(Received for publication, January 3, 1996)
From the
The enzyme farnesyl-diphosphate synthase (FPS; EC 2.5.1.1/EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This reaction is considered to be a rate-limiting step in isoprenoid biosynthesis. Southern blot analysis indicates that Arabidopsis thaliana contains at least 2 genes (FPS1 and FPS2) encoding FPS. The FPS1 and FPS2 genes have been cloned and characterized. The two genes have a very similar organization with regard to intron positions and exon sizes and share a high level of sequence similarity, not only in the coding region but also in the intronic sequences. Northern blot analysis showed that FPS1 and FPS2 have a different pattern of expression. FPS1 mRNA accumulates preferentially in roots and inflorescences, whereas FPS2 mRNA is predominantly expressed in inflorescences. The cDNA corresponding to the FPS1 gene was isolated by functional complementation of a mutant yeast strain deffective in FPS activity (Delourme, D., Lacroute, F., and Karst, F.(1994) Plant Mol. Biol. 26, 1867-1873). By using a reverse transcription-polymerase chain reaction strategy we have cloned the cDNA corresponding to the FPS2 gene. Analysis of the FPS2 cDNA sequence revealed an open reading frame encoding a protein of 342 amino acid residues with a predicted molecular mass of 39,825 Da. FPS1 and FPS2 isoforms share an overall amino acid identity of 90.6%. Arabidopsis FPS2 was able to rescue the lethal phenotype of an ERG20-disrupted yeast strain. We demonstrate that FPS2 catalyzes the two successive condensations of IPP with both DMAPP and geranyl diphosphate leading to FPP. The significance of the occurrence of different FPS isoforms in plants is discussed in the context of the complex organization of the plant isoprenoid pathway.
Higher plants synthesize a great variety of isoprenoid products
that are required not only for their normal growth and development, but
also for their adaptative responses to environmental
challenges(1) . Plant isoprenoid biosynthesis involves a
complex multibranched pathway. The ramifications leading to the
specific isoprenoid products emerge from a central pathway in which
acetyl-CoA is converted, via mevalonic acid and isopentenyl diphosphate
(IPP), ()to a series of prenyl diphosphates of increasing
size. These polyprenyl diphosphates serve as donors or intermediates in
the synthesis of the wide range of isoprenoid end
products(1, 2) . It is generally accepted that this
metabolic pathway must be stringently regulated to maintain the
appropriate cellular balance of isoprenoids under changing
physiological conditions. In spite of this, the major rate-limiting
steps in the pathway have not yet been clearly identified. It is likely
that the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase, which
catalyzes the synthesis of mevalonic acid, plays a relevant role in the
overall control of the isoprenoid biosynthetic
pathway(3, 4, 5, 6, 7) .
However, there is also general agreement that additional key enzymes
are involved in the control of the pathway to ensure the synthesis of
the necessary isoprenoid compounds required for many different purposes
in different parts of the plant at different stages of growth and
development(1) .
Farnesyl-diphosphate (FPP) synthase (FPS; EC 2.5.1.1./EC 2.5.1.10) catalyzes the sequential 1`-4 condensation of two molecules of IPP with both dimethylallyl diphosphate (DMAPP) and the resultant 10-carbon compound geranyl diphosphate (GPP), to produce the 15-carbon compound FPP(8) . In plants, FPP serves as a substrate for the first committed reactions of several branched pathways leading to the synthesis of compounds that are required for growth and development, such as phytosterols (membrane structure and function), dolichols (glycoprotein synthesis), ubiquinones, and heme a (electron transport), abscisic acid (growth regulator), or sesquiterpenoid phytoalexins (defense against pathogen attack). FPP is also a prenyl donor in protein prenylation, a mechanism that promotes membrane interactions and biological activities of a variety of cellular proteins involved in signal transduction, membrane biogenesis, and cell growth control(9, 10) . Therefore, changes in FPS activity could alter the flux of isoprenoid compounds down the various branches of the pathway and, hence, play a central role in the regulation of a number of essential functions in plant cells. The role of FPS in the control of the plant isoprenoid pathway is further supported by the observation that in mammals FPS is a regulated enzyme known to have an important role in the overall control of the sterol biosynthetic pathway(11, 12, 13, 14) .
