Plant Physiol. (1998) 118: 1411-1419
The First Step of Gibberellin Biosynthesis in Pumpkin Is
Catalyzed by at Least Two Copalyl Diphosphate Synthases Encoded by
Differentially Regulated Genes
Maria W. Smith*,
Shinjiro Yamaguchi1,
Tahar Ait-Ali2, and
Yuji Kamiya
Plant Hormone Function Team, Frontier Research Program, The
Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1,
Wako-shi, Saitama 351-0198, Japan
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ABSTRACT |
The
first step in gibberellin biosynthesis is catalyzed by copalyl
diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two
cDNAs, CmCPS1 and CmCPS2, that each
encode a CPS. Both recombinant fusion CmCPS proteins
were active in vitro. CPS are translocated into plastids and processed
by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the
first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS
transcripts were strictly regulated in an organ-specific and
developmental manner. Both transcripts were almost undetectable in
leaves and were abundant in petioles. CmCPS1 transcript
levels were high in young cotyledons and low in roots. In contrast,
CmCPS2 transcripts were undetectable in cotyledons but
present at significant levels in roots. In hypocotyls, apices, and
petioles, CmCPS1 transcript levels decreased with age
much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is
correlated with the early stages of organ development, whereas
CmCPS2 expression is correlated with subsequent growth.
In contrast, C. maxima ent-kaurene synthase transcripts
were detected in every organ at almost constant levels. Thus,
ent-kaurene biosynthesis may be regulated through
control of CPS expression.
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INTRODUCTION |
GAs are synthesized from a common cyclic precursor,
ent-kaurene (Graebe, 1987
). This precursor is produced in
two steps: (a) the cyclization of GGDP to CDP and (b) the cyclization
of CDP to ent-kaurene. In plants these reactions are
catalyzed by two distinct enzymes that have been purified separately
(Duncan and West, 1981; Saito et al., 1995). Formerly, the enzymes were
known as kaurene synthetase A and B (Duncan and
West, 1981). At present, they are named CPS and KS, respectively
(MacMillan, 1997).
Both CPS and KS are known to be localized in plastids (Railton et al.,
1984; Aach et al., 1995, 1997). Early experiments in which
[14C]GGDP and [3H]CDP
were simultaneously supplied as the substrates to partially purified
enzymes from Marah macrocarpus endosperm suggested
that CPS and KS might interact (Duncan and West, 1981). In these
experiments, CDP synthesized by CPS activity was utilized 13 times more
efficiently for ent-kaurene production than exogenously
supplied CDP. Thus, ent-kaurene biosynthesis was suggested
to be catalyzed by a one-to-one CPS/KS complex in which CDP could be
channeled from CPS to the KS catalytic site (Duncan and West, 1981). In
the fungus Phaeosphaeria sp. L487, ent-kaurene
biosynthesis is catalyzed by a single bifunctional CPS/KS enzyme
(Kawaide et al., 1997); thus, the presence of two activities in one
complex may be important for efficient ent-kaurene production.
ent-Kaurene and CDP are the first intermediates in GA
biosynthesis from GGDP, and in pea, GA levels are directly correlated with the rate of ent-kaurene production (Moore and
Coolbaugh, 1991). However, GGDP serves as a common precursor for
several large families of terpenoid natural products (Graebe, 1987;
Chappell, 1995; McGarvey and Croteau, 1995). Thus, the regulation of
GGDP conversion into ent-kaurene is important for the
partitioning of GGDP among different pathways and for the rate of GA
biosynthesis (Duncan and West, 1981; Moore and Coolbaugh, 1991).
Cloning of the CPS and KS genes from several
species provided a way to examine the regulation of
ent-kaurene biosynthesis by analyzing patterns of gene
expression (for review, see Sun and Kamiya, 1997). The first
KS gene was cloned from pumpkin (Cucurbita maxima
L.) immature seeds, which contain high ent-kaurene synthetic
activity (Yamaguchi et al., 1996). Thus, to compare expression patterns
and to analyze a possible interaction between CPS and KS, we undertook
the cloning of a pumpkin CPS gene.
Previously, studies of GA-deficient mutants resulted in the cloning of
CPS genes from Arabidopsis (GA1; Sun and Kamiya,
1994), maize (An1; Bensen et al., 1995b), and pea
(LS, Ait-Ali et al., 1997). However, analyses of mutants in
some species also suggested the presence of additional CPS
genes. In Arabidopsis, a mutant ga1-3 with a large deletion
in the locus encoding CPS is still able to produce low amounts of GAs
(Zeevaart and Talon, 1992). In a similar fashion, a maize deletion
mutant with a knock-out for the An1 gene encoding a putative
CPS was shown to accumulate ent-kaurene (Bensen et al.,
1995b). These data indicate that several CPS genes may be
expressed in a plant species.
