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Plant Physiol, November 2002, Vol. 130, pp. 1079-1089
UPDATE ON ISOPRENOID BIOSYNTHESIS
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INTRODUCTION |
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 TOP
 INTRODUCTION
 ISOPRENOID BIOSYNTHESIS. A TALE...
SIMPLER IS BETTER. ELUCIDATION...
THE MEP PATHWAY IN...
CONCLUDING REMARKS
LITERATURE CITED
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Plants synthesize an enormous
variety of metabolites that can be classified into two groups based on
their function: primary metabolites, which participate in nutrition and
essential metabolic processes within the plant, and secondary
metabolites (also referred to as natural products), which influence
ecological interactions between plants and their environment (Croteau
et al., 2000
). Isoprenoids (also called terpenoids) are the most
functionally and structurally varied group of plant metabolites.
Isoprenoids are synthesized in all organisms but are especially
abundant and diverse in plants, with tens of thousands of compounds
reported to date (Chappell, 1995, 2002; McGarvey and Croteau, 1995;
Croteau et al., 2000). Many isoprenoids are present in all plants and
act as primary metabolites with roles in respiration, photosynthesis,
and regulation of growth and development. However, the highest variety
of isoprenoids is secondary metabolites that function in protecting
plants against herbivores and pathogens, in attracting pollinators and
seed-dispersing animals, and as allelochemicals that influence
competition among plant species (Croteau et al., 2000; Chappell, 2002).
Many compounds with important commercial value as flavors, pigments,
polymers, fibers, glues, waxes, drugs, or agrochemicals are secondary
metabolites of isoprenoid origin. Each plant species synthesizes a
specific array of isoprenoid secondary metabolites, and most of them
(including rubber and the anticancer drug taxol) are produced only in a
few wild or semiwild plant species. Although genetic engineering
appears to be a powerful tool to direct the production of both primary and secondary isoprenoid products in plants, only a partial knowledge of the pathways involved in the biosynthesis of their precursors was
available until very recently.
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ISOPRENOID BIOSYNTHESIS. A TALE OF TWO PATHWAYS |
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TOP
INTRODUCTION
ISOPRENOID BIOSYNTHESIS. A TALE...
SIMPLER IS BETTER. ELUCIDATION...
THE MEP PATHWAY IN...
CONCLUDING REMARKS
LITERATURE CITED
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Despite their diversity of functions and structures, all
isoprenoids derive from the common five-carbon
(C5) building units isopentenyl diphosphate (IPP)
and its isomer dimethylallyl diphosphate (DMAPP), also called isoprene
units (Fig. 1). The simplest isoprenoids, like isoprene (a volatile product released from photosynthetically active tissues that participates in the formation of tropospheric ozone), contain a single C5 unit and are called
hemiterpenes. More complex isoprenoids are usually formed by
"head-to-tail" or "head-to-head" addition of isoprene units.
Monoterpenes are C10 isoprenoids that consist of
two isoprene units and are components of the essences of flowers,
herbs, and spices. The isoprenoids that derive of three isoprene units
are C15 sesquiterpenes, which can be found in
essential oils and act as antimicrobial phytoalexins and antifeedants.
The diterpenes (C20) include the side chain of
chlorophyll, phylloquinones and tocopherol, gibberellins, phytoalexins, and taxol. The triterpenes (C30), such as
phytosterols, brassinosteroids, and some phytoalexins, toxins, and
waxes, are generated by the joining of two C15
chains. The most prevalent tetraterpenes (C40) are carotenoids, which are pigments in many flowers and fruits, contribute to light harvesting, and protect the photosynthetic apparatus from photooxidation. Polyterpenes contain more than eight
isoprene units and include prenylated electron carriers (ubiquinone and
plastoquinone) and polyprenols such as rubber and dolichol (required
for protein glycosylation). The products of partial isoprenoid origin,
including cytokinins or prenylated proteins, are called meroterpenes
(McGarvey and Croteau, 1995; Croteau et al., 2000).
