Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway.

Abstract

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the first committed step of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis. In Arabidopsis, DXR is encoded by a single-copy gene. We have cloned a full-length cDNA corresponding to this gene. A comparative analysis of all plant DXR sequences known to date predicted an N-terminal transit peptide for plastids, with a conserved cleavage site, and a conserved proline-rich region at the N terminus of the mature protein, which is not present in the prokaryotic DXR homologs. We demonstrate that Arabidopsis DXR is targeted to plastids and localizes into chloroplasts of leaf cells. The presence of the proline-rich region in the mature Arabidopsis DXR was confirmed by detection with a specific antibody. A proof of the enzymatic function of this protein was obtained by complementation of an Escherichia coli mutant defective in DXR activity. The expression pattern of beta-glucuronidase, driven by the DXR promoter in Arabidopsis transgenic plants, together with the tissue distribution of DXR transcript and protein, revealed developmental and environmental regulation of the DXR gene. The expression pattern of the DXR gene parallels that of the Arabidopsis 1-deoxy-D-xylulose 5-phosphate synthase gene, but the former is slightly more restricted. These genes are expressed in most organs of the plant including roots, with higher levels in seedlings and inflorescences. The block of the 2-C-methyl-D-erythritol 4-phosphate pathway in Arabidopsis seedlings with fosmidomycin led to a rapid accumulation of DXR protein, whereas the 1-deoxy-D-xylulose 5-phosphate synthase protein level was not altered. Our results are consistent with the participation of the Arabidopsis DXR gene in the control of the 2-C-methyl-D-erythritol 4-phosphate pathway.

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Expression and Molecular Analysis of the Arabidopsis
DXR Gene Encoding 1-Deoxy-D-Xylulose 5-Phosphate
Reductoisomerase, the First Committed Enzyme of the
2-C-Methyl-D-Erythritol 4-Phosphate Pathway1
Lorenzo Carretero-Paulet, Iva ´n Ahumada2, Nuria Cunillera, Manuel Rodrı ´guez-Concepcio ´n, Albert Ferrer,
Albert Boronat, and Narciso Campos*
Departament de Bioquı ´mica i Biologia Molecular, Facultat de Quı ´mica, Universitat de Barcelona, C/Martı ´ i
Franque `s 1, 08028 Barcelona, Spain (L.C.-P., I.A., M.R.-C., A.B., Na.C.); and Departament de Bioquı ´mica i
Biologia Molecular, Facultat de Farma `cia, Universitat de Barcelona, Avinguda Diagonal 643, 08028 Barcelona,
Spain (Nu.C., A.F.)
1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the first committed step of the 2-C-methyl-d-erythritol
4-phosphate pathway for isoprenoid biosynthesis. In Arabidopsis, DXR is encoded by a single-copy gene. We have cloned
a full-length cDNA corresponding to this gene. A comparative analysis of all plant DXR sequences known to date predicted
an N-terminal transit peptide for plastids, with a conserved cleavage site, and a conserved proline-rich region at the N
terminus of the mature protein, which is not present in the prokaryotic DXR homologs. We demonstrate that Arabidopsis
DXR is targeted to plastids and localizes into chloroplasts of leaf cells. The presence of the proline-rich region in the mature
Arabidopsis DXR was confirmed by detection with a specific antibody. A proof of the enzymatic function of this protein was
obtained by complementation of an Escherichia coli mutant defective in DXR activity. The expression pattern of
?-glucuronidase, driven by the DXR promoter in Arabidopsis transgenic plants, together with the tissue distribution of DXR
transcript and protein, revealed developmental and environmental regulation of the DXR gene. The expression pattern of
the DXR gene parallels that of the Arabidopsis 1-deoxy-d-xylulose 5-phosphate synthase gene, but the former is slightly
more restricted. These genes are expressed in most organs of the plant including roots, with higher levels in seedlings and
inflorescences. The block of the 2-C-methyl-d-erythritol 4-phosphate pathway in Arabidopsis seedlings with fosmidomycin
led to a rapid accumulation of DXR protein, whereas the 1-deoxy-d-xylulose 5-phosphate synthase protein level was not
altered. Our results are consistent with the participation of the Arabidopsis DXR gene in the control of the 2-C-methyl-d-
erythritol 4-phosphate pathway.