In the present paper, for the first time to our knowledge, two
CPS genes from the same plant species, CmCPS1
and CmCPS2, were cloned and characterized. Functional
expression of the corresponding cDNAs demonstrated that both
CmCPS proteins had enzyme activity. No interaction between
the pumpkin CPS and KS proteins could be detected. The transcript
levels of the two CmCPS genes and the CmKS gene
were compared during seedling development and in adult plants. Our data
indicate that the CmCPS genes are strictly regulated in a
different organ-specific and developmental manner.
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MATERIALS AND METHODS |
Plant Material
Seeds of pumpkin (Cucurbita maxima L. cv Riesenmelone
gelb vernetzt) were obtained from van Waveren Pflanzenzucht (Rosdorf, Germany) courtesy of Professor Jan Graebe. Pumpkin seedlings were cultivated under continuous light (220 µmol
m
2 s1) at 25°C on
moist vermiculite. Adult (1-month-old) plants were grown under the same
conditions. Immature seeds were harvested from field-grown plants, as
described by Yamaguchi et al. (1996).
PCR Amplification of Pumpkin CPS cDNA Fragments
Degenerate primers were designed from the sequences SAYDTAWV (1F
forward primer), FNGGVPN (3R reverse primer), and KHFERNG (5R reverse
primer) (Ait-Ali et al., 1997). Double-stranded cDNA was synthesized
from poly(A+) RNA isolated from cotyledons of immature
pumpkin seeds, as described by Yamaguchi et al. (1996). PCR was carried
out as described by Ait-Ali et al. (1997). Two PCR fragments of 0.89 kb
(1F and 5R primers) and 0.64 kb (1F and 3R primers) were obtained and
subcloned into the pCRII vector, as described by the manufacturer (TA
Cloning, Invitrogen, San Diego, CA). We named the genes corresponding
to the 0.89- and 0.64-kb PCR fragments CmCPS1 and
CmCPS2, respectively.
Cloning of CmCPS1 cDNAs
Both 0.89- and 0.64-kb PCR fragments were used as the probes to
screen a cDNA library prepared from immature pumpkin seeds (Yamaguchi
et al., 1996). The probes were labeled using the ECL Direct Nucleic
Acid Labeling and Detection System (Amersham, Japan). Plaque lifts on
nylon membranes (Hybond-ECL, Amersham) were hybridized in the buffer
provided by the manufacturer at 42°C. Washing was repeated three
times at 42°C with a buffer containing 4 M urea, 0.5×
SSC, and 0.4% (w/v) SDS, and then three times at room temperature with
2× SSC buffer.
All positive clones that were selected by library screening carried the
CmCPS1 sequence. Only one of the clones had the full-length ORF; however, it also contained a putative unspliced intron. To obtain
a CmCPS1 cDNA for functional expression in Escherichia coli, the coding region was amplified by PCR using double-stranded cDNA prepared from hypocotyls and petioles of pumpkin seedlings with
the Gene Amplification RNA PCR Kit (Perkin-Elmer). Poly(A+)
RNA for cDNA synthesis was enriched using magnetic resin with oligo(dT)25 (Dynal, Oslo, Norway). For PCR,
end-specific primer linkers were used: forward,
5
-ATATAAGTCGACATGAAAGCTCTCTCTCTCTCTCGC-3 (the
SalI site upstream of the putative first ATG codon of the CmCPS1 ORF is underlined), and reverse,
5-ATATTAGCGGCCGCTTGACAATACAACATGGCTG-3 (the
NotI site is underlined). The amplified cDNAs were subcloned into both the expression vector pGEX-4T-3 (Pharmacia) and
the pBluescript SK(
) vector (Stratagene). The obtained constructs were used to transform E. coli strain JM109.
To determine which part of the ORF is dispensable for the CPS activity,
we subcloned several 5- and 3-truncated CmCPS1 cDNAs in
the pGEX-4T-3 (Pharmacia) for functional expression in E. coli. For cloning, 6 reverse (containing a NotI site)
and 15 forward (containing a SalI site) primers were
designed from the CmCPS1 ORF and used for PCR reactions with
the CmCPS1 cDNA as the template.
Cloning of CmCPS2 cDNAs
To isolate the full-length CmCPS2 cDNA,
CmCPS2, 5- and 3-RACE experiments were done with the
Marathon cDNA amplification kit (Clontech, Palo Alto, CA). We used cDNA
fractions prepared from male pumpkin flower buds. PCR reactions were
done with the specific primers designed from the sequence of the
0.64-kb fragment: forward, 5-GGGAATCCGGAGCGACTCCCCGGCG-3, and
reverse, 5-CGCCGGGGAGTCGCTCCGGATTCCCAGC-3. Twelve independently
obtained 5- and 3-RACE products were subcloned into the pCRII vector
(Invitrogen) and sequenced.