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After the discovery of the mevalonic acid (MVA) pathway in yeast and
animals in the 1950s, it was assumed that IPP was synthesized from
acetyl-CoA via MVA and then isomerized to DMAPP in all organisms (Chappell, 1995; McGarvey and Croteau, 1995). In many cases, however, the experimental data on the biosynthesis of specific isoprenoids in
plants and microorganisms could not be explained from the exclusive operation of the MVA pathway (for review, see Lichtenthaler et al.,
1997, 1999; Eisenreich et al., 1998, 2001; Rohmer, 1999). A few years
ago, an alternative MVA-independent pathway for the biosynthesis of the
isoprene building units was identified by labeling experiments in
bacteria (Flesch and Rohmer, 1988; Rohmer et al., 1993; Broers, 1994)
and plants (Schwarz, 1994). This pathway was originally named
non-mevalonate pathway or Rohmer pathway. After the
identification of the first steps of the pathway, its name was changed
to indicate the substrates (pyruvate/glyceraldehyde 3-phosphate [G3P]
pathway) or the first intermediate, deoxyxylulose (DX) 5-phosphate
(DXP pathway). However, it is becoming more accepted to name the
pathway after what is currently considered its first committed
precursor, methylerythritol 4-phosphate (MEP), following the same rule
used to name the MVA pathway.
Isoprenoids are synthesized in all living organisms, but experimental
evidence accumulated since the discovery of the MEP pathway has shown
that most organisms only use one of the two pathways for the
biosynthesis of their precursors. Thus, the MEP pathway is the only one
present in most eubacteria and the malaria parasite Plasmodium
falciparum, but it is absent from archaebacteria, fungi and
animals, which synthesize their isoprenoids exclusively through the
operation of the MVA pathway. By contrast, plants use both the MEP
pathway and the MVA pathway for isoprenoid biosynthesis, although they
are localized in different compartments (Fig. 1; Lichtenthaler et al.,
1997; Eisenreich et al., 1998, 2001; Lichtenthaler, 1999; Rohmer,
1999). The MEP pathway synthesizes IPP and DMAPP in plastids, whereas
the MVA pathway produces cytosolic IPP (Fig. 1). Mitochondrial
isoprenoids are synthesized from MVA-derived IPP that is imported from
the cytosol (Lichtenthaler, 1999). Some exchange of IPP or a common
downstream intermediate does also appear to take place between the
plastids and the cytoplasm (for review, see Eisenreich et al., 1998,
2001; Lichtenthaler et al., 1997; Lichtenthaler, 1999; Rohmer, 1999).
This limited exchange may explain in part why the MEP pathway was
completely overlooked until very recently, because labeled precursors
of the MVA pathway could be incorporated (although with very low
efficiency) into most plastid isoprenoids. The now uncovered MEP
pathway for the biosynthesis of isoprenoids may represent one of the
last evolutionarily conserved metabolic pathways which remained to be unraveled.
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SIMPLER IS BETTER. ELUCIDATION OF THE MEP PATHWAY IN Escherichia coli |
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TOP
INTRODUCTION
ISOPRENOID BIOSYNTHESIS. A TALE...
SIMPLER IS BETTER. ELUCIDATION...
THE MEP PATHWAY IN...
CONCLUDING REMARKS
LITERATURE CITED
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E. coli, the metabolically best studied bacterium, has served as a powerful model system for the elucidation of the MEP pathway (Fig. 2), which has been achieved thanks to multidisciplinary approaches that included organic chemistry, microbial genetics, biochemistry, molecular biology, and bioinformatics. However, the impressively fast identification of the genes involved in the pathway in bacteria and plants would not have been possible without recently developed genomic tools such as the availability of full genome sequences and expressed sequence tag (EST) collections. The elucidation of the MEP pathway is also a beautiful example of how genomics can be readily integrated with traditional approaches to identify whole metabolic pathways in distant organisms.
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1996 to 1998. From the Precursors to the Identification of the First Genes of the Pathway
Although evidences of a MVA-independent pathway for IPP
biosynthesis were found independently by different research groups (Flesch and Rohmer, 1988; Rohmer et al., 1993; Broers, 1994; Schwarz, 1994), Rohmer and collaborators were the first in publishing their work
in refereed journals. These authors used labeled precursors to study
the biosynthesis of the bacterial isoprenoids hopanoids, and they
observed labeling patterns that suggested the addition of a
C2 unit derived from pyruvate by decarboxylation
to a C3 triose phosphate (or a derivative) in a
transketolase type reaction (Flesch and Rohmer, 1988; Rohmer et al.,
1993). G3P and pyruvate were afterward identified as the direct
precursors of IPP by labeling experiments with E. coli
mutants defective in enzymes of the triose phosphate metabolism (Rohmer
et al., 1996). These experiments suggested that the first reaction of
the novel pathway involved the head-to-head condensation of
(hydroxyethyl) thiamin derived from pyruvate with the C1 aldehyde
group of G3P to yield DXP (Fig. 2), a compound that also serves as a
precursor in the biosynthesis of the vitamins B1
(thiamine) and B6 (pyridoxol) in bacteria and plastids (Fig. 1). Other studies in different research groups confirmed
the incorporation of labeled DX into bacterial and plant plastidial
isoprenoids (for review, see Lichtenthaler et al., 1997; Eisenreich et
al., 1998; Lichtenthaler, 1999; Rohmer, 1999).