Plants synthesize a large number of isoprenoid com-
pounds that are very diverse in structure and function
(Chappell, 1995; McGarvey and Croteau, 1995). Some
isoprenoids are essential in all plants. For instance,
chlorophylls and carotenoids are required as photo-
synthetic pigments, ubiquinone and plastoquinone as
electron carriers, sterols as structural components of
membranes, dolichols as oligosaccharide donors in
protein glycosylation, and abscisic acid, brassino-
steroids, cytokinins, and gibberellins as growth regu-
lators. In addition, a vast array of specific isoprenoid
compounds found in the different plant species are
involved in the interaction with other organisms or in
the response to environmental challenges. Despite
their diversity, all isoprenoids derive from isopente-
nyl diphosphate (IPP) and dimethylallyl diphosphate
(DMAPP), two readily interchangeable five-carbon
isomers that can be conceptually viewed as a single
isoprenoid building unit. In higher plants, the isopre-
noid building unit is formed by two pathways that
operate in different subcellular compartments (Eisen-
reich et al., 1998; Rohmer, 1999; Lichtenthaler, 2000).
In the cytosol-endoplasmic reticulum, the two isomers
are synthesized by the well-known mevalonate
(MVA) pathway. In plastids, IPP and DMAPP are
formed by the 2-C-methyl-d-erythritol 4-phosphate
(MEP) pathway. In the first reaction of this pathway,
1-deoxy-d-xylulose 5-phosphate (DXP) is synthesized
from pyruvate and d-glyceraldehyde 3-phosphate.
This step is catalyzed by 1-deoxy-d-xylulose 5-phos-
phate synthase (DXS), which is encoded by the DXS
gene. The following reaction, consisting in the con-
version of DXP to MEP, is catalyzed by 1-deoxy-d-
xylulose 5-phosphate reductoisomerase (DXR), the
product of the DXR gene. Because DXP is a precursor
not only of isoprenoids but also of the cofactors
1This work was supported by the Spanish Ministerio de Edu-
cacio ´n y Cultura (grant no. BIO2000–0334), by the Generalitat de
Catalunya (grant no. CIRIT 1999SGR–00032), and by the Agencia
Espan ˜ola de Cooperacio ´n Internacional (predoctoral MUTIS fel-
lowship to I.A.).
2Present address: Instituto de Biologı ´a Vegetal y Biotecnologı ´a,
Universidad de Talca, 2 Norte 685, Casilla 747, Talca, Chile.
* Corresponding author; e-mail campos@sun.bq.ub.es; fax 34–
93–4021219.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.003798.
Plant Physiology, August 2002, Vol. 129, pp. 1581–1591, www.plantphysiol.org © 2002 American Society of Plant Biologists1581

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thiamine pyrophosphate and pyridoxal phosphate
(Julliard and Douce, 1991; Julliard, 1992), the reaction
catalyzed by DXR is actually the first committed step
of the MEP pathway. Therefore, DXR could play an
important role in the control of plastid isoprenoid
biosynthesis. In Escherichia coli, three subsequent re-
actions catalyzed by the products of ygbP, ychB, and
ygbB genes, respectively, allow the synthesis of 2-C-
methyl-d-erythritol 2,4-cyclodiphosphate (Eisenreich
et al., 2001), which is converted to (E)-4-hydroxy-3-
methyl-but-2-enyl pyrophosphate by the gcpE gene
product (Hecht et al., 2002; Seemann et al., 2002a,
2002b; Wolff et al., 2002). Recent work has shown that
the lytB gene product is involved in the final step of
the MEP pathway consisting in the conversion of
(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate to
IPP and DMAPP (Hintz et al., 2001; Rohdich et al.,
2002).
The identification of the regulatory steps of the
MEP pathway is an issue of major importance in the
study of isoprenoid biosynthesis for both theoretical
and applied reasons. The fact that two separate but
likely coordinated pathways produce the same iso-
prenoid precursors adds further interest to the re-
search on this issue. So far, the unraveling of the
control of the MEP pathway has been focused in DXS
and DXR, the first two enzymes identified. Several
investigations support that DXS plays a role in the
control of plant isoprenoid biosynthesis. A positive
correlation was found between the levels of DXS
transcript and protein and the accumulation of vari-
ous plastid isoprenoid products in transgenic Arabi-
dopsis engineered to under- or overexpress DXS (Es-
te ´vez et al., 2001). A remarkable spatial and temporal
correlation was found between the level of DXS tran-
script and the synthesis of specific isoprenoid prod-
ucts in a variety of systems: lycopene in tomato (Ly-
copersicon esculentum) fruit during ripening (Lois et
al., 2000), apocarotenoids in roots from monocots
after colonization by mycorrhizal fungi (Walter et al.,
2000), terpenoid indole alkaloids in periwinkle (Ca-
tharanthus roseus) cell suspension culture upon hor-
monal induction (Chahed et al., 2000), and carote-
noids in pepper (Capsicum annuum) fruit during
chloroplast to chromoplast transition (Bouvier et al.,
1998). In addition, 1-deoxy-d-xylulose feeding in ma-
ture green tomato fruits induced expression of carot-
enoid biosynthetic genes and concomitant carotenoid
accumulation (Lois et al., 2000).