Next, cloning of CmCPS2 cDNAs containing the full-length ORF
was done from male flower bud cDNA by PCR with end-specific
primers: forward, 5-ATATATGAATTCCATGTCCTCCTCCTCCTCTCTCT-3
(the EcoRI site upstream of the putative first ATG codon of
the CmCPS2 ORF is underlined), and reverse,
5-ATAATTCTCGAGACAACATGGGTGTGTGGGTAGCTA-3 (the
XhoI site is underlined). The amplified cDNAs were
subcloned into the pGEX-4T-3 and the pCRII vectors. Also,
three 5-truncated CmCPS2 cDNAs were cloned into the
pGEX-4T-3 for functional assay. For cloning, three forward primers
containing an EcoRI site in frame with the CmCPS2
ORF were used in PCR reactions with the CmCPS2 cDNA as the
template.
DNA Sequencing
DNA sequencing was done with double-stranded DNA using a dye
primer cycle sequencing kit (Applied Biosystems) and an ABI373A DNA
Sequencer (Applied Biosystems). For sequencing of the CmCPS1 cDNA clones obtained from the library, a series of unidirectional deletions was constructed from both ends using the Exonuclease III mung
bean nuclease deletion kit (Stratagene).
Functional Analysis of the CPS Fusion Proteins
For each expression construct in pGEX-4T-3, at least five
independent clones were analyzed for in vitro activity. Recombinant fusion proteins were produced by E. coli cultures incubated
for 22 h at 20°C. Bacterial extracts in a CPS buffer (50 mM potassium phosphate, pH 8.0, 10% [w/v] glycerol, 2 mM DTT, and 5 mM MgCl2) were cleared by centrifugation at 12,000g for 20 min at
4°C. Rapid assays of CPS and KS activities were carried out as
described by Ait-Ali et al. (1997). Reactions were performed for 30 min at 30°C in 200 µL of the CPS buffer that contained either 1 kBq (75 GBq mmol1) of [3H]GGDP
(Amersham) or 1.5 kBq (74 GBq mmol1) of
[3H]CDP (a gift from Dr. T. Saito, Institute of
Physical and Chemical Research, Saitama, Japan).
The products of CPS enzymatic activity were identified by full-scan
GC-MS (GCQ, Finnigan MAT, San Jose, CA) as described by Kawaide
et al. (1995). For analysis of CPS activity, 5 µg of GGDP was
incubated with the bacterial extracts as described above. In the
absence of MBP-KS, CDP was hydrolyzed to copalol as described by Sun
and Kamiya (1994). Nonpolar products were purified from the reaction
mixtures as described by Kawaide et al. (1995). Spectra were compared
with that of the authentic ent-kaurene.
Immunodepletion and Immunoblotting
Five synthetic oligopeptides were designed from the
CmCPS1 sequence, as shown in Figure 1. A CmKS
oligopeptide was designed from the sequence EDDGYTSNRLMNTVK. The
oligopeptides were coupled to Imject maleimide-activated keyhole limpet
hemocyanin (Pierce). The anti-CPS1 and anti-KS polyclonal antibodies
were custom produced by Sawady Technology (Tokyo, Japan) in rabbits
against the mixture of the five CmCPS1 oligopeptides and the
CmKS oligopeptide, respectively.
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| Figure 1.
Sequence alignment of plant CPS. The deduced
CmCPS1 and CmCPS2 polypeptides are
compared with the deduced pea LS, Arabidopsis
GA1, and maize genes. Identical residues in two
or more sequences are shown in black boxes with white lettering;
biologically similar amino acids are shown by gray boxes. The terpene
cyclase motif "DXDDTA" is shown with plus signs, and the motif
"SAYDTAWVA" conserved among the CPS and KS enzymes is shown by
asterisks below the alignment. Sequences used to design the degenerate
primers for initial cloning are shown by arrows, and the sequences of
synthetic oligopeptides are shown by horizontal bars above the
alignment. The inverted triangle marks the limit of N-terminal
deletions in CmCPS1 and CmCPS2 that still
retain enzyme activity. Dashes indicate gaps introduced for
optimization of the alignment.
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Immunodepletion and immunoblotting were carried out as described by
Harlow and Lane (1988). The endosperm extracts were prepared from
immature pumpkin seeds, as described by Saito et al. (1995). Each
immunoprecipitation reaction was performed using 30 µL (80 µg of
total protein) of the endosperm extract in a total volume of 150 µL
of the CPS buffer containing 150 mM NaCl. Proteins were precipitated by addition of the whole anti-CPS1 antiserum (in the
control, this was replaced by the preimmune serum). Immune complexes
were collected using 50 µL of protein A-Sepharose beads (Pharmacia).
Protein A beads containing precipitated proteins were used for the
rapid assay of KS activity, as described above. The remaining
supernatants were concentrated 10 times using Molcut LGC (Nihon
Millipore, Yonezawa, Japan) and subjected to SDS-PAGE using 9% (w/v)
acrylamide gels. After the gel was blotted, the membranes (Hybond-ECL)
were incubated with a 5000-fold dilution of anti-CPS1 or anti-KS
antibodies. Detection was achieved using peroxidase-conjugated
anti-rabbit IgG (Promega) and enhanced chemiluminescence (ECL,
Amersham).
