(Received for publication, November 16, 1995; and in revised form, January 30, 1996)
From the
In plants, the dominant sterols are 24-alkyl sterols, which play
multiple roles in plant growth and development, i.e. as
membrane constituents and as precursors to steroid growth regulators
such as brassinosteroids. The initial step in the conversion of the
phytosterol intermediate cycloartenol to the 24-alkyl sterols is
catalyzed by S-adenosyl-L-methionine:-sterol-C-methyltransferase
(SMT), a rate-limiting enzyme for phytosterol biosynthesis. A cDNA
clone (SMT1) encoding soybean SMT was isolated from an etiolated
hypocotyl cDNA library by immunoscreening using an anti-(plasma
membrane) serum. The deduced amino acid sequence of the SMT1 cDNA
contained three conserved regions found in S-adenosyl-L-methionine-dependent methyltransferases.
The overall structure of the polypeptide encoded by the SMT1 cDNA is
most similar to the predicted amino acid sequence of the yeast ERG6 gene, the putative SMT structural gene. The polypeptide encoded by
the SMT1 cDNA was expressed as a fusion protein in Escherichia coli and shown to possess SMT activity. The growing soybean vegetative
tissues had higher levels of SMT transcript than mature vegetative
tissues. Young pods and immature seeds had very low levels of the SMT
transcript. The SMT transcript was highly expressed in flowers. The
expression of SMT transcript was suppressed in soybean cell suspension
cultures treated with yeast elicitor. The transcriptional regulation of
SMT in phytosterol biosynthesis is discussed.
In plants, the dominant sterols are 24-alkyl sterols. Sterols
such as cholesterol which are nonmethylated at C-24 are only present in
low amounts. It has been suggested that sterols may have two distinct
functions in higher plants (Haughan et al., 1988), as has been
found for fungi (Ramgopal and Bloch, 1983; Rodriguez and Parks, 1983;
Nes and Heupel, 1986). The first is as a structural component of
membranes, a function which requires relatively large amounts of
sterols. The second function is related to cell proliferation; trace
amounts of 24-alkyl sterols such as stigmasterol are required for cell
growth to proceed (Haughan et al., 1988). 24-Alkyl sterols
also serve as precursors to steroid growth regulators such as
brassinosteroids (Ikekawa, 1991). Biosynthesis of 24-alkyl phytosterols
can be divided into two stages: conversion of acetate to cycloartenol
and transformation of cycloartenol to 24-alkyl sterols. The
transformation of cycloartenol to 24-alkyl sterols includes four steps:
alkylation of the -bond in the side chain, removal of
methyl groups at C-4 and C-14, isomerization of the cyclopropyl group
into a
-double bond, and rearrangement of the
to
(Benveniste, 1986). The
transmethylation of the
-double bond in the
cycloartenol side chain is catalyzed by S-adenosyl-L-methionine:
-sterol-C-methyltransferase
(SMT). (
)Although several outcomes are possible for the
methylation reaction, 24(28)-methylene cycloartanol appears to be the
kinetically favored product in most, if not all, higher plants (Misso
and Goad, 1983; Scheid at al., 1982; Nes et al., 1993; Guo et al., 1995) (Fig. 1). The C-24 methylation step has
been implicated as a rate-limiting step in sterol transformations (Guo et al., 1995; Fang and Baisted, 1975; Parker and Nes, 1992;
Nes et al., 1991a, 1991b; Chappell et al., 1995). In
animals, instead of a C-24 methylation in the side chain of
cycloartenol, a reduction of the
-double bond in the
side chain of lanosterol takes place, leading to cholesterol
biosynthesis.
Figure 1: Kinetically favored C-24 methylation of cycloartenol catalyzed by SMT in higher plants (A). SMT can also catalyze the C-24 methylation of lanosterol to produce 24(28)-methylene-24,25-dihydrolanosterol in vitro (B). SAM, S-adenosyl-L-methionine.
The SMT is a membrane-bound enzyme and has been
localized in the endoplasmic reticulum in plant cells
(Hartmann-Bouillon and Benveniste, 1978). The enzyme recognizes the
sterols with a free 3-hydroxyl group, a
-double
bond, and the flat conformation that mimics the three-dimensional shape
of cycloartenol and catalyzes their methylation at C-24 position in in vitro assay (Nes et al., 1991a; Rahier et
al., 1984). So far, no SMT has been purified to homogeneity. Our
knowledge on this enzyme is based on in vivo studies on plants
fed with radiotracers and stable isotopes (Guo et al., 1995),
and in vitro studies using microsomal preparations as enzyme
source (Misso and Goad, 1983; Scheid et al., 1982; Nes et
al., 1991a). Cloning of the plant SMT genes and characterization
of the gene products would provide an alternative approach to address
some of the important questions on SMT such as the C-24 methylation
mechanism and developmental regulation of the enzyme.
