From the Institute of Biological Chemistry, Program in Plant Physiology, and Department of Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164-6340
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Grand fir (Abies grandis) has been
developed as a model system for the study of oleoresin production in
response to stem wounding and insect attack. The turpentine fraction of
the oleoresin was shown to contain at least 38 sesquiterpenes that
represent 12.5% of the turpentine, with the monoterpenes comprising
the remainder. Assays of cell-free extracts from grand fir stem with
farnesyl diphosphate as substrate indicated that the constitutive
sesquiterpene synthases produced the same sesquiterpenes found in the
oleoresin and that, in response to wounding, only two new products were synthesized, -cadinene and (E)-
-bisabolene. A
similarity based cloning strategy yielded two new cDNA species from
a stem cDNA library that, when expressed in Escherichia
coli and the gene products subsequently assayed, yielded a
remarkable number of sesquiterpene products. The encoded enzymes have
been named
-selinene synthase and
-humulene synthase based on the
principal products formed; however, each enzyme synthesizes three major
products and produces 34 and 52 total sesquiterpenes, respectively,
thereby accounting for many of the sesquiterpenes of the oleoresin. The deduced amino acid sequence of the
-selinene synthase cDNA open reading frame encodes a protein of 581 residues (at 67.6 kDa), whereas
that of the
-humulene synthase cDNA encodes a protein of 593 residues (at 67.9 kDa). The two amino acid sequences are 83% similar
and 65% identical to each other and range in similarity from 65 to
67% and in identity from 43 to 46% when compared with the known
sequences of monoterpene and diterpene synthases from grand fir.
Although the two sesquiterpene synthases from this gymnosperm do not
very closely resemble terpene synthases from angiosperm species
(52-56% similarity and 26-30% identity), there are clustered
regions of significant apparent homology between the enzymes of these
two plant classes. The multi-step, multi-product reactions catalyzed by
the sesquiterpene synthases from grand fir are among the most complex
of any terpenoid cyclase thus far described.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Conifer oleoresin is a mixture of turpentine (monoterpene (C10) and sesquiterpene (C15) olefins) and rosin (diterpene (C20) resin acids) that functions in insect defense and in wound sealing (1, 2). Grand fir (Abies grandis) has been developed as a model system for the study of both constitutive and wound-induced oleoresin formation (oleoresinosis). The composition of the monoterpene olefin and the diterpene resin acid fractions of grand fir oleoresin has been defined (3), and the induced biosynthesis of these natural products upon stem wounding has been described in detail (2, 4-6). The time course of induction of the monoterpene synthases involved in turpentine formation has been analyzed by immunoblotting techniques, and the process of induced oleoresinosis was thus shown to involve de novo synthesis of these enzymes (5). The cDNA sequence of a diterpene cyclase from grand fir (abietadiene synthase involved in resin acid biosynthesis (7)) has been reported (8), and several cDNA clones encoding monoterpene synthases from this conifer species have recently become available (9).
The sesquiterpenes of conifer turpentine have received relatively
little experimental attention because they constitute less than 10% of
the oleoresin. However, sesquiterpenoid phytoalexins are well known in
angiosperm species (10), suggesting that the sesquiterpenes of conifer
oleoresin may play a similar role in antibiosis and thus be of greater
significance than their lower concentration in resin might otherwise
indicate. Sesquiterpenes are produced in the cytosol/endoplasmic
reticulum compartment, whereas monoterpene and diterpene biosynthesis
are compartmentalized in plastids (11), which raises the additional
issue of coordinate regulation of oleoresin terpene biosynthesis at
several cellular sites. Only a single sesquiterpene synthase,
(E)--farnesene synthase, from a gymnosperm source,
maritime pine (Pinus pinaster), has been reported (12),
whereas several sesquiterpene synthases from angiosperms have been
described (13-15), and a number of genes encoding sesquiterpene
synthases involved in phytoalexin biosynthesis in angiosperms have been
isolated (16-18).