RNA Extraction and Gel-Blot Analysis
Total RNA was extracted using Trizol reagent (GIBCO-BRL) according
to the manufacturer's protocol. The CmCPS and
CmKS cDNAs containing the full-length ORF were used as the
templates for preparation of [32P]UTP-labeled
RNA probes using the Riboprobe Combination Systems kit (Promega) as
described by the manufacturer. RNA samples (40 µg per lane for total
RNA or 3 µg per lane of poly(A+) RNA) were subjected to
electrophoresis in 1% (w/v) agarose-2.2 M formaldehyde
gels. After the gel was blotted, the membranes (Hybond
N+) were hybridized for 16 h at 65°C in a
buffer containing 50% (v/v) formamide, 1× Denhardt's solution, 1%
(w/v) SDS, 5× SSPE, 0.1 M sodium phosphate (pH 7.0), and
0.2 mg/mL calf-liver RNA. The membranes were then washed in a 0.1× SSC
and 0.1% (w/v) SDS buffer at 68°C. For control of RNA loading,
membranes were rehybridized with an oligonucleotide probe complementary
to 18S rRNA (Gallo-Meagher et al., 1992). The signals were visualized
using a bio-imaging analyzer (BAS 2000, Fuji, Japan).
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RESULTS |
Cloning of Two Pumpkin CPS cDNAs
To clone pumpkin CPS sequences we used three degenerate PCR
primers, designed by Ait-Ali et al. (1997), that consisted of one
forward primer, 1F, and two reverse primers, 3R and 5R (Fig. 1). For reverse transcriptase-PCR, RNA
fractions were isolated from cotyledons of immature pumpkin seeds. PCR
reactions with each of the two different primer combinations generated
a single product: a 0.89-kb cDNA fragment with primers 1F and 5R and a 0.64-kb cDNA fragment with primers 1F and 3R. Sequencing showed that
the two PCR fragments were derived from different mRNA species; however, both fragments had high sequence homology with known CPS genes from other plants.
The two obtained PCR fragments were used as the probes for screening of
a pumpkin cDNA library prepared from cotyledons of immature seeds, as
described in Yamaguchi et al. (1996). All five cDNA clones obtained
from the library corresponded to the 1F/5R PCR fragment of 0.89 kb.
Among these cDNAs named CmCPS1, four were lacking the 5 end
and the full-length ORF of the fifth one was interrupted by a sequence
that may have been an unspliced intron (data not shown). The deduced
CmCPS1 sequence of 2.98 kb encodes an ORF of 823 amino acids
followed by a 506-bp 3-untranslated region.
To clone cDNAs corresponding to the 1F/3R PCR fragment of 0.64 kb, we
used 5- and 3-RACE with gene-specific primers designed from the
fragment sequence. RACE products of the appropriate sizes could be
amplified only from RNA fractions of male flower buds. Six independent
clones were analyzed for both the 3 and 5 ends of this sequence named
CmCPS2. CmCPS2 encodes an ORF of 828 amino acids
followed by a 156-bp 3-untranslated region.
Alignment of the putative CmCPS polypeptides with other
known CPS amino acid sequences is shown in Figure 1. CmCPS
and CmCPS2 have 78% identity with each other and show high
identity scores with the deduced CPS sequences of pea, Arabidopsis, and
maize (about 55%, 50%, and 42%, respectively). Both pumpkin
polypeptides carry the same conserved motifs as the other CPS,
including the aspartate-rich box "DXDDTA" that is present in
terpene cyclases catalyzing cyclization without removal of the
diphosphate group (Sun and Kamiya, 1994). The two CmCPS have
approximately 30% identity with the pumpkin KS (CmKS), and
all three sequences contain a motif, "SAYDTAWVA," that is conserved
among the other plant CPS and KS polypeptides (Yamaguchi et al.,
1998).
To show that the cloned CmCPS genes are present in the
pumpkin genome, genomic DNA blots were hybridized with the
CmCPS1 and CmCPS2 probes. Under high-stringency
conditions, each probe hybridized with one or two bands of
genomic DNA, and no cross-hybridization between probes could be
detected (data not shown). The observed patterns were consistent with
the restriction maps of the corresponding cDNAs. Thus, the pumpkin
genome contains at least two different CPS genes that
correspond to the cloned CmCPS1 and CmCPS2 cDNAs.
The CmCPS1 and CmCPS2 cDNAs Encode Proteins
with CPS Activity
Recombinant CmCPS proteins were produced by expression
in E. coli as GST-CPS fusions. The enzyme activities were
first measured in vitro by conversion of labeled GGDP into
ent-kaurene in the presence of the recombinant fusion of
maltose-binding protein with CmKS (MBP-KS) (Ait-Ali et al.,
1997). We used pumpkin endosperm extract as a positive control for the
enzyme assay (Table I). Both GST-CPS1 and
GST-CPS2 in the presence of MBP-KS converted labeled GGDP into a
nonpolar product (presumably, ent-kaurene). To confirm the
identity of the products, the n-hexane extracts from the
enzymatic reactions with the unlabeled GGDP were analyzed by combined
GC-MS, as described by Kawaide et al. (1997). As shown in Figure
2, the control extracts containing only
GST or GST fused to a truncated CPS1 (
102, 102 amino acids deleted
from the N terminus) did not metabolize GGDP, whereas the full-length
GST-CPS1 converted all GGDP into CDP. The mixture of GST-CPS1 and
MBP-KS converted all GGDP into ent-kaurene.