Earlier studies indicated that the yeast ERG6 gene probably encodes an SMT (McCammon at al., 1984; Gaber et al., 1989). Mutations in ERG6 locus, arising from mutagenesis or gene replacement, partially or totally abolished SMT activity and 24-alkyl sterol production in yeast cells. However, SMT activity of the ERG6 gene product has not been documented. No plant SMT gene has been cloned previously. Reported here is the isolation of a cDNA encoding SMT from soybean. The polypeptide encoded by the soybean cDNA was expressed in Escherichia coli and shown to be an active SMT enzyme. The expression of the SMT transcript was found to be developmentally regulated, and suppressed following elicitor treatment. Transcriptional regulation of SMT in sterol biosynthesis is discussed.
The cDNA clone Spm482 contained a 1.5-kilobase insert (Fig. 2). A 1101-bp open reading frame encoded a polypeptide of 367 amino acids with a calculated molecular mass of 41.5 kDa. The DNA sequence upstream of the proposed ATG translation initiation codon contains four stop codons within the translation frame. Hydropathy analysis of the deduced amino acid sequence showed no potential membrane-spanning sequence. The polypeptide was classified as a peripheral protein (with a P:I odds value 580.7) using the PC/Gene program SOAP (Klein et al., 1985).
Figure 2: DNA sequence and deduced amino acid sequence of the soybean SMT1. S-Adenosyl-L-methionine-dependent methyltransferase motifs are underlined and numbered. The GenBank accession number of the soybean SMT1 cDNA sequence is U43683.
A data base search revealed similarity of the polypeptide encoded by the soybean cDNA to several known methyltransferases, such as Herpetosiphon giganteus cytosine-specific DNA-methyltransferase (P25265), Rhodobacter sphaeroides phosphatidylethanolamine N-methyltransferase (Q05197), Streptomyces sp. aklanonic acid methyltransferase (L35154), and Rattus norvegicus dihydroxypolyprenylbenzoate methyltransferase (L20427). Further sequence comparison revealed that the deduced amino acid sequence contained three methyltransferase sequence motifs identified in diverse S-adenosyl-L-methionine-dependent methyltransferases (Fig. 3). The three motifs were arranged in the same order on the soybean polypeptide chain as on other methyltransferases and the amino acid sequences separating the three motifs had comparable length as those in other methyltransferases. Motif I was highly conserved in the soybean protein. The motif II in the soybean protein contained the invariant central aspartate residue. The conserved aromatic amino acid phenylalanine and tyrosine were found at positions -1 and +3 with respect to the central aspartate, as in other methyltransferases. The motif III was located at an interval of 19 residues C-terminal to motif II. The first half of this region was well conserved and the central glycine residue was present. However, the other half of the region did not match with the consensus described by Kagan and Clarke(1994). The presence of the three sequence motifs of S-adenosyl-L-methionine-dependent methyltransferases suggests that the soybean cDNA may encode an S-adenosyl-L-methionine-dependent methyltransferase.
Figure 3: The deduced amino acid sequence of the soybean SMT1 cDNA contains the S-adenosyl-L-methionine-dependent methyltransferase motifs identified by Kagan and Clarke (1994).
The yeast ERG6 gene product is the most closely related
sequence to the soybean polypeptide in current protein data bases
(Hardwick and Pelham, 1994). Overall, the two sequences share 47.1%
amino acid identity. It has been suggested, but not yet proven, that
the yeast ERG6 is the structural gene encoding S-adenosyl-L-methionine:-sterol-C-methyltransferase
(McCammon at al., 1984; Gaber et al., 1989). The high identity
of amino acid sequence between the soybean protein and the yeast ERG6 gene product indicated that the soybean cDNA may encode
an S-adenosyl-L-methionine:
-sterol-C-methyltransferase.
The cDNA was therefore designated as SMT1.
Figure 4:
Expression of the FLAG epitope-tagged
soybean SMT1 in E. coli. Crude extracts of the JM109 cells
were electrophoresed in an SDS-polyacrylamide gel, transferred to a
membrane, and probed with the anti-FLAG M2 antibodies (Kodak/IBI). Lane 1, the JM109 cells were transformed with the plasmid
carrying FLAG-epitope tagged SMT1 cDNA and grown in the absence of
isopropyl-1-thop--D-galactopyranoside and without M13/T7
phage infection. Lane 2, the transformed JM109 cells were
grown in the presence of 1 mM isopropyl-1-thop-
-D-galactopyranoside and infected
with M13/T7 phage.