The participation of DXR in the control of isopre-
noid accumulation in plants is also sustained by ex-
perimental results. Overexpression of DXR in trans-
genic peppermint (Mentha piperita) plants led to an
increase of essential oil monoterpenes in leaf tissue
compared with wild type (Mahmoud and Croteau,
2001). Conversely, partial DXR gene silencing in
some of the engineered peppermint plants led to a
reduction of essential oil accumulation. In agree-
ment, a positive correlation was found between the
accumulation of DXR transcript and apocarotenoids
in mycorrhizal roots from monocots (Walter et al.,
2000) or terpenoid indole alkaloids in periwinkle cell
suspension culture (Veau et al., 2000). In these two
systems, both DXS and DXR could have a regulatory
role because a parallel increase of DXS and DXR
transcripts was observed (Veau et al., 2000; Walter et
al., 2000). In contrast to these results, neither DXR
transcript nor DXR protein level increased in tomato
fruit during ripening, despite the massive carotenoid
accumulation, suggesting a non-limiting role for
DXR in this system (Rodrı ´guez-Concepcio ´n et al.,
2001).
So far, all investigations concerning expression of
genes encoding plant DXR have been restricted to the
analysis of transcript or protein levels in a variety of
systems in which the synthesis of specific isopre-
noids is induced developmentally or in response to
external stimuli (Veau et al., 2000; Walter et al., 2000;
Rodrı ´guez-Concepcio ´n et al., 2001). The expression
pattern of the DXR gene in the whole plant along
normal development has not been investigated yet,
nor other important related aspects as the subcellular
localization of the encoded product. We have chosen
Arabidopsis for this research. As a first step, we
determined the 5? end sequence of the Arabidopsis
DXR transcript. This allowed the isolation of a cDNA
encoding the entire Arabidopsis DXR, the study of
the intracellular targeting of the protein, and the
construction of DXR-GUS translational fusions. The
expression pattern of ?-glucuronidase (GUS) driven
by the DXR promoter, together with the accumula-
tion profile of DXR transcript and protein, indicates
developmental and environmental regulation of the
Arabidopsis DXR gene. The DXR protein is targeted
to plastids.
RESULTS
Sequence Analysis of Arabidopsis DXR
The cDNA sequences encoding Arabidopsis DXR
reported to date were incomplete at the 5? end (Lange
and Croteau, 1999; Schwender et al., 1999). To deter-
mine the 5? end of the DXR transcript, we performed
5?-RACE using total RNA from 12-d-old Arabidopsis
seedlings as a template. Sequencing of four clones
derivedfromthe major
showed a single 5? end that corresponds to the ade-
nine at position ?1 of the full-length cDNA sequence
deposited in GenBank (accession no. AF148852). This
information, together with previous sequence data,
allowed the design of PCR primers to amplify a
cDNA encoding the entire Arabidopsis DXR. The
protein predicted from this cDNA contains 477
amino acid residues and has a molecular mass of 52
kD. The alignment of the cDNA sequence with the
corresponding genomic sequence (clone MQB2, ac-
cession no. AB009053) revealed the organization of
the Arabidopsis DXR gene. This gene maps in chro-
amplificationproduct
Carretero-Paulet et al.
1582Plant Physiol. Vol. 129, 2002

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mosome 5 and contains 12 exons and 11 introns
extending over a region of 3.2 kb. A databank search
showed that no additional sequence homologous to
the DXR gene exists in the completely sequenced
Arabidopsis genome. In agreement, the pattern of
bands obtained in Southern-blot analysis performed
under high- and low-stringency conditions, with a
0.84-kb SalI-EcoRV cDNA fragment as a probe, per-
fectly fits to that predicted from the MQB2 clone
(data not shown). It can be concluded that Arabidop-
sis DXR is encoded by a single-copy gene and that
the probe used is DXR specific.
To characterize the sequence of the N-terminal re-
gion of Arabidopsis DXR, we aligned this sequence
with the equivalent region of the plant DXRs known
to date and the E. coli DXR. As shown in Figure 1, the
plant enzyme contains an extension of 73 to 80 resi-
dues that is not present in the prokaryotic sequence.