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Table I.
ent-Kaurene synthetic activity of E. coli extracts
containing GST-CPS fusion proteins of different sizes and the
full-length MBP-KS
Recombinant fusion proteins were incubated with 1 kBq (about 50,000 dpm) of [3H]GGDP for 30 min at 30°C. Values are
means ± SE from three assays shown. For each
expression construct, similar results were obtained with four to seven
independent clones.
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| Figure 2.
Mass chromatograms of products after incubation of
GGDP with recombinant fusion proteins. The indicated E. coli protein extracts (50 µg) were incubated with 5 µg of
GGDP. Fragment ion peak with a mass-to-charge ratio
(m/z) of 257 from each molecular ion (geranylgeraniol
[GGol] and copalol [Col], m/z 290;
ent-kaurene, m/z 272) was monitored.
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Functional expression in E. coli was used to analyze which
parts of the CmCPS sequences are dispensable for the enzyme
activity. Six C-terminally truncated GST-CPS1 fusion proteins were
prepared and all were inactive (the smallest examined deletion,
C-86, is shown in Table I). In contrast, N-terminal deletions within the first 99 residues (98 and 99) did not affect the activity of
GST-CPS1 (Table I). However, the deletion of the next residue I-100
(100) and further deletions (101) destroyed the enzyme activity.
Also, three N-terminal deletions of CmCPS2, 106, 107, and 108, were prepared that corresponded to 98, 99, and 100 of CmCPS1, respectively (Fig. 1). The first two deletions,
106 and 107, did not change the activity. As expected from the
homology between two CPS sequences, the third deletion of I-108
rendered the truncated GST-108-CPS2 inactive (Table I).
The N termini of the CPS proteins contain transit peptides cleaved in
the process of plastid import (Sun and Kamiya, 1997). The in vitro
translated CmCPS1 protein was imported by isolated pea
chloroplasts (data not shown) in a fashion similar to the Arabidopsis
GA1 protein (Sun and Kamiya, 1994). After chloroplast uptake, the
processed CmCPS1 protein was smaller than the full-length protein CmCPS1. The size of the putative CmCPS1
transit peptide was estimated at 10 kD.
Immunodepletion of Pumpkin Protein Extracts with Antibodies against
CmCPS1
To study the possible complex formation between the CPS and KS
proteins, polyclonal antibodies were raised against five oligopeptides designed from the CmCPS1 sequence (Fig. 1). The anti-CPS1
antibodies recognized a single 82-kD polypeptide of the putative CPS
protein in the endosperm of immature pumpkin seeds but did not react
with proteins from vegetative tissues (data not shown). Thus, endosperm extracts were used for immunodepletion experiments with anti-CPS1 antibodies and protein A-Sepharose beads.
As shown in Figure 3, the 82-kD CPS band
remained in the supernatant after precipitation with the preimmune
serum, but was depleted from the extracts by excess amounts of
anti-CPS1 antibodies. However, the supernatants that were completely
immunodepleted of the CPS protein still contained a putative KS protein
of 81 kD recognized by anti-KS antibodies. We could not detect any KS activity in the immune complexes precipitated by anti-CPS1 antibodies (data not shown). Thus, no coprecipitation of CPS and KS proteins was
observed in the extracts from immature pumpkin seeds.
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| Figure 3.
Immunodepletion of the pumpkin CPS protein with
the anti-CPS1 antibodies. Endosperm extracts from immature pumpkin
seeds (80 µg of total protein per reaction) were immunoprecipitated
as follows: lane 1, by addition of 5 µL of the preimmune serum; lanes
2, 3, and 4, by addition of 1, 2, and 5 µL of the anti-CPS1
antiserum, respectively. Precipitated immune complexes were removed
using protein A beads. The resultant supernatants were concentrated and
subjected to SDS-PAGE and immunoblotting with either anti-CPS1 (left)
or with anti-KS (right) antibodies (Ab).
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The Levels of CmCPS1, CmCPS2, and CmKS
Transcripts in Developing Pumpkin Seedlings
Pumpkin seedlings were collected daily from 3 to 10 d after
imbibition and separated into roots (including root tips), hypocotyls, cotyledons, and apical parts containing meristems with small (less than
3 mm) leaf primordia and some remaining hypocotyl tissues. In addition,
petioles and first leaves were collected from 7- and 10-d seedlings.
Root tips were collected from 7-d seedlings.