It has been reported that plant SMT can methylate a
variety of -sterols, such as cycloartenol,
lanosterol, and desmosterol, at the C-24 position in in vitro assay (Scheid et al., 1982; Nes et al., 1991a).
SMT activity of the crude extract of the transformed E. coli expressing the soybean protein was also assayed using desmosterol
as substrate. In the incubation condition of 50 µM desmosterol and 50 µM [methyl-
H]AdoMet (130,000
cpm/reaction), the extracts of the transformed E. coli cells
showed an SMT specific activity of 4.8 pmol/min/mg protein.
The
product from the incubation of transformed E. coli cell
lysates with lanosterol and AdoMet was analyzed using GC-MS. A peak
corresponding to a more polar compound than lanosterol was detected in
samples prepared from reaction mixtures containing the extracts of
induced E. coli cells, as shown in Fig. 5. The peak was
absent in samples prepared from reaction mixtures containing the
extracts of uninduced E. coli cells. The metabolite has a
molecular mass of 440 (the substrate lanosterol molecular mass is 426).
Examination of the mass spectrum of the metabolite indicated that the
methylation of lanosterol did not occur in the sterol nucleus. In the
side chain of lanosterol, the only potential methylation site is the
-double bond (Fig. 1). Transmethylation of
the double bond catalyzed by SMT could produce
24(28)-methylene-24,25-dihydrolanosterol and
-24-methyl lanosterol (Scheid et al., 1982).
This reaction could also produce
-24-methyl
lanosterol (Misso and Goad, 1983). These compounds differ in the
position of a double bond in the side chain and have similar mass
spectra. The mass spectrum of the metabolite (Fig. 5C)
was compared with that of 24(28)-methylene-24,25-dihydrolanosterol
methylation (Nes et al., 1991a). The mass spectra of the two
compounds were identical except for two minor peaks at m/z 384
and m/z 399 atomic mass units that were missing in the mass
spectrum of the metabolite, possibly due to low levels of signals.
Although we cannot identify the double bond location in the side chain
of the methylated lanosterol based on mass spectrum, methylation at the
C-24 position in the lanosterol side chain is demonstrated. Therefore,
it was concluded that the soybean cDNA SMT1 encodes the S-adenosylL-methionine:
-sterol-C-methyltransferase.
Figure 5:
GC-MS analysis of the metabolite from the
incubation of lanosterol and S-adenosyl-L-methionine
with cell lysates prepared from E. coli cells transformed with
the plasmid carrying the soybean SMT1 cDNA. A, GC chromatogram
of the sterol from control incubation (soybean SMT1 not expressed). B, GC chromatogram of the sterol from SMT incubation. C, the mass spectrum of the metabolite recorded on a GC-MS HP
tabletop 5890 mass detector. The GC (capillary, 30 m:HP-1701) was
operated from 130 to 280 °C (15 °C min),
then held at 280 °C for another 15 min.
Figure 6: Gel blot analysis of soybean genomic DNA. Soybean DNA (10 µg) digested with the indicated restriction enzymes was hybridized with the entire SMT1 cDNA sequence (A), 5`-terminal region (192 bp) (B) and 3`-terminal region (306 bp) (C). DNA length markers are given at right in kilobases.
Figure 7: Gel blot analysis of total RNA extracted from various soybean tissues. A, total RNA (10 µg) was hybridized with the entire SMT1 cDNA sequence. B, the same blot was probed with 18 S ribosomal cDNA. The mature plants have flowered and podded.
Figure 8: RNA gel blot analysis of SMT expression in elicitor-treated soybean cell suspension cultures. A, total RNA (10 µg) from cells at various times after treatment was hybridized with SMT cDNA. B, the same blot was probed with 18 S ribosomal cDNA.
Identification of the soybean SMT1 cDNA was accomplished using both molecular and biochemical criteria. Analysis of the deduced amino acid sequence of the SMT1 cDNA revealed three conserved regions found in S-adenosyl-L-methionine-dependent methyltransferases. The methyltransferase motifs may contribute to the binding of the substrate S-adenosyl-L-methionine and/or the product S-adenosyl-L-homocysteine (Kagan and Clarke, 1994). The high identity of deduced amino acid sequences in the entire length between the soybean cDNA and the putative yeast SMT structural gene ERG6 suggested that the soybean cDNA might also encode SMT. This supposition was confirmed by expression of the cDNA in E. coli. Extracts from transformed bacteria had C-24 methylation activity on lanosterol. Although the natural substrate of plant SMT is cycloartenol, SMT can use lanosterol and other related sterols as methyl group acceptors in in vitro assays (Scheid et al., 1982; Nes et al., 1991a). Therefore, showing SMT activity on lanosterol provides biochemical evidence for positive identification of the soybean cDNA.