Once this information was available, three independent approaches led
to the identification of the first gene of the MEP pathway, encoding DXP synthase (DXS; Sprenger et al., 1997; Lange et al., 1998;
Lois et al., 1998). Synthesis of DXP according to the mechanism described above required an acyloin condensation reaction whereby pyruvate is decarboxylated. This type of reaction was well documented as a secondary activity of thiamine diphosphate-dependent
transketolases or the E1 component of pyruvate dehydrogenase or
pyruvate decarboxylase. Taking advantage of the recent advent of full
genomic sequence information for E. coli, Sprenger et al.
(1997) and Lois et al. (1998) independently found a bacterial gene
encoding a product with homology to transketolase and E1. Expression of
the corresponding protein in E. coli and determination of
its ability to form only DXP from pyruvate and G3P confirmed that it
encoded a DXS enzyme (Sprenger et al., 1997; Lois et al., 1998).
DXS-like sequences were found widespread in bacteria and plants but
were absent from animal and yeast genomes. The Arabidopsis homolog had
been previously described as CLA1, a plastid-targeted protein of
unknown function encoded by a nuclear gene whose disruption caused an
albino phenotype (Mandel et al., 1996). Following a homology-based
approach, Lange et al. (1998) identified another plant
transketolase-like sequence in a cDNA library from peppermint
(Mentha piperita) oil gland secretory cells, which are
highly specialized for monoterpene production and are therefore an
enriched source of transcripts from genes involved in isoprenoid
biosynthesis. The identified gene encoded a protein with DXS activity
that was most similar to Arabidopsis CLA1, suggesting a role in the
biosynthesis of plastid isoprenoids essential for photosynthesis and
chloroplast function.
Rohmer et al. (1996) had proposed that an intramolecular rearrangement
of DXP followed by an unspecified reduction process could produce MEP
in the next reaction of the pathway (Fig. 2). Subsequent experiments
showed that chemically synthesized ME was directly incorporated into
E. coli isoprenoids (Duvold et al., 1997). A genetic
strategy based on this information succeeded in identifying the
bacterial gene encoding DXP reductoisomerase (DXR), the enzyme that
converts DXP into MEP (Kuzuyama et al., 1998; Takahashi et al., 1998).
Because MEP is only known to be a precursor for isoprenoids, these
authors hypothesized that E. coli auxotrophic mutants
requiring ME should be specifically affected in MEP and isoprenoid
biosynthesis. After isolating mutants that grew on minimal medium with
ME but not in the absence of this compound, they identified
yaeM (now designated dxr or ispC) as the gene complementing ME auxotrophy in all the mutants and
demonstrated that its product was a DXR enzyme involved in isoprenoid
biosynthesis (Kuzuyama et al., 1998; Takahashi et al., 1998).
1999 to 2000. Bioinformatics and Comparative Genomics Identify New Candidate Genes
For the identification of the next gene of the MEP pathway,
Rohdich et al. (1999) incubated radiolabeled MEP with E. coli cell extracts and purified enzyme fractions and observed that a radioactive product was produced when the reaction mixture contained a nucleotide 5'-triphosphate (CTP was the preferred substrate). On the basis of NMR spectroscopy data, the structure of the new metabolite was assigned as 4-diphosphocytidyl ME (CDP-ME; Fig. 2). A
database search with CDP and pyrophosphorylase as keywords retrieved a
gene encoding a bacterial enzyme that catalyzes the formation of
CDP-ribitol from ribitol 5-phosphate and CTP. Subsequent database
searches with this sequence uncovered a number of similar genes from
organisms with the MEP pathway, including Arabidopsis (in which the
corresponding protein encompassed a putative plastid leader sequence).