Plant
FPS has been purified and characterized from different species (1, 15, 16) and, recently, cDNA sequences
encoding this enzyme have been cloned from Arabidopsis thaliana(17) and Lupinus albus(18, 19) .
Comparison of the amino acid sequences of FPS from a variety of
organisms, ranging from bacteria to higher eukaryotes, has shown that
all the FPS known so far contain five distinct regions with high
similarity at the amino acid level(19, 20) . These
regions are also conserved in other prenyltransferases, including
geranylgeranyl-(C), hexaprenyl-(C
), and
heptaprenyl-(C
) diphosphate
synthases(20, 21) . Two of these regions are the
aspartate-rich domains that have been shown to play a role in the
catalytic reactions of the enzyme, most likely acting as binding sites
for the metal ion-complexed pyrophosphate moieties of IPP and the
allylic substrates(22, 23) .
As a first step toward a better understanding of the role of FPS in the biosynthesis of isoprenoids in plants, we have undertaken the characterization of the genes encoding Arabidopsis FPS. In this paper we report the isolation and characterization of the Arabidopsis FPS1 and FPS2 genes. The FPS1 gene encodes the FPS isoform previously described(17) . We have also isolated the cDNA corresponding to the FPS2 gene and shown that it encodes a functional FPS.
Yeast
strains were grown in YPD medium (1% (w/v) yeast extract, 2% (w/v)
bactopeptone, and 2% (w/v) glucose) or minimal medium (0.16% (w/v)
yeast nitrogen base without amino acids and
(NH)
SO
, 0.5% (w/v)
(NH
)
SO
, and 1% (w/v) glucose).
Unless otherwise stated, yeast cells were grown at 28 °C either in
liquid culture or on agar plates (media supplemented with 15 g of agar
per liter). When required to supplement auxotrophies, uracil (50
µg/ml), tryptophan (50 µg/ml), or ergosterol (4 µg/ml in
liquid culture or 80 µg/ml in agar plates) were added to the growth
media. Ergosterol was supplied by dilution of a stock solution (4
mg/ml) in a mixture of Tergitol Nonidet P-40, ethanol (1:1). E.
coli cells were grown in LB medium (1% (w/v) bactotryptone, 0.5%
(w/v) yeast extract, and 5% (w/v) NaCl) with tetracycline (15
µg/ml) and with or without ampicillin (100 µg/ml).
Plasmid pNCFPS2 contains the FPS2 cDNA under the control of the strong yeast phosphoglycerate kinase gene (PGK) promoter. To construct pNCFPS2, a SacII-SacI fragment from plasmid pcNC2 (see below) was blunt ended with the Klenow fragment of deoxyribonuclease I and cloned into pDD62, cleaved with NotI, and blunt ended with the Klenow fragment of deoxyribonuclease I in the presence of deoxynucleotides. The transcription polarity of the insert was examined by restriction analysis. Plasmid pDD62 was derived from plasmid pFL61(27) , and contains the selectable marker TRP1 instead of URA3 in the BglII site. The yeast strains were transformed by the lithium acetate procedure(28) .
Total RNA from different tissues of Arabidopsis was
isolated (32) , and poly(A) RNA was obtained
by oligo(dT)-cellulose according to the manufacturer's
recommendations (Amresco). For Northern analysis, 30 µg of Arabidopsis total RNA from each sample was fractionated by
electrophoresis in 1% (w/v) agarose gels containing 2.2 M formaldehyde and blotted to Hybond-N nylon membranes (Amersham).
Hybridization with the indicated
P-labeled probes was for
18 h at 42 °C in 50% (v/v) formamide, 1 M NaCl, 50 mM sodium phosphate, pH 6.5, 7.5
Denhardt's, 1% SDS,
10% (w/v) dextran sulfate, and 500 µg/ml denatured salmon sperm
DNA. Filters were washed twice at room temperature in 2
SSC,
0.1% SDS and at 40 °C twice in 1
SSC, 0.1% SDS, once in 0.1
SSC, 0.1% SDS, and once in 0.1
SSC. To ascertain that
equivalent amounts of RNA were present in each lane, filters were
reprobed with a
P-labeled 900-bp BamHI-EcoRI fragment of the gene for the 25 S
cytoplasmic rRNA. The probe used was obtained from plasmid pTA250 which
contains a wheat rRNA gene repeating unit(33) .