Genomic DNA hybridization analysis indicates that the soybean genome may contain additional sequence(s) with similarity to the cloned SMT1 cDNA. Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor (Hymowitz and Singh, 1987). Duplicated DNA sequences occur widely in the soybean genome (Zhu et al., 1994). However, whether the additional genomic sequence(s) encode an active SMT enzyme remains to be studied. Alternatively, these sequences may represent unrelated gene segments.
The soybean SMT1 was cloned by immunoscreening a cDNA expression library using an anti-(plasma membrane) serum. It has been shown that plant SMT is localized in endoplasmic reticulum membranes (Hartmann-Bouillon and Benveniste, 1978) and yeast SMT is associated with lipid particles (Zinser et al., 1993). However, recognition of the soybean SMT by anti-(plasma membrane) serum may not necessarily indicate the association of SMT with plasma membranes. It is more likely that the plasma membrane preparation used for raising antibodies contained some contaminating endoplasmic reticulum membranes, since the plasma membranes prepared by aqueous-polymer two-phase partitioning only have a purity of about 95% (Shi et al., 1995 and Refs. contained therein).
Transcript levels of the soybean SMT were found to be regulated developmentally. Young roots, leaves, and stems had higher levels of steady state SMT transcripts as compared with those of mature tissues such as old leaves and roots of the plants with pods, reflecting a high rate of sterol biosynthesis in the growing vegetative tissues. These results agree with previous reports that the growing soybean vegetative tissues (shoots and roots) have very high sterol content on the basis of dry weight (Fenner et al., 1986). These results are also consistent with the observation that old leaves accumulate much more cycloartenol than young leaves do in transgenic tobacco plants overexpressing hydroxymethylglutaryl-CoA reductase (Chappell et al., 1995). High expression of SMT transcript was detected in soybean flowers, indicating active synthesis of sterols in flowers. Young pods and immature seeds had very low levels of SMT transcripts. It has been reported that soybean seeds have the highest sterol concentration on the basis of dry weight and total lipids shortly after pollination and sterol concentration then steadily decreases during seed development and maturation (Katayama and Katoh, 1973; Kajimoto et al., 1982). The low levels of SMT transcripts in immature seeds found in this study may indicate a low rate of sterol biosynthesis in soybean seeds during maturation.
Biochemical and in vivo radiolabeling studies have suggested that SMT is a rate-limiting enzyme and may regulate the biosynthesis of 24-alkyl sterols (Guo et al., 1995; Fang and Baisted, 1975; Parker and Nes, 1992; Nes et al., 1991a, 1991b). Recently, it has been shown that transgenic tobacco plants overexpressing the hydroxymethylglutaryl-CoA reductase accumulate a large amount of cycloartenol, providing further evidence for the role of SMT in the regulation of phytosterol biosynthesis (Chappell et al., 1995). In this study, analysis of SMT transcript implicates the possibility of transcriptional control of SMT in regulating sterol biosynthesis. The transcript of SMT was highly expressed in the growing soybean vegetative tissues where sterol biosynthesis is very active (Fenner et al., 1986). Low levels of SMT transcript expression was associated with low levels of sterol content on the basis of dry weight in immature soybean seeds (Katayama and Katoh, 1973; Kajimoto et al., 1982). Furthermore, suppression of SMT transcripts was observed in soybean cell suspension cultures treated with elicitor. Suppression of sterol biosynthesis and alteration of sterol composition caused by elicitor treatment and pathogen infection have been demonstrated in various plants such as maize, parsley, potato, tobacco, and T. divaricata (Vögeli and Chappell, 1988; Haudenschild and Hartmann, 1995; Brindle et al., 1988; van der Heijden et al., 1989; Jennings et al., 1970).
The -double bond transmethylation catalyzed by the
SMT could produce several products with a double bond at various
positions. The kinetically favored outcome of the reaction appears to
be a 24(28)-sterol in maize and sunflower (Misso and Goad, 1983; Scheid et al., 1982; Nes et al., 1993; Guo et al.,
1995). It has been proposed that a single SMT may be responsible for
the production of three C-24-methylated cycloartenol
[24(28)-methylene cycloartanol,
-24-methyl
cycloartenol, and
-24
-methyl cycloartenol]
in the C-24 methylation reaction, and the product distribution may
result from kinetic, thermodynamic, and allosteric control mechanisms
acting on the development of the enzyme-substrate complex and
subsequent catalysis (Janssen and Nes, 1992). At the present time,
mechanism of the C-24 methylation is poorly understood, partially due
to lack of knowledge of SMT protein structure. The availability of SMT
cDNA and active SMT enzyme expressed in E. coli will permit
the establishment of the SMT protein structure. This might prove useful
in elucidating the interactions of enzyme, AdoMet, and sterol
substrate, and the C-24 methylation process.