Activity assays with the recombinant product of the E. coli
gene (ygbP, also designated ispD) demonstrated that it encoded a CDP-ME synthase (CMS) that specifically produced CDP-ME from MEP and CTP (Fig. 2). Furthermore, incubation of
radiolabeled CDP-ME with pepper (Capsicum annuum)
chromoplasts resulted in the incorporation of radioactivity into
carotenoids, suggesting that this metabolite was an intermediate of the
MEP pathway (Rohdich et al., 1999).
The identification of the E. coli dxs, dxr, and
ygbP genes provided sequence information that established
the basis for a comparative genomics procedure that eventually led to
the elucidation of the entire MEP pathway: the bioinformatic search for
genes that were conserved in eubacteria and plants (the latter showing a N-terminal extension that could serve as a plastid targeting signal)
but absent in archaebacteria, yeast, and animals (which synthesize
their isoprenoids exclusively from MVA). Thus, whole genome comparisons
to identify genes after the distribution of the identified MEP pathway
genes retrieved the next two genes of the MEP pathway, ychB
and ygbB; Herz et al., 2000; Lüttgen et al., 2000). A
procedure similar to that developed for ygbP was used to
study the activity of the encoded proteins and their involvement in the
MEP pathway. The purified recombinant enzyme encoded by the E. coli ychB gene was shown to be a CDP-ME kinase (CMK) that
catalyzes the ATP-dependent phosphorylation of CDP-ME to CDP ME
2-phosphate (CDP-MEP). This compound was then converted into ME
2,4-cyclodiphosphate (ME-cPP) by the enzyme ME-cPP synthase (MCS),
encoded by the E. coli ygbB gene (Fig. 2). As expected for
MEP pathway enzymes, the plant homologs showed putative plastid signal
peptides. In addition, incorporation experiments with pepper chromoplasts suggested that both CDP-MEP and ME-cPP were intermediates of the MEP pathway (Herz et al., 2000; Lüttgen et al., 2000). A
plant gene homologous to ychB had previously been retrieved in a bioinformatic approach designed to identify ESTs encoding metabolite kinases in a cDNA library from peppermint oil gland secretory cells (Lange and Croteau, 1999a). Although these authors proposed that the encoded protein could phosphorylate isopentenyl monophosphate to IPP in the putative last step of the MEP pathway, further experiments with the recombinant enzymes from E. coli and tomato (Lycopersicon esculentum) showed that
they catalyzed the phosphorylation of CDP-ME to CDP-MEP at a much
higher rate, indicating that this is the true metabolic role of the
enzyme (Rohdich et al., 2000a).
2000 to 2001. Strains Engineered to Synthesize IPP from MVA Demonstrate the Branching of the Pathway and Confirm the Role of the Previously Identified Genes
Although the results described above strongly suggested that
ygbP (ispD), ychB
(ispE),and ygbB (ispF) encoded enzymes
directly involved in the MEP pathway, a clear-cut demonstration was
provided by the development of a neat experimental system originally
designed for the cloning of unknown MEP pathway genes in E. coli (Kuzuyama et al., 2000a, 2000b; Takagi et al., 2000; Campos
et al., 2001a). To rescue lethal mutants in the MEP pathway genes,
E. coli cells were genetically engineered with a recombinant
MVA operon containing heterologous genes for the last three enzymes of
the MVA pathway: MVA kinase, MVP kinase, and MVPP decarboxylase (see
Fig. 1). These cells do not synthesize MVA, but they can take it from
the growth medium and use it as an alternative source of IPP, which
could be then converted to DMAPP by the E. coli IPP
isomerase encoded by the idi gene (Hahn et al., 1999). By
using this system Rodríguez-Concepción et al. (2000)
demonstrated that idi is the only gene encoding an enzyme
with IPP isomerase activity in E. coli and showed that this
enzyme plays a role in isoprenoid biosynthesis in vivo. However, idi is not an essential gene in E. coli (Hahn et
al., 1999; Rodríguez-Concepción et al., 2000). The work
with strains harboring the MVA operon supported previous evidence from
labeling experiments (Giner et al., 1998; Charon et al., 2000)
demonstrating that the MEP pathway branched at some point after MEP
leading to the separate synthesis of IPP and DMAPP
(Rodríguez-Concepción et al., 2000). The MVA operon
system was also used by two independent groups to provide genetic
evidence that the enzymes encoded by ygbP, ychB,
and ygbB catalyze reactions of the MEP pathway before the
proposed branching, because the disruption of these genes was
lethal, indicating that they were not acting in the proposed
branches to IPP or DMAPP) and could be rescued with MVA (Kuzuyama et
al., 2000a, 2000b; Takagi et al., 2000; Campos et al., 2001a).
