From the Département des Sciences biologiques,
Université du Québec à Montréal, C.P. 8888 Succursale Centre-ville, Montréal, Québec H3C 3P8, Canada,
the § Plant Biochemistry Laboratory and Centre for
Structural-Functional Genomics, Concordia University, Montréal,
Québec H3G 1M8, Canada, and the ¶ Department of Plant
Sciences, University of Western Ontario, London,
Ontario N6A 5B7, Canada
Received for publication, September 16, 2002, and in revised form, November 3, 2002
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ABSTRACT |
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In plants, O-methylation of phenolic
compounds plays an important role in such processes as lignin
synthesis, flower pigmentation, chemical defense, and signaling.
However, apart from phenylpropanoids and flavonoids, very few enzymes
involved in coumarin biosynthesis have been identified. We report here
the molecular and biochemical characterization of a gene encoding a
novel O-methyltransferase that catalyzes the methylation of
7,8-dihydroxycoumarin, daphnetin. The recombinant protein displayed an
exclusive methylation of position 8 of daphnetin. The identity of the
methylated product was unambiguously identified as
7-hydroxy-8-methoxycoumarin by co-chromatography on cellulose TLC and
coelution from high performance liquid chromatography, with
authentic synthetic samples, as well as by UV, mass spectroscopy,
1H NMR spectral analysis, and NOE correlation signals of
the relevant protons. Northern blot analysis and enzyme activity assays
revealed that the transcript and corresponding enzyme activity are
up-regulated by both low temperature and photosystem II excitation
pressure. Using various phenylpropanoid and flavonoid substrates, we
demonstrate that cold acclimation of rye leaves increases
O-methyltransferase activity not only for daphnetin but
also for the lignin precursors, caffeic acid, and 5-hydroxyferulic
acid. The significance of this novel enzyme and daphnetin
O-methylation is discussed in relation to its putative role
in modulating cold acclimation and photosystem II excitation pressure.
Low temperature is one of the most important environmental factors
limiting the productivity and distribution of plants. Exposure of
plants to low, nonfreezing temperatures, a process known as cold
acclimation, induces the genetic system required for increased freezing
tolerance. Knowledge of the molecular, physiological, and biochemical
changes that occur during this process could lead to the improvement of
plant productivity. This complex process has been extensively studied
and several cold-responsive genes have been isolated from a range of
dicotyledonous and monocotyledonous species (1, 2). Although the
functions of some of these genes are known (3-6), the details of the
processes responsible for their regulation and detection of temperature
changes are still incomplete.
Previous studies have shown that development of freezing tolerance in
winter cereals, such as wheat and rye, is correlated with an increase
in their photosynthetic capacity (7). Thus, growth at low temperature
not only induces freezing tolerance, but also results in an increased
resistance to low temperature-induced photoinhibition of
photosynthesis, and requires adjustment to a combination of light and
low temperature. The common photosynthetic response of plants to low
temperature and normal light is rationalized in terms of photosystem II
(PSII)1 excitation pressure,
which is a measure of the redox state of the first electron acceptor,
quinone A (7-9). It has been shown that cold-acclimated rye and wheat
grown at 5 °C/250 µmol m Methyltransferases are essential enzymes for directing intermediates
into specific biosynthetic pathways (11). Enzymatic O-methylation, catalyzed by
S-adenosyl-L-methionine-dependent OMTs, is a ubiquitous reaction that takes place in almost all organisms
including bacteria, fungi, plants, and mammals. In plants, O-methylation of phenolic compounds such as
phenylpropanoids, coumarins, and flavonoids, play an important role in
processes such as structural support, flower pigmentation, chemical
defense, and signaling (12). Of the large number of plant OMTs that are involved in secondary metabolism (11), only a few involved in coumarin
biosynthesis have been enzymatically characterized (13-15). In
addition, a cDNA encoding the 5-OMT for bergaptol (a linear furanocoumarin), has been cloned (16).
Simple plant coumarins are the cyclization products of their
corresponding o-hydroxycinnamic acids (17). They are widely distributed in plants, although members of the Apiaceae, Rutaceae, and
Moraceae are particularly rich sources of coumarins. Several members of
these families are used as spices and vegetables in human diet, as well
as for medicinal purposes (18). Coumarins are considered to be
components of the general defense response of plants to abiotic and
biotic stresses. In addition, various substituted coumarins exhibit
antimicrobial or anti-inflammatory activity and act as inhibitors of
numerous enzyme systems (17). Furthermore, coumarins exhibit numerous
effects of medicinal value (18).
We report here the molecular and biochemical characterization of a rye
cDNA, ScOMT1 encoding a novel enzyme that exclusively methylates the 7,8-dihydroxycoumarin (daphnetin) at position 8 to yield
7-hydroxy-8-methoxycoumarin. This enzyme is regulated by both
photosystem II excitation pressure and low temperature. The possible
role of this enzyme in the modulation of photosystem II excitation
pressure and cold acclimation is discussed.