To examine the possible role of sesquiterpenes in conifer defense
against stem boring insects and their associated fungal pathogens, it
is first necessary to examine in greater detail the origin of these
oleoresin constituents. In this paper, we describe the sesquiterpene
composition of grand fir oleoresin, and the cell-free biosynthesis of
these terpenoids from the common isoprenoid intermediate farnesyl
diphosphate in extracts from wounded (induced) and nonwounded control
(constitutive) sapling stems. In addition, we report on the use of a
general cloning strategy (9, 19) in the isolation and functional
expression of two cDNA species encoding sesquiterpene synthases
from this gymnosperm. These multiple product enzymes, termed
-selinene synthase and
-humulene synthase based on their
corresponding major products, synthesize 34 and 52 sesquiterpene
olefins, respectively, and thus constitute the most mechanistically
complex terpenoid cyclases to be described thus far from any
source.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plant Materials, Substrates, and Reagents--
Two-year-old
grand fir (A. grandis Lindl.) saplings were purchased from
the Forestry Research Nursery, University of Idaho, Moscow, ID.
Saplings were grown in standard potting mix (Sals Inc., Puyallup, WA)
with a 16-h photoperiod (200-300 microeinsteins/m2·s)
and a 26 °C day/15 °C night temperature cycle and were fertilized (15:30:15 (N:P:K)) weekly and watered daily. The syntheses of [1-3H]farnesyl diphosphate (125 Ci/mol) (13),
[1-3H]geranylgeranyl diphosphate (120 Ci/mol) (7), and
[1-3H]geranyl diphosphate (250 Ci/mol) (20) have been
reported previously. The sources of authentic sesquiterpene standards
were as follows: -longipinene, cyclosativene, sativene, and
-ylangene were from Abies balsamea oleoresin;
-selinene was from Abies alba oleoresin;
-,
-, and
-himachalene,
- and
-amorphene,
-muurolene,
guaia-6,9-diene,
-cadinene,
-selinene, germacrene A,
-ylangene,
-longipinene, (E)-
- and
-bisabolene
were gifts from Larry Cool (University of California, Berkeley);
germacrene B and black pepper oleoresin containing
-cubebene and
-copaene were gifts from Robert Adams (Baylor University);
-humulene was from allspice seeds and as a gift from Ron Binder
(USDA, Albany, CA); sibirene and longicyclene were from Pinus
sibirica oleoresin; longifolene was purchased from Sigma;
(E)-
-farnesene was from parsley leaf oil; germacrene D
was from the essential oil of caraway and Nepeta mussini;
-caryophyllene was a gift from Rudolf Hopp (Haarmann and Reimer
GmbH);
-bisabolene was isolated from carrot roots;
-humulene was
purchased from Fluka Chemical Co.;
-cubebene was from the essential
oil of Valencia orange peels;
-selinene and seli-3,7(11)-diene were
from celery seed;
-cadinene was a gift from Margaret Essenberg
(Oklahoma State University);
-guaiene was from patchouli essential
oil; germacrene C was isolated from tomato leaf oil; bicyclogermacrene was from the essential oil of marjoram; and
-gurjunene was from ginger root. All other biochemicals and reagents were purchased from
Sigma or Aldrich, unless otherwise noted.
Oleoresin Isolation and Analysis-- Grand fir sapling stems were sectioned into 2-3-mm discs and extracted overnight with pentane (3.0 ml/g tissue) at room temperature. The pentane extract was decolorized with activated charcoal, washed with water, and passed over a column of MgSO4 and silica gel (Mallinckrodt, type 60A) to remove any traces of water and to bind oxygenated metabolites, thereby providing the turpentine fraction. The oxygenated metabolites were then released from the column by rinsing with diethyl ether. Capillary GLC1 (flame ionization detector) was utilized for identification and quantification of the turpentine monoterpene and sesquiterpene olefin components (Hewlett-Packard model 5890 with cool (40 °C) on-column injection, detector at 300 °C, and H2 carrier at 14 p.s.i.; column: 0.25 mm inner diameter × 30 m fused silica with 0.25-µm film of FFAP (Alltech) programmed from 35 to 50 °C at 50 °C/min (5 min hold) then to 230 °C at 10 °C/min). Capillary GLC-MS was employed to confirm identifications by comparison of retention times and 70 eV mass spectra to those of authentic standards (Hewlett-Packard model 6890 gas chromatograph coupled to a Hewlett-Packard model 5872 mass spectrometer with cool (40 °C) on-column injection, and He carrier at 0.7 ml/min; column: 0.25 mm inner diameter × 30 m fused silica with 0.25-µm film of 5MS (Hewlett-Packard) or polyethylene glycol ester (AT1000, Alltech) and programmed from 40 to 50 °C at 50 °C/min (5 min hold) then to 230 °C at 10 °C/min).