2001 to 2002. Identification of the Last Two Genes of the Pathway
Although the described system with the MVA operon was a good
genetic tool for the discovery of the rest of the MEP pathway genes,
they were first described by bioinformatic approaches of comparative
genomics (Cunningham et al., 2000; Campos et al., 2001b). The E. coli genes annotated as gcpE (ispG) and
lytB (ispH) were putatively ascribed to the MEP
pathway because they were conserved in plants and eubacteria with this
pathway but were absent from archaebacteria, yeast, and animal genomes.
In addition, the corresponding plant gene products contained an
N-terminal domain that could act as a plastid targeting signal.
Directed deletion of gcpE (Altincicek et al., 2001b; Campos
et al., 2001b) or lytB (Altincicek et al., 2001a) in
E. coli strains engineered with the MVA operon resulted in
cells that were able to grow only when the medium was supplemented with
MVA, demonstrating that both genes were required specifically for IPP
biosynthesis in E. coli. Subsequent studies (Hecht et
al., 2001; Seemann et al., 2002a, 2002b; Wolff et al., 2002)
contributed to reveal that the gcpE gene product encoded an
enzyme (hydroxymethylbutenyl 4-diphosphate [HMBPP] synthase
[HDS]) that catalyzes the formation of HMBPP from ME-cPP (Fig. 2).
The role of lytB is less clear, but it appears to encode an
enzyme (IDS) that directly converts HMBPP into a 5:1 mixture of IPP and
DMAPP (Fig. 2; Rohdich et al., 2002). Therefore, the activity of this
enzyme could be identified as responsible for the branching, which had
been previously predicted by biochemical and genetic approaches (Giner
et al., 1998; Charon et al., 2000; Rodríguez-Concepción
et al., 2000). The branching is an important difference with the MVA
pathway, in which IPP and DMAPP are generated sequentially, the latter
arising from the former in a reaction catalyzed by IPP isomerase (Fig.
1).
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THE MEP PATHWAY IN PLANTS |
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TOP
INTRODUCTION
ISOPRENOID BIOSYNTHESIS. A TALE...
SIMPLER IS BETTER. ELUCIDATION...
THE MEP PATHWAY IN...
CONCLUDING REMARKS
LITERATURE CITED
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The recent development of genomic tools is revolutionizing the
study of plant metabolism. As described above, the MEP pathway is a
good example of how bioinformatics and comparative genomics have made
relatively fast and simple to identify the genes potentially involved
in a metabolic pathway in different organisms based only on sequence
information. Searches on The Arabidopsis Information Resources
database (http://www.Arabidopsis.org) indicate that genes encoding
proteins with homology to all the E. coli MEP pathway enzymes are present in Arabidopsis (Table
I). The ChloroP algorithm (http://www.cbs.dtu.dk/services/ChloroP) predicts that all of these proteins contain a putative plastid targeting peptide of variable
length (Table I), consistent with their predicted role in plastid
isoprenoid biosynthesis. Functional genomics approaches consisting of
the generation and screening of collections of T-DNA and transposon
insertion mutants have led to the identification of Arabidopsis mutants
defective in the genes encoding DXS, DXR, and CMS (Budziszewski et al.,
2001). All of these mutants have a seedling-lethal albino phenotype,
confirming that the MEP pathway is essential for plant life. With the
increasing availability on public on-line databases of plant functional
genomics tools (including collections of ESTs and DNA microarrays), it
will soon become possible to even deduce accurate gene expression data
that may provide some clues as to their biological role. However, only functional analysis of each proposed plant protein ortholog with biochemical and genetic approaches will ascertain its contribution to
the biosynthesis of plastid isoprenoids.
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Plant Genes and Enzymes
Genes and ESTs corresponding to all the MEP pathway enzymes (Table
I) can be found in the availableArabidopsis databases. The most
abundant ESTs are those from the genes encoding DXS and HDS (about
0.2
of all the ESTs in the collections), followed by IDS (0.1).