Results of RNA gel-blot analyses are shown in Figure
4A. Although the nucleotide sequences of
CmCPS1 and CmCPS2 shared about 80% identity,
no cross-hybridization was observed between each probe and the
heterologous CmCPS mRNA produced by in vitro transcription (data not shown). The CmCPS1 probe hybridized with two RNA
species of about 2.7 and 1.8 kb. RNase A treatment of blots after
hybridization with the CmCPS1 probe did not remove any of
these signals. Also, both RNAs were detected on blots of
poly(A+) RNA (data not shown). Thus, the two RNAs are
likely to be mRNA species specifically recognized by the probe. Most
probably, the higher band represented the full-length CmCPS1
transcript. The identity of the lower 1.8-kb band was not clear. This
band was absent in some organs (e.g. cotyledons), and we speculate that it may be an alternatively spliced form or a specific RNA degradation product.
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| Figure 4.
A, Transcript levels of CmCPS1,
CmCPS2, and CmKS in growing pumpkin seedlings.
Seedlings were collected daily from 3 to 7 d after imbibition and
divided into roots, hypocotyls, cotyledons, and apical parts. Three
different blots of total RNA (40 µg/lane) were hybridized with the
antisense RNA probes prepared from the coding regions of CmCPS1,
CmCPS2, and CmKS cDNAs. As a loading control,
the same blots were rehybridized with the labeled oligonucleotide probe
recognizing 18S rRNA. Numbers on the top indicate days after
imbibition. P, Petiole; Rt, root tip. Leaves were divided by size into
the following three groups: S, small (less than 1.5 cm in length); M,
medium (1.5-3 cm); and L, large (more than 3 cm). Arrows and numbers
at the left indicate locations and sizes (in kb), respectively, of the
mRNA species hybridizing with the probes. B, The growth of pumpkin
seedlings from 3 to 7 d after imbibition. The length ( ) and
fresh weight ( ) of hypocotyls and cotyledons were measured for 15 seedlings daily.
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Both CmCPS1 and CmCPS2 transcripts were present
in hypocotyls, petioles, and apices and undetectable in leaves. In
cotyledons CmCPS1 transcript levels were very high at 3 and
4 d after imbibition. In contrast, CmCPS2 transcripts
were almost undetectable in cotyledons of any age. In roots
CmCPS1 transcript levels were very low, whereas CmCPS2 transcripts were observed at higher levels.
As shown in Figure 4B, for the whole period from 3 to 7 d, the
hypocotyls and cotyledons underwent very rapid growth both in length
and fresh weight. However, CmCPS1 transcript levels decreased below detection in 5-d cotyledons and decreased drastically in 4-d hypocotyls (Fig. 4A). Thus, in seedlings older than 4 d after imbibition, CmCPS1 expression did not correlate with
organ growth. In contrast, CmCPS2 transcript levels in
growing hypocotyls and roots did not change significantly from 3 to
7 d. Similar results were observed for the apical parts. Although
the apices were still actively producing new leaves at 10 d after
imbibition, CmCPS1 transcript levels decreased drastically,
whereas CmCPS2 transcript levels decreased only slightly.
In contrast to the expression patterns observed for the
CmCPS genes, CmKS transcripts were detected in
all organs, and the transcript levels changed only slightly with time.
The Levels of CmCPS1, CmCPS2, and CmKS
Transcripts in Different Organs of Adult Pumpkin Plants
To analyze the expression levels in adult organs, we collected
1-month pumpkin plants that had eight fully expanded true leaves. As
shown in Figure 5A, plants were divided
into four regions each containing leaves, petioles, and stems from two
internodes. Regions were numbered from the first formed (lowest) to the
last formed. At this age, first true leaves started to turn yellow and
were collected as senescing material. The uppermost parts of plants, including shoot apices, youngest small leaves, and petioles, were pooled together. In addition, tendrils and male flower buds were collected from the same plants. Cotyledons of immature pumpkin seeds
were obtained from a different study (Yamaguchi et al., 1996).
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| Figure 5.
A, Scheme showing the division of adult (1-month)
pumpkin plants into four regions. Thick vertical bars show the region
borders and numbers correspond to the region number. Ap, Apical part;
Sc, senescing leaf. B, Transcript levels of CmCPS1,
CmCPS2, and CmKS in 1-month pumpkin plants.
Three different blots of total RNA (40 µg/lane) were hybridized with
the antisense RNA probes prepared from the coding regions of
CmCPS1, CmCPS2, and CmKS cDNAs. As
loading controls, the same blots were rehybridized with the labeled
oligonucleotide probe recognizing 18S rRNA. Numbers on the top indicate
region numbers. T, Tendrils; Fb, male flower buds; Is, cotyledons of
immature seeds. Arrows and numbers at the left indicate locations and
sizes (in kb), respectively, of the mRNA species hybridizing with the
probes.
|
|
Results of RNA gel-blot analyses are shown in Figure 5B. Both
CmCPS transcripts were observed in young apical parts,
petioles, male flower buds, and cotyledons of immature seeds. The
relative amounts of CmCPS1 transcripts in immature seeds
were extremely high (much higher than in the other tissues). In
contrast, the relative amounts of CmCPS2 transcripts
were higher in male flower buds than in immature seeds.