Enzyme Isolation and Assay--
Grand fir saplings in active
growth were used as the enzyme source for determination of constitutive
terpenoid synthases and of terpenoid synthases induced by stem wounding
by a standard protocol (6). Stems from control saplings and saplings 8 days after wounding (usually 10 saplings) were harvested by removing the top and lateral growth and cutting at about 5 cm from the base. The
stems were chopped into 5-7-cm segments, frozen in liquid N2, and following removal of any needles were ground to a
powder in a liquid N2-chilled number 1 Wiley mill. The
frozen powder was added to chilled extraction buffer (5 ml/g fresh
tissue weight) consisting of 10 mM dibasic potassium
phosphate and 1.8 mM monobasic potassium phosphate (pH
7.3), 140 mM NaCl, 20 mM -mercaptoethanol, 10 mM MgCl2, 5 mM
MnCl2, 10% (v/v) glycerol, and 1% (w/v) each of
polyvinylpyrrolidone (Mr 40,000) and
polyvinylpolypyrrolidone. The extract was stirred for 30 min at
0-4 °C and then clarified by centrifugation at 5000 × g and filtration through Miracloth (Calbiochem). Partial
purification of the extract was achieved by chromatography on
O-diethylaminoethyl-cellulose (Whatman DE52) as described
previously (21).
cDNA Isolation, 5-RACE, and Expression of Sesquiterpene
Synthases--
Construction of the wound-induced fir stem cDNA
library has been described (8), and the details of hybridization probe generation and library screening are reported elsewhere (9). In
summary, hybridization probes for terpenoid synthases were generated by
PCR using degenerate oligonucleotide primers designed from conserved
amino acid sequences (designated in boldface in Fig. 7) of
several monoterpene, sesquiterpene, and diterpene synthases from
angiosperm species (19). DNA from a
phage cDNA library, constructed from mRNA isolated from wounded grand fir sapling stems
(8), was purified and used as template for PCR reactions (26). Four
unique, 110-bp fragments were amplified, cloned, and shown to be
cyclase-like in sequence, and they were designated probes 1, 2, 4, and
5. Upon screening of the cDNA library, probes 4 and 5 hybridized,
respectively, to two unique cDNA species designated ag4.30 and ag5.9; the location of each probe is
doubly underlined in the sequences illustrated in Fig. 7.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Oleoresin Sesquiterpenes--
Grand fir has been utilized as a
model system for the study of induced oleoresin production in conifers
in response to wounding and insect attack (19). The monoterpene and
diterpenoid components of the oleoresin have been defined, and several
of the responsible monoterpene and diterpene synthases have been
purified and characterized and the corresponding cDNAs isolated to
provide tools for examining the regulation of this defense response
(6-9, 21). The third component of the oleoresin, the sesquiterpenes,
has not been examined in any detail as it comprises the smallest
fraction of the defensive secretion. Capillary GLC-MS analysis of grand
fir stem turpentine revealed a minimum of 38 sesquiterpenes
constituting approximately 12.5% of this material, with the remaining
87.5% composed of previously identified monoterpenes (3). The six
major sesquiterpenes present are -cubebene,
-copaene,
-caryophyllene,
-muurolene,
-cadinene and
(E,E)-germacrene B, representing 62% of the
total sesquiterpene fraction (Fig. 1).
-Selinene, guaia-6,9-diene,
-amorphene, sibirene,
-humulene,
longifolene,
-,
-, and
-himachalene,
-longipinene,
-bisabolene,
-ylangene, sativene, and cyclosativene were also identified (~33% of the sesquiterpene fraction), with the remaining minor fraction (~5%) composed of some 20 as yet unidentified
sesquiterpene olefins.
|
Sesquiterpene Synthases--
To examine the sesquiterpene
synthases of grand fir, a soluble enzyme extract from non-wounded
(control) sapling stems was prepared by methods previously employed in
the study of monoterpene and diterpene synthases from this tissue (7,
31). These preparations catalyzed the divalent metal
ion-dependent conversion of [1-3H]farnesyl
diphosphate, the universal precursor of sesquiterpenoids (32, 33), to a
labeled olefin fraction (12 × 2 ml assays yielded ~1.6 nmol of
product) that upon radio-GLC analysis (Fig.