These ESTs are widely distributed in the available Arabidopsis
collections (which are made from a variety of tissues and developmental
stages) suggesting that the corresponding genes are expressed
throughout the plant. From all of the tentative orthologs of the
E. coli MEP pathway enzymes that can be found in the
Arabidopsis genome, only DXS might be encoded by more than one gene
(Table I). Arabidopsis cla1 mutants defective in DXS (At4g15560) show an albino phenotype and a very early arrest of chloroplast development that can be rescued with DX (Mandel et al.,
1996; Araki et al., 2000; Estévez et al., 2000). However, mutant
plants can still accumulate low levels of plastid isoprenoids such as
chlorophylls and carotenoids, suggesting either an import of cytosolic
MVA-derived isoprenoid precursors to the plastids or the presence of
extra DXS enzymes (Araki et al., 2000; Estévez et al., 2000). Two
other Arabidopsis proteins, predicted from genomic and EST sequences
and tentatively named DXS2 (At3g21500) and DXS3 (At5g11380), show
homology to DXS (Fig. 3). Only a few ESTs
from these genes have been found in green siliques (three ESTs from
DXS2) and roots (one EST from DXS3), suggesting
that their expression is low and may be restricted to certain tissues or developmental stages. By contrast, the gene encoding DXS is widely
expressed in the Arabidopsis plant, as deduced from the number and
distribution of ESTs (Table I) and the analysis of mRNA and protein
accumulation (Estévez et al., 2000). The differential expression
pattern could explain why DXS-deficient seedlings show a block in
plastid isoprenoid synthesis (which causes the albino phenotype) that
is not rescued by the other two putative DXS isoforms. Although both
DXS2 and DXS3 contain N-terminal sequences predicted by the ChloroP
program to target them to plastids (Table I; Fig. 3), it is not known
whether they are functional DXS enzymes with a role in the MEP pathway.
The deduced mature proteins lack stretches of amino acids that are
present in all the bacterial and plant DXS enzymes (Lois et al., 1998,
2000), and a conserved His residue essential for DXS activity (Querol
et al., 2001) is not present in the DXS3 protein (Fig. 3). A functional
analysis is therefore needed to confirm the predictions generated by
the sequence-based analysis and to demonstrate their biological
function.
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The rest of the Arabidopsis MEP pathway enzymes (Table I) appear to be
encoded by a single gene, and functional data supporting their role in
plastid isoprenoid biosynthesis are available for DXR (Lange and
Croteau, 1999b; Schwender et al., 1999; Carretero-Paulet et al., 2002),
CMS (Rohdich et al., 2000b; Okada et al., 2002), and HDS (Querol et
al., 2002). A CMK ortholog from tomato has also been described (Rohdich
et al., 2000a). The most obvious difference between plant and E. coli MEP pathway enzymes is the presence of N-terminal extensions
of variable sequence and length (Table I), which have been shown to
function as plastidial signal peptides for plant DXS (Araki et al.,
2000; Lois et al., 2000), DXR (Rodríguez-Concepción et
al., 2001; Carretero-Paulet et al., 2002), and HDS (Querol et al.,
2002). The mature proteins produced after cleavage of these peptides
are similar to the bacterial enzymes except in the case of HDS (GCPE),
which contains a large plant-specific domain (Querol et al., 2002). The
mature Arabidopsis HDS protein is able to complement the lethal
deletion of the gcpE gene in E. coli, but it is
possible that because of this extra domain, the plant protein may have
distinct regulatory or catalytic functions. Most of the work on the
characterization of the MEP pathway enzymes has been done with the
E. coli proteins, including the resolution of the crystal
structure of the enzymes DXR (Reuter et al., 2002; Yajima et al.,
2002), CMS (Kemp et al., 2001; Richard et al., 2001), and MCS (Kemp et
al., 2002; Richard et al., 2002; Steinbacher et al., 2002). By
contrast, only limited knowledge about the catalytic properties of the
plant enzymes is available (for review, see Eisenreich et al.,
2001).