In leaves and tendrils, both CmCPS transcripts were almost
undetectable by northern-blot analysis. However, CmCPS1
transcripts could be detected in young leaves by reverse
transcriptase-PCR (data not shown). In stems high CmCPS1
transcript levels were observed mainly in the younger organs (third and
fourth regions), whereas CmCPS2 transcript levels were
generally very low.
In stems and petioles CmCPS1 transcript levels were much
higher in the younger organs than in the older ones. In contrast, CmCPS2 transcript levels in petioles did not change as much
as those of CmCPS1, were high in the second region, and were
still detectable in the first region.
In contrast to the organ-specific expression of the CmCPS
genes, CmKS transcripts were observed in all of the organs.
CmKS expression levels did not change much from younger to
older organs and could be detected even in senescing leaves.
| |
DISCUSSION |
Two Genes Encoding Functional CPS Enzymes Are Present in Pumpkin
Previous reports indicated that several plant species
taxonomically distant from pumpkin may have more than one CPS
catalyzing the first step of ent-kaurene biosynthesis (Sun
and Kamiya, 1997). In maize a partial cDNA fragment for a putative
second CPS (An2) has been cloned (Bensen et al.,
1995a), but, to our knowledge, the corresponding protein has not yet
been tested for the CPS activity. We have cloned from pumpkin two
different cDNAs encoding enzymatically active CPS proteins. The deduced
CmCPS polypeptides share 78% identity with each other and
about 40% to 50% identity with the other known plant CPS
proteins.
No Complex Formation Could Be Detected between the CPS and KS
Enzymes
Endosperm extracts of Marah macrocarpus seeds were used
for studies that suggested a possible interaction and substrate
channeling between the CPS and KS enzymes (Duncan and West, 1981).
However, the same authors reported that no biochemical evidence could
be found for a possible heterodimer or heterooligomer of the native CPS
and KS partially purified from seed endosperm extracts.
Our data indicate that CmCPS1 may be the main CPS species
present in pumpkin seeds, since CmCPS1 cDNA was highly
represented in the library prepared from immature seeds and the
CmCPS1 transcript levels in this material were extremely
high (Fig. 5). Moreover, antibodies against CmCPS1 protein
recognized in seed extracts a putative CPS protein (Fig. 3). A putative
KS was also detected in this material. However, immunodepletion and
immunoprecipitation experiments using the anti-CPS1 antibodies failed
to detect a coprecipitation of pumpkin CPS and KS (Fig. 3). Addition of
the substrate (GGDP) did not facilitate interaction (M.W. Smith,
unpublished results). Also, no complex formation could be detected in
vitro between the GST-CPS1 and MBP-KS recombinant fusion proteins or by
the in vivo yeast two-hybrid assay (M.W. Smith, unpublished results).
We conclude that if the pumpkin CPS and KS do interact, they do not
form a stable complex.
Putative Transit Peptides of the CmCPS Proteins
ent-Kaurene biosynthesis is known to occur in plastids;
for pea and wheat, it is shown to be located in the proplastid stroma of rapidly dividing vegetative tissues (Aach et al., 1995, 1997). Both
the Arabidopsis GA1 protein (Sun and Kamiya, 1994) and the CmCPS1 protein (M.W. Smith, unpublished results) were
imported by isolated pea chloroplasts. During import the 10-kD transit peptides were cleaved from the CPS N termini. Functional analyses of
N-truncated CmCPS1 and CmCPS2 proteins showed
that the sizes of transit peptides cannot exceed the first 99 and 107 amino acids, respectively (Table I). The region of high sequence
homology among CPS proteins, which begins at position 100 of
CmCPS1 (108 of CmCPS2, Fig. 1), is indispensable
for the CPS activity of both pumpkin proteins. Our data indicate that
the calculated size of the 99-residue polypeptide, which could be
truncated from the N terminus of CmCPS1 without activity
loss, is close to the size of the CmCPS1 transit peptide
estimated by chloroplast import experiments. However, further
experiments are required to determine the precise N termini of
mature CPS proteins.
The Two CmCPS Genes Are Differentially Regulated in an
Organ-Specific and Developmental Manner
Previously, developmental control of CPS gene
expression was shown in pea, in which the LS transcript
levels were regulated during seed development (Ait-Ali et al., 1997).
In Arabidopsis the GA1 gene expression patterns were
analyzed using promoter-reporter fusions, and the promoter activity was
shown to be restricted to specific cell and tissue types (Silverstone
et al., 1997). In contrast, CmKS expression in pumpkin
seedlings was not organ specific (Yamaguchi et al., 1996).
The expression patterns of the CmCPS and CmKS
genes were analyzed in pumpkin plants using gel-blot analyses of total
RNA. Both CmCPS genes showed organ-specific expression
patterns. High levels of the two CmCPS transcripts were
observed in male flower buds and cotyledons of immature seeds (Fig.