2a) was shown to contain the
same spectrum of sesquiterpenes found in the oleoresin. Enzyme extracts
were similarly prepared from sapling stems 9 days after wounding and
were assayed as before (2 × 2 ml assays yielded 1.8 nmol of
product). Radio-GLC analysis of the olefin fraction revealed the
presence of an apparently single component with retention time very
similar to that of -cadinene (Fig. 2b). Partial
purification of the extract from induced saplings to eliminate traces
of endogenous oleoresin, followed by preparative-scale assay, provided
sufficient material for capillary GLC-MS analysis. This higher
resolution method revealed that the product derived from farnesyl
diphosphate by the induced enzyme(s) consisted of two components that
were identified as
-cadinene and (E)-
-bisabolene. Differential loss of the
-cadinene synthase activity during storage (data not shown) suggested that
-cadinene and
(E)-
-bisabolene were the products of two different
enzymes. Boiled controls, and control reactions without farnesyl
diphosphate, confirmed that both the constitutive and inducible
sesquiterpene synthase activities observed were enzymatic and
substrate-dependent. The Km value for
[1-3H]farnesyl diphosphate with the partially purified
inducible sesquiterpene synthases was determined to be about 0.4 µM. It is of interest, and of probable physiological
significance,2 that the
constitutive sesquiterpene synthase activities differ from the
wound-induced enzyme activities in product composition. A similar
phenomenon has been previously observed with the constitutive and
wound-inducible monoterpene and diterpene synthases of this tissue (6,
21).
|
cDNA Isolation and Expression--
A similarity-based PCR
cloning strategy for terpenoid synthases (9) yielded two different
truncated cDNA species, ag4.30 and ag5.9,
upon screening a grand fir stem cDNA library. The full-length forms, ag4 and ag5, were acquired by a 5-RACE
technique, and both of these, and the original cDNA isolates, were
sequenced completely on both strands. Sequence similarity to other
terpenoid synthases of plant origin (see below) and the apparent lack
of an encoded plastidial transit peptide characteristic of both
monoterpene and diterpene synthases (8, 9, 34) suggested that the two
new sequences represented sesquiterpene synthases. The sequences of
ag5.9 and ag5 were identical to each other;
however, comparison of the ag4.30 cDNA to ag4
indicated six nucleotide differences resulting in three amino acid
changes from ag4.30 to ag4 at positions 47 (Glu
to Gln), 437 (Val to Leu), and 447 (Gly to Asp) (see Fig. 7). These
differences may represent alleles in the tetraploid genome or members
of a small gene family but are more likely to simply reflect
polymorphic variation within the 120 individual trees used in cDNA
library construction.
|
|
|
|
Sesquiterpene Synthase Characterization--
The
Km values for [1-3H]farnesyl
diphosphate with -humulene synthase and
-selinene synthase were
estimated to be about 4.5 and 1.5 µM, respectively. The
metal ion requirements of
-humulene synthase and
-selinene
synthase were also evaluated, as cofactor specificity is often
characteristic of the different terpenoid synthase types (39).
-Selinene synthase shows a distinct preference for Mg2+;
the maximum rate with Mn2+ is less than 10% of that with
Mg2+ at saturation. By contrast,
-humulene synthase can
utilize Mg2+ or Mn2+ with comparable velocities
in the cyclization reaction. For both enzymes, the
Km value for Mg2+ is about 125 µM and for Mn2+ about 25 µM.
Neither of the sesquiterpene synthases requires K+ or other
monovalent cation for activity. The monoterpene synthases from conifers
require Mn2+ or Fe2+ for activity, but
Mg2+ fails to support catalysis (9, 40), and these enzymes
also exhibit an absolute requirement for a monovalent cation, with K+ preferred (9, 39).
