|
Plant Physiol. (1999) 119: 507-510
Arabidopsis ent-Kaurene Oxidase Catalyzes Three Steps
of Gibberellin Biosynthesis
Chris A. Helliwell*,
Andrew Poole,
W. James Peacock, and
Elizabeth
S. Dennis
Commonwealth Scientific and Industrial Research Organization, Plant
Industry, G.P.O. Box 1600, Canberra, ACT 2601, Australia
 |
ABSTRACT |
The Arabidopsis GA3
cDNA was expressed in yeast (Saccharomyces cerevisiae)
and the ability of the transformed yeast cells to metabolize
ent-kaurene was tested. We show by full-scan gas chromatography-mass spectrometry that the transformed cells produce ent-kaurenoic acid, and demonstrate that the single
enzyme GA3 (ent-kaurene oxidase) catalyzes the three
steps of gibberellin biosynthesis from ent-kaurene to
ent-kaurenoic acid.
| |
INTRODUCTION |
GAs are an important group of plant growth regulators with roles
in a number of plant growth and developmental processes (Hooley, 1994 ).
Considerable progress has been made in isolating and characterizing the
genes encoding enzymes of GA biosynthesis (Hedden and Kamiya, 1997), in
particular the enzymes that synthesize ent-kaurene and the
dioxygenases that catalyze the late steps of GA biosynthesis. The
intermediate steps that oxidize ent-kaurene to
GA12 are catalyzed by a number of Cyt P450
monooxygenases. Two Cyt P450 genes implicated in GA biosynthesis have
been isolated. The maize Dwarf3 gene encodes a member of the
CYP88 family of Cyt P450 functions (Winkler and Helentjaris, 1995), but
although the dwarf3 mutant responds to GA, the point of the
lesion in GA biosynthesis is unknown. The Arabidopsis GA3
gene encodes a Cyt P450 protein, which is a member of the CYP701 family
(Helliwell et al., 1998). The ga3 mutant accumulates
ent-kaurene and shows a growth response to
ent-kaurenoic acid but not ent-kaurene, and only
a slight response to ent-kaurenol (Helliwell et al., 1998).
These data are consistent with GA3-encoding ent-kaurene oxidase, which has been proposed to catalyze the
three-step oxidation of ent-kaurene to
ent-kaurenoic acid (Fig. 1). A
direct demonstration of this activity has not been made.
The pea lh-2 mutant is also blocked in
ent-kaurene oxidation. Using extracts from embryos, Swain et
al. (1997) demonstrated that the mutant was not able to oxidize the
radiolabeled intermediates ent-kaurene,
ent-kaurenol, or ent-kaurenal, but was able to
oxidize ent-kaurenoic acid. Extracts from wild-type plants
could metabolize all four substrates. These data suggest that a single
enzyme catalyzes these three reactions, although the mutation could be
in a gene encoding a regulatory protein affecting all three steps.
Other data also support the proposal that the oxidation of
ent-kaurene to ent-kaurenoic acid is catalyzed by
a single enzyme. Coolbaugh et al. (1978) showed that in wild cucumber
(Marah macrocarpus) the inhibition of each of the
three steps from ent-kaurene to ent-kaurenoic
acid by ancymidol had the same kinetics, whereas inhibition of
oxidation of ent-kaurenoic acid to
ent-7 -hydroxykaurenoic acid was greater, suggesting
that it is catalyzed by a different enzyme.
In this paper we describe the expression of the Arabidopsis
GA3 cDNA in yeast (Saccharomyces cerevisiae) and
show that the GA3 protein does catalyze the three-step oxidation of
ent-kaurene to ent-kaurenoic acid.
| |
MATERIALS AND METHODS |
Expression in Yeast
The entire GA3 cDNA was amplified by PCR and inserted
between the glyceraldehyde 3-phosphate dehydrogenase promoter and
glyceraldehyde 3-phosphate dehydrogenase terminator in the pYE22
multiple cloning site vector, a modification of the pYE2211 vector
(Ashikari et al., 1989) in which a polylinker containing
EcoRI, SalI, KpnI, NotI,
and BamHI restriction sites was inserted in place of the glyceraldehyde 3-phosphate dehydrogenase open reading frame. Plasmids were prepared from single colonies after the transformation of Escherichia coli and the cDNA insert was sequenced to
confirm that the fragment would encode the precise protein encoded by the GA3 cDNA. The sequenced plasmid was then used to
transform the yeast (Saccharomyces cerevisiae) strain G1315
by a lithium chloride method (Cullin and Pompon, 1988). The
transformation mixture was plated on a minimal medium consisting of
0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) Glc and
2% (w/v) agar to select for transformants. Yeast RNA was extracted by
the hot acidic-phenol method (Ausubel et al., 1993). Yeast microsomes were prepared using an enzymatic digestion method (Pompon et al., 1996).