Regulation of the Metabolic Flow through the MEP Pathway
Despite the impressive progress in the elucidation of the MEP
pathway in bacteria and plants, much work is still ahead to analyze the
contribution of the different enzymes to the control of the flux of
intermediates through the pathway that will eventually determine the
supply of IPP and DMAPP for the synthesis of plastid isoprenoid end
products. The first studies have been carried out with DXS and DXR
(Mandel et al., 1996; Bouvier et al., 1998; Lange et al., 1998; Lange
and Croteau, 1999b; Schwender et al., 1999; Araki et al., 2000; Chahed
et al., 2000; Estévez et al., 2000, 2001; Lois et al., 2000; Veau
et al., 2000; Walter et al., 2000; Mahmoud and Croteau, 2001;
Rodríguez-Concepción et al., 2001; Carretero-Paulet et
al., 2002). To date, DXS is the only enzyme of the MEP pathway that has
been shown to have a limiting role for isoprenoid biosynthesis in all
the systems analyzed, including Arabidopsis (Estévez et al.,
2001), tomato (Lois et al., 2000), and bacteria (Harker and Bramley,
1999; Miller et al., 1999, 2000; Kuzuyama et al., 2000c; Matthews and
Wurtzel, 2000). The role of DXR is less clear. Overexpression studies
suggest that DXR activity is not limiting for isoprenoid biosynthesis
in bacteria (Miller et al., 2000). The dramatic accumulation of
carotenoids that takes place during tomato fruit ripening does not
require increased levels of DXR transcripts and encoded
protein either (Rodríguez-Concepción et al., 2001). By
contrast, overexpression of DXR in peppermint led to increased
isoprenoid synthesis (Mahmoud and Croteau, 2001), and a positive
correlation was found between enhanced isoprenoid biosynthesis and
accumulation of transcripts encoding both DXS and DXR in monocot roots
(Walter et al., 2000) and periwinkle (Catharanthus roseus)
cell cultures (Veau et al., 2000). The distribution of DXR
and DXS transcripts in the Arabidopsis plant is similar,
with highest levels in light-grown seedlings and inflorescences
(Carretero-Paulet et al., 2002). However, DXS expression
precedes that of DXR in some organs, such as developing inflorescences, suggesting that DXR instead of DXS might be limiting for the onset of plastid isoprenoid biosynthesis in this case (Carretero-Paulet et al., 2002). Together, the results support a
general regulatory role for DXS in controlling the metabolic flux
through the MEP pathway, whereas DXR activity may be limiting or not
depending on the species, organ, and/or developmental stage. It is
likely that other enzymes of the MEP pathway may also contribute to
regulate the supply of intermediates for plastid isoprenoid biosynthesis, but this remains to be established.
Coordination with Related Metabolic Pathways
The MEP pathway produces plastidial IPP and DMAPP precursors that
are then used as building blocks for the production of isoprenoid end
products by many different pathways (Fig. 1). A central question is how
the downstream pathways are coordinated with the MEP pathway (and among
them) to make sure that the required precursors will be supplied when
needed. Expression of some of the MEP pathway genes has been shown to
either precede or parallel the activation of specific pathways for the
production of monoterpenes in peppermint oil gland secretory cells
(Lange et al., 1998), monoterpenoid indole alkaloids in periwinkle cell
cultures (Veau et al., 2000), apocarotenoids in monocot roots (Walter
et al., 2000), and carotenoids in pepper and tomato fruit (Bouvier et
al., 1998; Lois et al., 2000). In the last case, it has been shown that
the expression of tomato DXS can be regulated by changes in
the carotenoid composition of the fruit (Lois et al., 2000).
Furthermore, changes in the levels of MEP pathway intermediates in
tomato fruit fed with DX or treated with fosmidomycin (a specific
inhibitor of DXR activity; Fig. 1) induced the expression of
DXS but also of PSY1, the gene encoding the
committed enzyme that catalyzes the first step of the carotenoid
pathway in fruit (Lois et al., 2000; Rodríguez-Concepción et al., 2001). These results suggest a significant coordination between
both the MEP pathway and the carotenoid pathway through the control of
the expression of key genes, which may contribute to a fine regulation
of carotenoid accumulation. Interference with this balanced regulation
by overexpression of PSY1 under the 35S promoter
in transgenic tomato led to the production of dwarf plants because the
geranylgeranyl diphosphate available for gibberellin synthesis was
redirected into the carotenoid pathway (see Fig. 1; Fray et al., 1995).
This exemplifies how our limited knowledge on the mechanisms by which
the MEP pathway and the downstream pathways are coordinated represents
an important obstacle to modify precisely the production of specific
isoprenoid end products.