Data analysis with the ChloroP program (Emanuels-
son et al., 1999) predicted a transit peptide for plas-
tids in all plant DXR sequences (Fig. 1). In 11 of the 14
transit peptides, the processing site was predicted at
the N terminus of a conserved Cys-Ser-X motif,
where X means any of the hydrophobic residues Ala,
Val, or Met. The regions at the N- or C-terminal side
of the putative processing site have different struc-
tural features. At the N-terminal side, the sequence is
poorly conserved but enriched in Ser residues, fea-
tures that are typical of plastid transit peptides (von
Heijne et al., 1989). In contrast, the extended region at
the C-terminal side (positions 50–80 of Arabidopsis
DXR) is highly conserved and particularly rich in Pro
residues (Fig. 1). The number of Pro residues in this
region ranges from 6 to 8. The consensus motif
P(P/Q) PAWPG(R/T) A can be defined in the Pro-
rich region of plant DXR (positions 60–68 of the
Arabidopsis sequence). The collective sequence anal-
ysis suggests that all plant DXRs have a transit pep-
tide for plastids, are processed at a conserved cleav-
age site, and contain an extended Pro-rich region at
the N terminus of the mature protein, which is not
present in prokaryotic DXR.
Functional Analysis and Subcellular Localization of
Arabidopsis DXR
To demonstrate that the isolated Arabidopsis
cDNA encodes a functional DXR, we complemented
an E. coli lethal mutant defective in the dxr gene
(Rodrı ´guez-Concepcio ´n et al., 2000). As expected,
this mutant can be rescued by expression of plasmid-
encoded E. coli DXR (EcDXR, Fig. 2). In addition, the
mutant was rescued by expression of either a short
derivative of Arabidopsis DXR (AtDXR-S, residues
Figure 1. Multiple sequence alignment of the N-terminal region of plant DXR. The sequence of Arabidopsis DXR was
aligned to the other plant DXR sequences known to date, deduced either from complete cDNA clones or expressed sequence
tag (EST) entries. Only those EST sequences confirmed by at least two independent entries were considered. Sequence
alignment was performed with the ClustalW 1.8 program (http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.html)
and optimized by visual inspection. The origin of the DXR sequence is indicated on the left and the number of amino acid
residues on the right. Residues are written in white inside black boxes if they are identical in all plant sequences, in white
inside gray boxes if two alternative residues are found in equivalent positions, or in black over white background if a lower
conservation is observed. Gaps in the sequence are represented with a dash. The last residue of the transit peptide predicted
by the ChloroP program (Emanuelsson et al., 1999) in the different sequences is indicated by an asterisk above the
corresponding letter. The putative cleavage site deduced from the collective analysis of all plant DXR sequences is indicated
with an arrowhead. The Arabidopsis peptide used for antibody production is marked with a line on the top. The cDNA and
EST sequences are accessible at the GenBank with the following accession numbers: Arabidopsis (AF148852), Artemisia
annua (AF182287), periwinkle (AF250235), Glycine max (EST 1, BE804032; EST 2, BE211397; and EST 3, BG839054),
tomato (AF331705), Lycopersicon hirsutum (EST, AW617386), Medicago truncatula (EST 1, BG456710; and EST 2,
BG450566), peppermint (AF116825), Oryza sativa (AF367205), Solanum tuberosum (EST, BE924278), and Zea mays
(AJ297566). For reference, the N-terminal sequence of the DXR from E. coli is represented at the bottom.
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81–477),
N-terminal region, or a longer version of the protein
(AtDXR-L, residues 57–477), which lacked the pre-
dicted transit peptide and only the first seven resi-
dues of the mature protein (Fig. 2). We conclude that
the two forms of the Arabidopsis protein have DXR
activity. However, AtDXR-L led to a much more
vigorous growth than AtDXR-S, as estimated by the
colony size (Fig. 2). The same observation was made
with dxr mutants generated in two different genetic
backgrounds (E. coli strains MC4100 and JC7623).
Our results suggest that the N-terminal Pro-rich re-
gion of Arabidopsis DXR (residues 57–80) contains
elements that are important for activity or stability, at
least when expressed in E. coli.