Figure 4: Nucleotide sequence of the Arabidopsis FPS2 cDNA and amino acid alignment of Arabidopsis FPS1 and FPS2. Nucleotides are numbered (right) by assigning position +1 to the first base of the ATG codon. The 5`-end sequence obtained from the RACE clones is shown in italic. A putative polyadenylation signal is double underlined. Stop codons are denoted by an asterisk. Amino acid positions are indicated on the left. Identical residues are represented by dots. The five regions (I to V) that are present in many prenyltransferases are shaded and the amino acid residues within these regions that are present in all the FPS known so far are shown below. Intron positions are indicated by open triangles.
Figure 1: Southern blot analysis of Arabidopsis FPS genes. Genomic DNA from Arabidopsis (8 µg) was digested with the restriction enzymes indicated at the top, electrophoresed and transferred onto nitrocellulose membranes. Filters were hybridized with a 340-bp NotI-HindIII cDNA fragment from plasmid pDD71, which contains the cDNA encoding the Arabidopsis FPS1 isoform(17) , under conditions of high (A) and low stringency (C), or a 800-bp XhoI-HindIII fragment from the FPS2 gene, shown in Fig. 2, under conditions of high stringency (B). Numbers on the right indicate the mobility of DNA size standards.
Figure 2: Restriction and structural maps of Arabidopsis FPS1 and FPS2 genomic clones. A, restriction map of the genomic regions containing the FPS1 and FPS2 genes. FPS1 and FPS2 transcription units are represented by solid boxes. The cloned regions contained in recombinant plasmids are indicated below the restriction maps. The 800-bp XhoI-HindIII probe from pgNC102 used in genomic Southern blot analysis is indicated by a double arrowhead line. Restriction sites are as follows: B, BamHI; E, EcoRI; EV, EcoRV; H, HindIII. B, structural organization of the FPS1 and FPS2 genes. Exons are represented by boxes and are numbered from the 5`-end of the genes. Lines between boxes correspond to introns. Coding regions are represented by solid boxes.
To clone the Arabidopsis FPS genes, a
730-bp EcoRI-PstI cDNA fragment from clone pDD71 was
used to screen an Arabidopsis genomic library under low
stringency conditions. Eleven positive clones were isolated. These
clones were classified in two distinct groups since restriction
endonuclease mapping and Southern hybridization analyses showed that
they contained DNA inserts corresponding to two different genomic
regions. Clones gNC10 and
gNC24 were selected for further
characterization as representatives of each group. Two genomic
fragments from each clone hybridizing to the cDNA probe were subcloned.
Sequence analysis revealed that plasmids pgNC241 and pgNC242 (Fig. 2A) contained overlapping inserts including the
entire coding region of the FPS1 gene as well as 5`- and
3`-flanking regions. Plasmids pgNC101 and pgNC102 (Fig. 2A) contained overlapping fragments with a
sequence different although highly similar to that of the FPS1 gene, which corresponds to a second FPS gene (FPS2), as
was later verified.
Southern blot analysis of Arabidopsis genomic DNA, performed under high stringency conditions using as a probe a 800-bp XhoI-HindIII fragment from the FPS2 gene (Fig. 2), revealed a simple pattern of bands (Fig. 1B) which accounted for a subset of genomic fragments previously detected at low stringency by the FPS1 probe (Fig. 1C). It was concluded that these fragments derived from the FPS2 gene. Interestingly, the bands specifically detected by the FPS1 and FPS2 probes (Fig. 1, A and B) accounted for most of the bands identified by the FPS1 probe under low stringency conditions (Fig. 1C). However, one additional weakly hybridizing fragment was detected in each lane. Taken together, these results indicated that Arabidopsis contains two genes encoding FPS (FPS1 and FPS2) and a genomic sequence that might correspond to a gene encoding either an additional FPS isoform or a closely related prenyltransferase. The nucleotide sequences of the FPS1 and FPS2 genes (data not shown) have been deposited in the GenBank data base with accession numbers L46367 and L46350, respectively.