The unique compartmentalization of isoprenoid biosynthesis in plants
involves the existence of additional plant-specific regulatory mechanisms. Although the MEP pathway and the MVA pathway are
independent pathways that are physically separated, they usually
coexist within the plant cell (Fig. 1). In fact, a limited exchange of
isoprene building units (IPP and DMAPP) or a common downstream
intermediate takes place between compartments, and some isoprenoid end
products are built from precursors supplied by both the MEP pathway and the MVA pathway (for review, see Eisenreich et al., 1998, 2001; Lichtenthaler et al., 1997; Lichtenthaler, 1999; Rohmer, 1999). Although the extent of this crossflow depends on the plant species, it
has been estimated to be below 1% in intact plants under physiological conditions (Eisenreich et al., 2001). In experiments carried out with
seedlings, the rate of exchange of intermediates appears not to be high
enough to fully rescue a block of one of the two pathways. Thus, the
specific inhibition of MVA-derived isoprenoid biosynthesis with
mevinolin (Fig. 1) in radish (Raphanus sativus) seedlings
cannot be overcome by the delivery of common isoprenoid intermediates
from the plastidial MEP pathway (Schindler et al., 1985). Arabidopsis
mutant seedlings defective in MEP pathway genes (Mandel et al., 1996;
Araki et al., 2000; Estévez et al., 2000; Budziszewski et al.,
2001) similarly show an albino phenotype likely because the block in
the synthesis of plastid isoprenoids required for photosynthesis and
photoprotection (such as chlorophylls, carotenoids, tocopherol, and
plastoquinone) cannot be rescued by the import of cytosolic MVA-derived
intermediates. The same phenotype is observed when seeds from
Arabidopsis (Fig. 4) or tomato
(Rodríguez-Concepción et al., 2001) are germinated in the
presence of fosmidomycin, a specific DXR inhibitor that causes a
general block in plastid isoprenoid biosynthesis (Zeidler et al.,
1998). However, the dynamics and the regulation of the crossflow of
common intermediates between cell compartments may vary dramatically in
different species, cell types, and/or developmental stages. This is an
area of intensive research that will benefit from the availability of
specific inhibitors such as mevinolin and fosmidomycin (Fig. 1) to
block any of the two pathways for isoprenoid synthesis in a given
plant, organ, or stage of development. For instance, treatment of
tomato mature green fruit with fosmidomycin inhibited subsequent
carotenoid accumulation (Zeidler et al., 1998;
Rodríguez-Concepción et al., 2001), resulting in fruit of
yellow-orange color instead of red when ripe (Fig. 4C). These results
and previous experiments of treatment with mevinolin
(Rodríguez-Concepción and Gruissem, 1999) support that
the MVA pathway does not contribute significantly to carotenoid
biosynthesis in tomato fruit. Future experiments should establish how
the crossflow of MEP- or MVA-derived isoprenoid intermediates is
modulated under physiological conditions and the nature of the
transport system for prenyl diphosphate compounds between cytoplasm and
plastids.
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INTRODUCTION
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CONCLUDING REMARKS
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The joint contribution of genomics integrated with traditional
biochemical and genetic approaches has led to the impressively fast
elucidation of the MEP pathway for the biosynthesis of plastid isoprenoids, a metabolic milestone that represents a huge step forward
toward understanding (and manipulating) isoprenoid biosynthesis in
plants. Nevertheless, we still lack fundamental knowledge on the
regulatory mechanisms that control the flow of intermediates through
the pathway and the coordination with related metabolic pathways. The
benefits that the characterization of the MEP pathway can represent go
beyond metabolic engineering. The MEP pathway, which is absent from
humans but is present in pathogenic bacteria (many of which are
acquiring resistance to currently available antibiotics) and in the
malaria parasite Plasmodium falciparum, constitutes an ideal
target for the development of novel antimalarial and antibacterial
agents (Jomaa et al., 1999; Altincicek et al., 2001c; Hintz et al.,
2001). Plants are promising test systems for the development of such
inhibitors of the MEP pathway, which could also serve as herbicides
(Zeidler et al., 2000; Lange et al., 2001).
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ACKNOWLEDGMENTS |
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We thank Drs. Narciso Campos and Michel Rohmer for the critical reading of the manuscript and our laboratory members for stimulating discussions. We also thank Monsanto for the gift of fosmidomycin.
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FOOTNOTES |
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Received April 15, 2002; returned for revision June 18, 2002; accepted July 10, 2002.
1 This work was supported by the Spanish Ministerio de Ciencia y Tecnología (grant no. BIO1999-0503-C02-01 and "Ramon y Cajal" program) and by Generalitat de Catalunya (grant no. CIRIT 2001SGR-00109).
* Corresponding author; e-mail mrodrigu{at}sun.bq.ub.es; fax 34-93-402-1219.
www.plantphysiol.org/cgi/doi/10.1104/pp.007138.
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