Enzyme Assays
Single colonies of transformed yeast and untransformed controls
were used to inoculate 50-mL cultures in a yeast peptone dextrose medium containing 1% (w/v) Bacto yeast extract, 2% (w/v) Bacto (Difco, Detroit, MI) peptone, and 2% (w/v) Glc. After growing overnight, 0.5 mL of each culture was removed; the yeast was pelleted and resuspended in 0.5 mL of a reaction mixture containing 100 mM Tris-HCl pH 7.5, 0.5 mM NADPH, and 0.5 mM FAD, according to the method of Hazebroek et al. (1993).
The substrates added were 25 µg of ent-kaurene, 5 µg of
ent-kaurenol, or 20 µg of
[17,17-2H2]-ent-kaurenal.
The substrates were dissolved in 100% methanol before they were added
to the reaction mixture; the final methanol concentration in the
reaction mixture was 5%. The reactions were incubated for 1 h at
30°C, with shaking at 150 rpm. At the end of the incubation the
reaction mixture was extracted, once with 0.5 mL of hexane and twice
with 0.5 mL of ethyl acetate. The organic fractions were then pooled
and dried using a Speed-Vac (Savant Instruments, Farmingdale, NY)
before derivatization for GC-MS. Assays with yeast microsomal fractions
were carried out as described above with 100 µg of microsomal protein
replacing the yeast cells.
Analysis of ent-Kaurene Metabolites by GC-MS
For analysis by GC-MS some metabolites of ent-kaurene
require methylation or trimethylsilylation. Dried samples were
dissolved in 50 µL of methanol and methylated with excess
diazomethane (about 200 µL), after which the samples were redried.
Trimethylsilylation was carried out by the addition of 5 µL each of
pyridine and N,O-bis(TMS)trifluoroacetamide plus 1%
trimethylchlorosilane (Alltech Associates, Deerfield, IL).
Samples were injected onto a BPX-5 column (25-m × 0.22-mm i.d.
[SGE, Austin, TX]) with a 0.25-µm-thick 5% phenyl (equivalent) polysilphenylene-siloxane stationary phase and analyzed in a full-scan mode. A second injection of the samples was made onto a HP-1 column (25-m × 0.2-mm i.d., [Hewlett-Packard]) with a 0.33-µm
dimethyl polysiloxane stationary phase. Both columns were nonpolar. The HP-1 column achieved better separation of the ent-kaurenoic
acid and the ent-kaurenol in particular and also further
confirmed the identities of all metabolites. GC conditions were as
described by Green et al. (1997). We co-injected all of the sample (1 µL) with a series of hydrocarbons derived from Parafilm (Gaskin et al., 1971) for KRI determination, and used authentic standards (from
L.N. Mander, Australian National University, Canberra, and J.D.
Metzger, Ohio State University, Columbus) for a comparison. We also
compared the full scans with a PC-based spectral library (Gaskin and
MacMillan, 1991).
| |
RESULTS |
Yeast colonies were picked after a transformation with the
GA3 cDNA construct. We then performed RNA gel-blot analysis
to identify the transformed yeast cell line with the highest
expression of the GA3 mRNA (Fig.
2) and used this cell line for the
subsequent analysis of GA3 enzyme activity.
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| Figure 2.
RNA gel blot of total RNA from nontransformed
yeast and yeast transformed with the GA3 expression
construct probed with a GA3-specific probe. WT, Wild
type.
|
|
Yeast Expressing GA3 Are Able to Metabolize ent-Kaurene
to ent-Kaurenoic Acid
Yeast cells expressing the GA3 mRNA and control cells
of untransformed yeast were incubated with ent-kaurene.
Immediately after the incubation the cells were extracted and prepared
for GC-MS. Authentic ent-kaurene, ent-kaurenol,
ent-kaurenal, and ent-kaurenoic acid, derivatized
where appropriate, were also injected to generate KRIs and ion spectra
for these compounds.
Ion current peaks in the extracts from GA3-expressing and
nontransformed yeast were initially compared with a library of spectra. ent-Kaurene could be identified in the extracts from both
the nontransformed and the GA3-expressing yeast. In the
extracts from GA3-expressing yeast, peaks were present that
were putatively assigned as ent-kaurene,
ent-kaurenol, ent-kaurenal, and
ent-kaurenoic acid (Fig. 3).
None of these peaks was present in the extracts from nontransformed
yeast. In experiments in which the yeast cells expressed a different
P450 cDNA, pumpkin CYP88A2 (C.A. Helliwell and E.J. Dennis, unpublished
results), only ent-kaurene could be detected. KRIs were
calculated for the putatively identified peaks and compared with those
of authentic standards; this calculation was carried out using data
from both the BPX-5 and HP-1 columns (Table
I). We found that the KRIs for the
putative peaks did not differ significantly from those of the authentic
compounds. Confirmation of the identity of the putative peaks was
provided by a comparison of the relative abundances of characteristic
ions of the putative and reference compounds (Table I). The HP-1 column gave the best separation of the compounds, particularly of
ent-kaurenol and ent-kaurenoic acid.
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| Figure 3.