|
Sequence Analysis--
The -selinene synthase cDNA encodes
a protein that is 581 amino acids in length with a predicted molecular
weight of 67,625, and the
-humulene synthase cDNA encodes a
protein of 593 residues with a predicted molecular weight of 67,937 (Fig. 7). The
-humulene synthase
sequence contains a stop codon in frame with the putative initiation
methionine at
21 bp of the 89-bp 5
-untranslated region, whereas the
-selinene synthase sequence is truncated at
12 bp. The nucleotide
sequence surrounding the putative starting ATG of both sesquiterpene
synthase genes is conserved and resembles that which surrounds the
initiating methionine of other plant genes (41). These data support the
proposed location of the initiation sites and, thus, the identification
of both cDNAs as sesquiterpene synthases, since the predicted
molecular weights are appropriate for this class of cytosolic enzymes
(16, 18) which lack a plastidial targeting peptide found in both
monoterpene synthase and diterpene synthase preproteins (8, 9, 34, 42).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The analysis of the sesquiterpene fraction of grand fir oleoresin
reported here for the Rocky Mountain ecotype agrees well with a
previous analysis of this material from the coastal ecotype (38) with
but minor differences between the former (19% germacrene B without
detectable -elemene) and the latter (8%
-elemene without detectable germacrene B). The discrepancy is likely the result of
misidentification due to methodology (identification of
-elemene by
retention time only) and has been rectified by recent re-analysis of
the oleoresin of the coastal
ecotype.3 Upon stem wounding,
two sesquiterpene synthase activities are induced, one for the
increased production of a prominent constitutive component
(
-cadinene; see Fig. 1) and one for the production of a very minor
sesquiterpene of the constitutive oleoresin
((E)-
-bisabolene). This situation is reminiscent of that
observed with the constitutive and inducible monoterpene synthases of
grand fir (4, 6, 21).
Although -selinene synthase and
-humulene synthase are capable of
producing monoterpenes when presented with geranyl diphosphate, several
lines of evidence indicate that these enzymes are, in fact,
sesquiterpene synthases. First, the corresponding cDNA species do
not appear to encode preproteins bearing a plastidial transit peptide
characteristic of monoterpene (and diterpene) synthases but rather
mature proteins of a size typical of this class of cytosolic enzymes.
Second, the divalent and monovalent ion requirements do not resemble
those of the monoterpene synthases but rather those of other
sesquiterpene synthases. Finally, the acyclic monoterpenes (ocimenes)
produced by
-selinene synthase and
-humulene synthase from
geranyl diphosphate are not found in the turpentine fraction of grand
fir oleoresin (3, 22, 38). The accumulated evidence therefore clearly
supports the identification of these enzymes as sesquiterpene
synthases. Since sesquiterpene biosynthesis occurs in the cytosol where
the precursor farnesyl diphosphate is also synthesized, whereas the
monoterpene synthases are compartmentalized within plastids where the
precursor geranyl diphosphate also arises (11, 34, 55, 56), the ability
of the sesquiterpene synthases to produce monoterpenes in
vitro may simply represent the adventitious utilization of a
substrate that is never encountered in vivo and against
which there is no evolutionary pressure to discriminate. It now seems
likely that the adventitious utilization of geranyl diphosphate by the
sesquiterpene synthases accounts, in part, for the relatively high
level of limonene synthase activity observed in crude stem extracts of
grand fir (21).
The ability of terpene synthases to produce multiple products has been
well documented (14, 39, 40, 47, 57) and may be a consequence of the
unusual electrophilic reaction mechanisms employed by this enzyme type
(11, 35, 58) that may also represent an evolutionary adaptation for the
production of the maximum number of terpene products using the minimum
genetic and enzymatic machinery (59). Nevertheless, the production of
34 different sesquiterpenes by -selinene synthase and 52 discrete sesquiterpenes by
-humulene synthase, by variations upon several different cyclization routes, is quite remarkable. The reaction cascade
catalyzed by
-humulene synthase is particularly complex in
generating (by deprotonation) stable olefinic end products corresponding to many of the proposed carbocationic intermediates of
each cyclization route (Fig. 5). Significantly, the essential elements
of these cyclization schemes have been delineated by Arigoni and
collaborators (32, 60, 61) via a series of elegant in vivo
labeling studies directed toward the biosynthesis of longifolene and
sativene in the fungi Helminthosporium (victoria
or sativum) or the gymnosperm Pinus ponderosa.
Additionally, in vivo studies with (5R)- and
(5S)-[5-3H]mevalonate provided evidence, based
upon the observation of isotopically sensitive branching (62), that the
formation of (
)-longifolene and (
)-sativene was catalyzed by a
single enzyme. The isolation and functional expression of the
-humulene synthase cDNA reported here provides direct and
unequivocal proof for this earlier, prescient biosynthetic proposal
(60, 61).