The alignment of the nucleotide sequence of the FPS1 gene with that of the FPS1 cDNA showed that the gene consists of 12 exons and 11 introns (Fig. 2B). Comparison of these two sequences revealed several single-base differences. Because of two of these changes, Ser-177 (TCC) and Thr-283 (ACC) in the predicted amino acid sequence of the FPS1 protein previously reported (17) are converted to Ala (GCC) and Pro (CCC), respectively, in the protein encoded by the FPS1 gene. These changes presumably represent DNA polymorphisms associated with the different Arabidopsis ecotypes used. The organization of exons and introns of the FPS2 gene was initially deduced by comparing its sequence with that of the FPS1 gene, and further confirmed after alignment with the sequence of the FPS2 cDNA (see below). The FPS2 gene consists of 11 exons and 10 introns. The two genes have a very similar structure, although it is worth noting that exon 4 in the FPS2 gene corresponds to exons 4 and 5 in the FPS1 gene (Fig. 2B). In both genes, introns are located at equivalent positions relative to the coding sequences. All exon-intron junctions follow the GT/AG rule(36) . The alignment of the sequences of the FPS1 and FPS2 genes revealed that they share a high level of similarity not only in the coding region (87% overall identity) but also in the intronic sequences (identity higher than 57%).
Figure 3: Northern blot analysis of Arabidopsis FPS1 and FPS2 mRNA. A, total RNA samples from different tissues of Arabidopsis (30 µg/lane) was electrophoresed in 1% agarose-formaldehyde gels and transferred onto nylon membranes. Filters were hybridized with the FPS1 and FPS2 gene-specific probes shown in B. Exposure times were 9 days for FPS1 and 21 days for FPS2. B, map of the 3`-region of the FPS1 and FPS2 genes. The last exon of each gene is represented by a box. The 3`-untranslated regions are represented by open boxes. Lines correspond to the genomic regions flanking the 3`-end of the genes. The FPS1 (370-bp BglII-HindIII fragment) and FPS2 (450-bp BglII-KpnI fragment) gene-specific probes are indicated by double arrowhead lines.
To check the size of the protein encoded by the FPS2 cDNA, the FPS2 transcript was synthesized in vitro from plasmid pcSPNC2 and translated in a wheat germ cell-free system. A single protein migrating with an apparent molecular mass of about 41 kDa was generated from FPS2 mRNA (data not shown). The apparent molecular mass of this protein is in good agreement with the predicted molecular mass of FPS2 (39,825 Da).
The Arabidopsis FPS1 and FPS2 isoforms are composed of 343 and 342 amino acid residues, respectively. The alignment of the amino acid sequence of FPS1 and FPS2 is shown in Fig. 4. The two proteins are highly conserved throughout their sequence, showing an overall amino acid identity of 90.6% and a similarity of 94.5%. Both enzymes contain the five conserved regions, designated I to V (Fig. 4), which appear to be common not only to all the FPS isoforms previously reported (19) but also to other prenyltransferases(20, 21) . Regions II and V correspond to the two aspartate-rich domains that have been shown to be involved in enzyme catalysis(22, 23) .
Figure 5:
Confirmation of the FPS activity of the Arabidopsis FPS2 isoform. A, functional
complementation of the mutant yeast strain CC25 with plasmid pNCFPS2.
Strain CC25 and strain CC25[pNCFPS2] were streaked onto YPD
plates or YPD plates supplemented with 80 µg/ml ergosterol and
incubated at 36 °C for 3 days. B, identification of the
FPS reaction products in CC25, CC25[pNCFPS2], and NC1
strains. Cell-free extracts from each strain were incubated in the
presence of [C]IPP and DMAPP. The reaction
products obtained were analyzed by TLC after enzymatic hydrolysis. The
radioactivity was detected only in the geraniol and farnesol fractions,
and was measured as described under ``Experimental
Procedures.'' The amount of GPP and FPP produced is expressed as
percentage with respect to the sum of counts in the geraniol and
farnesol fractions, which was considered as 100%. Results are the
average of three experiments. Variation between measurements was
between 5 and 12%.