The intracellular targeting of Arabidopsis DXR was
studied by transient expression of a construct encod-
ing the entire DXR protein fused to the N terminus of
an optimized version of the soluble green fluorescent
protein (GFP). Leaves from 15-d-old light-grown
Arabidopsis seedlings were microbombarded with
this construct. The DXR-GFP fusion protein accumu-
lated in the chloroplasts of leaf cells as shown by its
colocalizationwith chlorophyll
(Fig. 3, A–C). In a second approach, we analyzed the
distribution of DXR in cells from Arabidopsis leaves,
using a polyclonal antibody (Ab-AtDXR1) raised
againstpeptideEAPRQSWDGPK,
sponds to the N-terminal extended region of Arabi-
dopsis DXR (Fig. 1, residues 71–81). Immunogold
particles were found in chloroplasts (Fig. 3D) and
which lacked theentireplant-specific
autofluorescence
which corre-
rarely outside this organelle. These observations,
which to our knowledge are the first experimental
evidence of plastid localization of plant DXR, are in
agreement with the proposed role of this protein in
the synthesis of plastid-derived isoprenoids.
Expression Analysis of the Arabidopsis DXR Gene
As a first step to evaluate the importance of the
DXR gene in the control of the MEP pathway, we
analyzed the distribution of the DXR transcript and
protein in Arabidopsis, studied the accumulation of
these molecules in response to particular conditions
and characterized the expression pattern of a chi-
meric construct containing the GUS reporter gene
under control of the DXR promoter. Northern-blot
analysis of total RNA from different tissues revealed a
transcript of about 1.8 kb (Fig. 4A), in agreement with
the size of full-length DXR cDNA. This transcript is
present in all tissues analyzed, but accumulates at
higher levels in seedlings and inflorescences (Fig. 4A).
Lower transcript levels were observed in leaves of
adult plants and roots of 15-d-old seedlings. The low-
est transcript level was detected in stems. The DXR
transcript is also present at a high level in the light-
grown cell suspension line T87 derived from Arabi-
dopsis leaves (Axelos et al., 1992; Fig. 4A). A good
correlation in tissue distribution was found between
the 1.8-kb transcript and a 46-kD protein detected by
western blot with the Ab-AtDXR1 antibody (Fig. 4B).
The DXR protein accumulates at higher levels in
Figure 2. Complementation of E. coli dxr mutant with Arabidopsis DXR. The E. coli dxr::TET mutant EcAB1–2 (Rodrı ´guez-
Concepcio ´n et al., 2000) was transformed with a control expression plasmid without DXR insert (pBADM1), a pBADM1
derivative coding for a long form of Arabidopsis DXR (pBAD-AtDXR-L), a pBADM1 derivative coding for a short form of
Arabidopsis DXR (pBAD-AtDXR-S), or a expression plasmid coding for the DXR from E. coli (pTAC-EcDXR). Transformants
were plated in Luria-Bertani medium containing 6 ?g mL?1tetracycline to select for the dxr::TET mutant, 100 ?g mL?1
ampicillin to select for the plasmid, 100 ?M isopropyl-?-D-thiogalactoside to induce expression of EcDXR, and 0.02% (w/v)
L-Ara to induce expression of AtDXR-S and AtDXR-L. After 17 h at 37°C, the colony size was 2 to 3 mm for the mutant
transformed with pTAC-EcDXR or pBAD-AtDXR-L and 0.3 to 0.4 mm for the mutant transformed with pBAD-AtDXR-S.
Carretero-Paulet et al.
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15-d-old seedlings, inflorescences, and fruits, and at
lower levels in cauline leaves and stems. The protein
is barely detectable in rosette leaves of adult plants.
The apparent molecular mass of the immunodetected
DXR is consistent with processing at the conserved
site described above. The detection of this protein by
western blot confirms that the N-terminal Pro-rich
sequence is present in the mature form of the Arabi-
Figure 3. Targeting and subcellular localization of plant DXR. Leaves of 15-d-old Arabidopsis seedlings were microbom-
barded with a construct encoding Arabidopsis DXR fused to the N terminus of GFP. Cells expressing the fusion protein were
studied by laser confocal microscopy. The images show green fluorescence of DXR-GFP (A), red autofluorescence of
chlorophyll (B), and the superimposed green and red fluorescence (C). Bars in A through C indicate 10 ?m. Arabidopsis DXR
was immunolocalized in leaves of 15-d-old seedlings with the Ab-AtDXR1 polyclonal antibody. The electron micrograph (D)
shows localization of 15-nm gold particles in chloroplast. C, Cytoplasm; S, stroma; St, starch granule; T, thylakoid. Bar in
D indicates 0.1 ?m.
Arabidopsis 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase
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