5B). Our data are consistent with the results of Silverstone et al.
(1997) showing that GA1 promoter activity is high in
Arabidopsis inflorescence meristem, anthers, and immature seeds. It is
interesting that CmCPS1 transcript levels in cotyledons of
immature seeds were much higher than in any other pumpkin organ (Fig.
5B). Also, very high CmCPS1 transcript levels were observed
in cotyledons early in the development of pumpkin seedlings (Fig. 4A).
It is unclear whether the CmCPS1 gene is specifically
expressed in young cotyledons or whether the CmCPS1
transcripts remain from the extremely high levels found in seeds.
Among vegetative organs the levels of the two CmCPS
transcripts were high in petioles but undetectable in all types of leaf material by RNA gel-blot analysis (Figs. 4A and 5B). However, GA1 promoter activity was detected in Arabidopsis leaf
vascular tissues (Silverstone et al., 1997). We could detect
CmCPS1 transcripts in young pumpkin leaves by reverse
transcriptase-PCR (S. Yamaguchi, unpublished data). Thus,
CmCPS transcript levels in pumpkin leaves may be extremely
low and/or the CPS expression may be restricted to certain
cell types. However, high levels of CmKS transcripts were
present in leaves (Figs. 4A and 5B). Our data are consistent with the
biochemical evidence showing that only KS, not combined CPS/KS,
activity is found in fractions of mature chloroplasts (Railton et
al., 1984). The experiments of Aach et al. (1995, 1997)
showed that combined CPS/KS activity is observed only in proplastids of
the growing plants. Leaves are known to produce large amounts of GGDP
for biosynthesis of carotenoids and phytol. We speculate that CPS
expression in leaf cells should be strictly regulated and kept at very
low levels to prevent the overproduction of ent-kaurene and
competition for the same substrate pool among the terpene-biosynthetic
pathways.
During vegetative organ development, the transcript levels of both
CmCPS genes decreased, albeit in a different manner. In the
growing hypocotyls and apices of seedlings, CmCPS1
transcript levels decreased with age much faster than those of
CmCPS2. Measurements showed that all seedling organs
underwent very rapid growth for the whole period from 3 to 7 d
(Fig. 4B). Thus, CmCPS1 gene expression was related to early
organ development. We speculate that in pumpkin seedlings
CmCPS1 expression may be high in dividing cells, which is
similar to what was found in wheat shoots, where combined CPS/KS activity was predominantly in the meristematic tissues (Aach et al.,
1997). However, even in meristematically active pumpkin shoot apices,
CmCPS1 transcript levels rapidly decreased with age. In contrast, CmCPS2 transcripts were observed for a long time
in apices and stayed at almost constant levels in growing roots and hypocotyls (Fig. 4A). Thus, CmCPS2 expression seemed to be
related more to the growth than to the age of organs.
Although the CmCPS1 gene seems to encode a CPS enzyme highly
expressed in seeds and young organs, CmCPS1 gene
expression cannot be attributed to the early stages of plant
ontogenesis, because significant levels of the transcripts were present
in the young organs of adult plants. The different expression patterns
of the two CmCPS genes suggest that they may play
complementary roles in providing GA precursors required for plant
growth.
The presence of at least two CPS genes may be a general
feature of higher plants. Our data show that in pumpkin both
CmCPS genes are much more strictly regulated than the
CmKS gene. Thus, the first cyclization reaction catalyzed by
CPS enzymes may be the key regulatory step in ent-kaurene
biosynthesis.
| |
FOOTNOTES |
1
Present address: Developmental, Cell and
Molecular Biology Group, Department of Botany, Box 91000, Duke
University, Durham, NC 27708-1000.
2
Present address: Department of Molecular
Genetics, John Innes Centre, Colney Lane, Norwich NR4 7UJ, UK.
*
Corresponding author; e-mail smimasha{at}postman.riken.go.jp; fax
81-048-462-4691.
Received May 21, 1998;
accepted September 12, 1998.
The accession numbers for the sequences reported in this article are
AF049905 (CmCPS1) and AF049906 (CmCPS2).
| |
ABBREVIATIONS |
Abbreviations:
CDP, copalyl diphosphate.
CPS, copalyl
diphosphate synthase(s).
GGDP, geranylgeranyl diphosphate.
GST, glutathione S-transferase.
KS, ent-kaurene synthase.
MBP, maltose-binding protein.
ORF, open reading frame.
RACE, rapid amplification of cDNA ends.
| |
ACKNOWLEDGMENTS |
We thank Dr. Hiroshi Kawaide for help with the GC-MS
identification of CDP and ent-kaurene, Drs. Mariken Rebers
and Barbara Brockman for valuable advice, and Ms. Yukiji Tachiyama for
technical assistance. We also gratefully acknowledge Drs. Tai-ping Sun, Gerard Bishop, Andy Phillips, and Alasdair Gordon for critical reading
and discussion of the manuscript.
| |
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Abstract
Introduction