Because the FPS
activity in CC25 strain is impaired in the condensation step of GPP
with IPP to produce FPP, it was not possible to ascertain whether FPS2
could actually catalyze the two sequential reactions involved in the
synthesis of FPP from IPP and DMAPP. To address this question, we
checked whether plasmid pNCFPS2 also complemented a disrupted FPS gene.
A haploid yeast strain bearing a disrupted FPS gene copy is not viable,
even in the presence of ergosterol(26) . Haploid strain NC1,
constructed as described under ``Experimental Procedures,''
having a disrupted copy of the yeast FPS and harboring plasmid pNCFPS2,
showed a wild type phenotype whatever the growth conditions tested.
When cell free extracts from strain NC1 were assayed for FPS activity
the major reaction product was FPP (Fig. 5B). Strain
NC1 also synthesized FPP when GPP was used instead of DMAPP as allylic
primer (data not shown), thus confirming the ability of FPS to use
either C or C
allylic primers. Taken together,
these results unequivocally demonstrate that the Arabidopsis FPS2 cDNA encodes a functional FPS isoform which is able to
catalyze the two successive condensations of IPP with both DMAPP and
GPP leading to FPP formation.
The multibranched isoprenoid biosynthetic pathway in plants represents one of the most complex metabolic pathways known(1, 2) . One of the most challenging aspects of plant isoprenoid biosynthesis is the identification of the enzymes that catalyze the rate-limiting steps in the pathway. It is widely assumed that 3-hydroxy-3-methylglutaryl-CoA reductase, the enzyme that synthetizes mevalonic acid, plays a relevant role in the overall control of plant isoprenoid biosynthesis(3, 4, 5, 6, 7) . However, it is also accepted that mevalonic acid synthesis is not the only limiting step in isoprenoid biosynthesis, and that additional key enzymes are involved in the control of the flux through the pathway to maintain the appropriate cellular balance of isoprenoids under different physiological conditions(1) . FPS is considered to play a relevant role in the control of plant isoprenoid biosynthesis, since FPP is the starting point of different branched pathways leading to the synthesis of key isoprenoid end products. As a first step to study the role of FPS in the control of plant isoprenoid biosynthesis, we have undertaken the molecular characterization of FPS in A. thaliana.
The results presented here demonstrate that Arabidopsis contains a small FPS gene family consisting of at least two genes (FPS1 and FPS2) that encode closely similar FPS isoforms. The Arabidopsis FPS1 and FPS2 genes have been cloned and characterized. The two genes have a very similar organization with regard to intron positions and exons sizes, and share a high level of sequence similarity not only in the coding region but also in the intronic sequences. These observations indicate that these two genes have arisen from a recent duplication of an ancestral FPS gene. In spite of this, FPS1 and FPS2 have a different pattern of expression. By using gene-specific probes we have shown that, although the two genes are expressed in all the tissues analyzed, FPS1 mRNA is present mainly in roots and inflorescences, whereas FPS2 mRNA is detected at a lower level and accumulates preferentially in inflorescences. It is worth noting that the 3`-untranslated region of the Arabidopsis FPS2 transcript contains one copy of the AUUUA motif (position +1068 in the FPS2 cDNA sequence). This sequence has been shown to act as an mRNA instability determinant (for review, see (39) ). However, it remains to be determined whether this motif actually participates in modulating the Arabidopsis FPS2 transcript levels.
