesis.

The CrIDI1 long isoform exhibits a N-terminal TP that

targets the pseudo-mature protein to both mitochondria and

plastids including stromules at an apparent similar effi-

ciency (Fig. 5a–m). This constitutes the main difference

with A. thaliana in which the long isoforms are preferen-

tially addressed to either plastid (AtIDI1) or mitochondria

(AtIDI2), respectively (Sapir-Mir et al. 2008). These spe-

cific localizations have been confirmed under our experi-

mental conditions and in our plant system (Supplemental

Fig. 1) confirming that the dual localization of the CrIDI1

long isoform is dependant of the protein, per se and not

to the cell type. Fusion/deletion experiments of CrIDI1

revealed that the first 76 residues are sufficient to target

the protein to both mitochondria and plastids (Fig. 6a–n)

in agreement with bioinformatic predictions (Table 1).

Interestingly, we also noted that the first 18 residues of the

TP, corresponding to a specific part of the CrIDI1 TP as

compared to those of Arabidopsis orthologs (Fig. 1), are

solely responsible of the mitochondrial targeting (Fig. 7a–

e). By contrast, the remaining part of the CrIDI1 (residues

19–76) is unable to drive a fusion protein to plastids

(Fig. 7f–j), suggesting that the whole TP is required to

plastid targeting. Dual targeting to mitochondria and

plastid is a common feature, observed for an increasing

number of proteins (Mitschke et al. 2009). There are two

basic ways in which a single gene can provide a product

targeted to both organelles. It includes the ‘‘twin’’ targeting

sequences represented by a mitochondrial and a plastidial

targeting sequence positioned in tandem at the N-terminus

of the protein, and the ambiguous targeting sequence that is

recognized as an import signal by both organelles (Peeters

and Small 2001; Pujol et al. 2007; Mitschke et al. 2009).

The N-terminal TP of CrIDI1 constitutes an intermediate

situation since the first 18-residues are sufficient to mediate

import in mitochondria while the first 76-residues

(including the first 18-residues) are required to mediate

additional import into plastids. It could therefore allow a

dual targeting albeit a preferential targeting to either

plastids or mitochondria could also be hypothesized

depending on the TP phosphorylation status since it has

been described to increase the efficiency of plastid import

by several fold (Waegemann and Soll 1996; Martin et al.

2006). From a physiological point of view, the presence of

CrIDI1 in plastids could be linked to the synthesis of MEP

derived isoprenoid and its mitochondrial localization to the

production of other specific isoprenoids such as ubiqui-

none (Lutke-Brinkhaus et al. 1984). In addition, we can

hypothesize that the dual targeting of CrIDI1 to plastids

and mitochondria could be required to maintain IDI

activity and the associated isoprenoid biosynthesis in these

two organelles since the sole CrIDI1 gene seems to be

expressed at detectable level within C. roseus organs. Such

interpretation could also be extrapolated to the peroxisomal

localization of CrIDI1. Nevertheless, we cannot rule out

the existence of an organelle specific targeting for the

CrIDI1 long isoform that could depend on the cell type

housing the enzyme. For instance, in IPAP cells where the

MEP pathway is most active, a preferential targeting to

plastids could occur to allow the production of high amount

of the isoprenoid precursor of MIA. Finally, our combined

approaches of BiFC assays and migration of recombinant

protein in native PAGE allowed us to establish that CrIDI1

is capable of self-interaction within the three subcellular

compartment housing this enzyme, including plastids,

mitochondria and peroxisomes (Fig. 9a–q). Our results do

not fit with the hypothesis that IDIs mainly operate as

monomers based upon the elucidation of the crystal

structure of the E. coli type 1 IDIs and from the purification

Plant Mol Biol (2012) 79:443–459 457

123

of IDIs from Cinchona robusta cells (Ramos-Valdivia et al.

1997; Durbecq et al. 2001). However, our results corrob-

orate the analysis of the yeast proteome complexes dem-

onstrating the propensity of type 1 IDIs to self interact

in multi-protein complexes (Gavin et al. 2002). Albeit

apparently not required for the catalytic activity, the

CrIDI1 self-interactions could be required for the control of

the metabolic flux. All together, our results demonstrated

that two CrIDI1 isoforms encoded by a single gene display

a unique triple localization in plastids, mitochondria and

peroxisomes under self-interacting complexes suggesting

that distinct mechanisms of compartmentalization of the

isoprenoid biosynthetic pathways could occur within plants

synthesizing high amount of specialized isoprenoids (e.g.

C. roseus) and plants mainly accumulating’’housekeeping’’

levels of non-specialized isoprenoids and low amount of

specialized isoprenoids (e.g. A. thaliana).

Acknowledgments This work was financially supported by the

‘‘Ministere de l’Enseignement Superieur et de la Recherche’’ (MESR)

and by a grant from the University of Tours. Gregory Guirimand

and Anthony Guihur were financed by MESR fellowships. We thank

Pr. Jorg Kudla (University of Munster, Germany) for providing us the

BiFC plasmids. We also thank M. A. Marquet, M. F. Aury, E. Danos

and E. Marais for help in maintaining cell cultures.

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