Relative abundances of the base peak ions (as in
Table I) of ent-kaurenal (286),
ent-kaurenoic acid (316), and
ent-kaurenol (270) between 15 and 18 min of retention
time on the HP-1 column for extracts of GA3-expressing
(A) and nontransformed (B) yeast after feeding of
ent-kaurene.
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|
Table I.
Identification of metabolites after feeding
ent-kaurene to yeast transformed with the Arabidopsis GA3 clone
|
|
Extending the length of incubation time increased the abundance of the
ent-kaurenoic acid ions for the GA3-expressing
yeast (data not shown). ent-Kaurenoic acid accumulation was
approximately linear over a 2-h incubation. The abundances of the
intermediates ent-kaurenol and ent-kaurenal were
approximately 5-fold lower than ent-kaurenoic acid and did
not vary greatly over the 2-h incubation, presumably because these
intermediates were metabolized to ent-kaurenoic acid.
In experiments in which microsomes (approximately 0.2 mg of protein)
prepared from GA3-expressing and nontransformed yeast were
assayed, no metabolism of ent-kaurene was observed in
preparations from nontransformed cells. In the incubations of the
microsomes from GA3-expressing cells,
ent-kaurenol was detected but not ent-kaurenal or
ent-kaurenoic acid.
Yeast Expressing GA3 Metabolize
ent-Kaurenol and ent-Kaurenal
To confirm that the GA3-expressing yeast cells
were catalyzing all three steps of the GA biosynthetic pathway from
ent-kaurene to ent-kaurenoic acid, both
GA3-expressing and nontransformed yeast cells were incubated
with ent-kaurenol or
[17,17-2H2]-ent-kaurenal
(Table II). Neither
ent-Kaurenoic acid nor
[17,17-2H2]-ent-kaurenoic
acid was detected in extracts from the nontransformed yeast incubated
with ent-kaurenol or
[17,17-2H2]-ent-kaurenal.
The extracts from the GA3-expressing yeast incubated with
ent-kaurenol or
[17,17-2H2]-ent-kaurenal
contained ent-kaurenoic acid or
[17,17-2H2]-ent-kaurenoic
acid, respectively.
[17,17-2H2]-ent-Kaurenal
was not detected in reactions where it was included as a substrate,
which could be due to nonenzymatic oxidation or metabolism by the yeast
cells to a product other than ent-kaurenoic acid.
View this table:
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|
Table II.
Summary of intermediates detected after a 1-h
incubation of yeast expressing the GA3 cDNA with ent-kaurene,
ent-kaurenol, and ent-kaurenal
|
|
| |
DISCUSSION |
We have expressed the Arabidopsis GA3 gene
CYP701A3 in yeast and shown that the transformed cells were able to
carry out the three-stage oxidation of ent-kaurene to
ent-kaurenoic acid. The cells were also able to catalyze the
oxidation of the intermediates ent-kaurenol and
ent-kaurenal to ent-kaurenoic acid. These data showed that all three oxidation steps were enzymatically catalyzed and
not due to spontaneous oxidation. The results confirmed previous evidence that GA3 encodes ent-kaurene oxidase.
These earlier studies were based on growth responses to fed
intermediates and measurements of ent-kaurene accumulation
in the ga3-1 mutant (Helliwell et al., 1998).
Our results demonstrated that yeast was a suitable system for analyzing
the function of this GA biosynthetic enzyme. It may now be possible to
determine which step of GA biosynthesis is catalyzed by the maize
Dwarf3 protein using this expression system. In the case of
ent-kaurene oxidase, whole-yeast cells were a better system
than the purified microsomal fraction of the cells. Whole cells
expressing GA3 consistently gave metabolism of
ent-kaurene to ent-kaurenoic acid, whereas the
microsomes carried out the single-step conversion to
ent-kaurenol only. The ability of the microsomes to
metabolize ent-kaurenol and ent-kaurenal was not tested. A possible explanation for the difference in activity between
whole cells and microsome preparations is that ent-kaurene oxidase was unstable in the microsome preparations. The
ent-kaurene oxidase from the fungus Gibberella
fujikuroi has been reported to lose activity rapidly in assays
using crude lysates (Ashman et al., 1990). Another possibility is that
in purification of the microsomal fraction, a cofactor essential for
the oxidation of ent-kaurenol was lost, but the oxidation of
ent-kaurene to ent-kaurenol could still proceed.
Our successful expression of the GA3 protein in a functional form in
yeast is the first direct demonstration, to our knowledge, of the
activity of a Cyt P450 enzyme of the GA biosynthesis pathway.
| |
FOOTNOTES |
*
Corresponding author; e-mail chrish{at}pican.pi.csiro.au; fax
61-2-6246-5000.
Received September 15, 1998;
accepted November 3, 1998.
| |
ABBREVIATIONS |
Abbreviations:
KRI, Kovat's retention index.
TMS, trimethylsilyl.
| |
ACKNOWLEDGMENTS |
We thank Ying Luo for technical assistance and Professors L.N.
Mander and J.D. Metzger for providing GA intermediates.
| |
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Top
Abstract
Introduction