It has
been previously shown that FPS1 is an active form of the enzyme (17) . At the protein level, Arabidopsis FPS1 and FPS2
are very similar (90.6% identity), with amino acid changes distributed
throughout their sequence (Fig. 4). This suggested that FPS2
might represent an active form of the enzyme. This was demonstrated by
the complementation of the mutant yeast strain CC25 with plasmid
pNCFPS2, which carries the Arabidopsis FPS2 cDNA under the
control of the yeast PGK promoter. Strain CC25 is auxotrophic
for ergosterol at 36 °C since it carries the leaky mutation erg20-2 in the FPS gene that impairs the C to C
elongation step. This results in a concomitant
accumulation of GPP which is dephosphorylated by endogenous
phosphatases and excreted to the growth medium as
geraniol(26) . Strain CC25 was initially chosen because it
allowed a rapid assay of the functionality of the FPS2. However, due to
the nature of the erg20-2 mutation, it remained
formally possible that the Arabidopsis FPS2 could catalyze the
synthesis of FPP from IPP and GPP, but not the preceding condensation
of IPP with DMAPP to form GPP. To rule out this possibility, we
generated the haploid strain NC1, which has a disrupted copy of the FPS
gene (erg20 mutation) and harbors plasmid pNCFPS2. It has been
shown that the disruption of the FPS gene is lethal for yeast even in
the presence of exogenously supplied ergosterol(26) . However,
strain NC1 showed a wild type phenotype, thus indicating that plasmid
pNCFPS2 encodes an enzyme which is able to catalyze the two successive
condensations of IPP with both DMAPP and GPP leading to FPP formation.
The presence of FPS activity was further confirmed by an in vitro assay using cell free extracts obtained from strain NC1.
In contrast to the controversy surrounding the subcellular location of the enzymes involved in the synthesis of IPP in plants, there is general agreement that the enzymes utilizing IPP are distributed in three subcellular compartments, namely cytosol, mitochondria, and plastids(1, 40) . The cytosol is the only cell compartment where plant FPS has been detected(1, 15, 40) . In animal cells, the major site of FPP synthesis is also the cytosol. However, it has recently been reported that in mammals FPS activity is also present in mitochondria (41) and peroxisomes(42) . This raises the question that in plants FPS might be present in cell compartments other than the cytosol. The alignment of the primary sequence of Arabidopsis FPS1 and FPS2 with that of the known FPS from other organisms (bacteria, fungi, plant, and animals) (19, 20) shows that the two Arabidopsis FPS isoforms lack amino-terminal extensions that could represent transit peptides to plastids and mitochondria. Furthermore, the N-terminal sequence of Arabidopsis FPS1 and FPS2 has no features of transit peptides for targeting into these organelles(43) . However, it cannot be ruled out that other forms of the enzyme, resulting from the use of alternative promoters or from alternative splicing processes, might be targeted to different subcellular locations. In addition, we cannot exclude that organellar forms of FPS could be encoded by additional genes not yet characterized.
One of the more intriguing findings arising out of the molecular biology studies of plant isoprenoid biosynthesis is the occurrence of gene families encoding key enzymes of this metabolic pathway. For example, the number of genes encoding 3-hydroxy-3-methylglutaryl-CoA reductase varies from the two genes described in Arabidopsis(44, 45) to at least 11 genes found in potato(5, 46) . At least five geranylgeranyl diphosphate synthase genes have been reported to occur in Arabidopsis(47) . It has been described that vetispiradiene synthase, a sesquiterpene cyclase found in Hyosciamus muticus, is encoded by a gene family of six to eight members(48) . Our results indicate that Arabidopsis also contains a small FPS gene family consisting of at least two genes. Although the complexity of the FPS gene family in plants has only been studied in Arabidopsis, it is tempting to speculate that FPS gene families with similar or even greater complexity may also be found in other plant species. The occurrence of FPS isozymes raises the question about the role of each individual FPS isoform in the isoprenoid biosynthetic pathway. The differential expression of FPS1 and FPS2 might be indicative of an specialized function of each FPS isoform in directing the flux of pathway intermediates into specific isoprenoid end products. This assumption is consistent with the recent hypothesis proposing that specific classes of isoprenoids are synthesized by discrete metabolic channels within the pathway, through the formation of multienzyme complexes (metabolons), which are independently regulated(49, 50) . The results presented in this paper lend further support to the view that plant isoprenoid biosynthesis is a complex metabolic pathway which is regulated by sophisticated control mechanisms. We are currently applying different molecular and cellular approaches to identify the specific function of each FPS isoform in the organization of the plant isoprenoid pathway.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L46349[GenBank], L46350[GenBank], and L46367[GenBank].