Sequence comparison between the -selinene synthase and the
-humulene synthase indicates that the two enzymes are very similar, but with the similarity decreasing toward the carboxyl terminus of the
proteins. This observation is consistent with the conclusions drawn
from domain swapping experiments with related sesquiterpene synthases
(54) which suggest that the amino-terminal regions of the proteins are
involved in the initial, common steps of the cyclization reactions and
that the more carboxyl-terminal regions are responsible for determining
the specific product outcome. The two gymnosperm sesquiterpene
synthases clearly resemble in sequence the angiosperm terpenoid
synthases (roughly 55% similarity and 30% identity), with levels of
conservation similar to those observed between the angiosperm
sesquiterpene and diterpene synthases and the monoterpene synthases of
this plant class (9, 18). The regions of highest similarity between the
various terpenoid synthases are clustered and likely represent those
elements responsible for common cyclization chemistry (e.g.
ionization, charge stabilization, and deprotonation). The more variable
regions likely impart the specific shape of the active site that
enforces substrate and intermediate conformation and thus dictates the
specific product outcome. The crystal structures of two sesquiterpene
cyclases have recently been described for pentalenene synthase from
Streptomyces UC5319 (63) and epi-aristolochene
synthase from tobacco (64). Both enzymes have been shown to possess
very similar fold structures related to farnesyl diphosphate synthase
(52) and to consist of mostly antiparallel
-helices that form a
large central cavity. Modeling studies with other terpenoid cyclases
have been initiated (64), and it should soon be possible to compare the
predicted structures of the multiple product sesquiterpene synthases of grand fir to these defined single product synthases and to perhaps reveal the structural basis for fidelity (or the lack thereof) in these
cyclization reactions.
The cDNAs encoding -selinene synthase and
-humulene synthase
provide tools for evaluating the transcriptional regulation of
sesquiterpene biosynthesis in the context of constitutive oleoresin formation and, along with bacterial expression systems, the means for
examining structure-function relationships in these mechanistically fascinating catalysts. These cDNAs should also provide access to
genomic clones to allow comparison of intron/exon structure of these
genes to their angiosperm counterparts (40, 65). Grand fir is the first
plant from which cDNA species encoding representative monoterpene,
sesquiterpene, and diterpene synthases have been isolated (8, 9).
Although the sequence comparisons are in themselves instructive, the
availability of these clones should permit a more highly refined
understanding of oleoresinosis and lead to the manipulation of this
defensive secretion in the protection of conifer species against the
devastating environmental and economic effects of bark beetle predation
(1, 2, 66, 67).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Larry Cool, Ron Binder, Robert Adams, Margaret Essenberg, and Rudolf Hopp for the gifts of sesquiterpene reference standards; Jack Alexander of the Harvard Arnold Arboretum for the gift of A. alba tissue; Joyce Tamura-Brown for typing the manuscript; and Thom Koehler for raising the trees. We thank Eva Katahira for technical assistance and Gerhard Munske of the Laboratory for Biotechnology and Bioanalysis for DNA sequencing. We are especially grateful to Larry Cool (Forest Products Laboratory, University of California, Berkeley) for expert assistance in the identification of the sesquiterpenes from grand fir oleoresin.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM 31354, Dept. of Agriculture NRI Grant 97-35302-4432, the Tode Foundation, and Project 0268 from the Agricultural Research Center, Washington State University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U92266 and U92267.
The first two co-authors share equal credit in this
investigation.
§ Present address: Plant Biology Division, S. R. Noble Foundation, Ardmore, OK 73402.
¶ Feodor Lynen Fellow of the Alexander von Humboldt Foundation.
To whom correspondence should be addressed: Institute of
Biological Chemistry, Washington State University, Pullman, WA
99164-6340. Tel.: 509-335-1790; Fax: 509-335-7643; E-mail:
croteau{at}mail.wsu.edu.
1 The abbreviations used are: GLC, gas-liquid chromatography; MS, mass spectrometry (spectra); aa, amino acid(s); bp, base pair(s); PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends.
2 C. L. Steele, S. Katoh, J. Bohlmann, and R. Croteau, manuscript submitted for publication.
3 L. Cool, personal communication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|