Plant Material and Growth Conditions
Seeds of winter rye (Secale cereale L., Gramineae,
cv. Musketeer) were germinated in coarse vermiculite and grown at
temperatures of either 20/16 or 5/5 °C (day/night) with a 16-h
photoperiod in controlled environment chambers (Conviron, Manitoba,
Canada) as described previously (9). Growth irradiance was adjusted to
50 or 250 µmol m Enzyme Substrates
S-Adenosyl-L-[14CH3]methionine
(AdoMet; specific activity 55 Ci/mol) was obtained from American
Radiolabeled Chemicals Inc. (St. Louis, MO) and unlabeled AdoMet was
from Roche Molecular Biochemicals (Montreal, Quebec). The
phenolic substrates and reference compounds used were from our
laboratory collection. The 7,8-dihydroxycoumarin (daphnetin) was
purchased from Extrasynthèse (Lyon, France).
Chemical Synthesis
Daphnetin (7,8-Dihydroxycoumarin)--
Daphnetin was synthesized
according to the method of Molyneux and Jurd (19), in which a mixture
of pyrogallol and malic acid was heated in concentrated sulfuric acid
under nitrogen. The gummy residue, which separated upon the addition of
ice-water, was dissolved in methanol, which on cooling and
concentration, yielded daphnetin: m.p. 256-257 °C; UV
O-Methylation of Daphnetin--
Because none of the methylated
derivatives of daphnetin are available commercially, both methylated
isomers were synthesized as follows. A mixture of
trimethylsilyldiazomethane in hexane and
N,N-diisopropylamine in dioxane-MeOH was stirred for 20 h at room temperature. The mixture was poured into 2 N HCl,
extracted with EtOAc, and then washed with saturated aqueous NaCl.
Solvent evaporation and chromatography of the residue on a silica gel column, using toluene-acetone (10:1, v/v) as eluent, gave rise to
7-hydroxy-8-methoxycoumarin, 8-hydroxy-7-methoxycoumarin, and 7,8-dimethoxycoumarin.
Spectroscopic Data
UV spectra were obtained from the diode array detector during
HPLC and are given as 7-Hydroxy-8-methoxycoumarin--
m.p. 163-164 °C; UV
8-Hydroxy-7-methoxycoumarin--
m.p. 173-175 °C; UV
The chemical structures of both methylated isomers were unambiguously
verified by NOESY. In the spectrum of 7-hydroxy-8-methoxycoumarin, NOE
(nuclear Overhauser effect) correlation signals were observed between
8-OCH3 at Construction and Screening of the cDNA Library
Poly(A)+ RNA from plants grown at
20 °C/800 µmol m Northern Blots and DNA Sequencing
Total RNA (10 µg) samples were separated on
formaldehyde-agarose gels as previously described (9). After
electrophoresis, RNA was transferred to nitrocellulose membranes and
hybridized with ScOMT1 32P-labeled cDNA
insert. Filters were washed at 65 °C with several buffer changes of
decreasing sodium saline citrate concentration (5× to 0.1) and
autoradiographed on Kodak X-Omat RP films with intensifying screens at
The cDNAs were completely sequenced using a Li-Cor automated
sequencing device (DNA Sequencing Facility, Centre for Applied Genomics, Toronto, ON). A computer-aided search of protein and DNA
sequences was carried out with the FASTA and TFASTA programs of the
Genetic Computer Group sequence analysis software Wisconsin package,
version 10.0.
Expression of the Recombinant Protein
The ScOMT1 open reading frame was amplified using the
primers 5'-CGGTGATGGAGGATCCCAACGATG-3' and
3'-AGTTTCACTTGCCATGGTATTTTC-5' containing recognition sites for
BamHI and KpnI, respectively. After PCR
amplification, the ScOMT1 cDNA was digested with
BamHI and KpnI and subcloned into the
BamHI and KpnI site of pTrc-His vector (Novagen)
containing a polyhistidine tag. Luria-Bertani (LB) medium containing
100 µg/ml ampicillin was inoculated with Escherichia coli
carrying the ScOMT1 construct. The bacterial cells were
grown to an A600 of 0.5 at 37 °C and were
induced by the addition of isopropyl- Purification of the Recombinant Protein
The ScOMT1 fusion protein was purified by affinity
chromatography on Ni-nitrilotriacetic acid resin (Qiagen) then desalted on PD10 columns (Amersham Biosciences). Alternatively, the
pellets derived from 2-liter cultures were re-suspended in 100 ml of
phosphate-buffered saline containing 2 mM
phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, and
10% glycerol, pH 7.5 (standard buffer). After sonication and centrifugation at 10,000 × g for 10 min at 4 °C,
the clear supernatant was brought to 40% ammonium sulfate saturation
and the pellet obtained was dissolved in the standard buffer adjusted
at pH 8.5, desalted on PD10 columns, then chromatographed on a
DEAE-cellulose column previously equilibrated with the standard buffer,
pH 8.5. The enzyme was eluted with the standard buffer containing 250 mM NaCl. The enzymatically active fraction was collected
and used for subsequent analyses.
Protein Extraction and Quantification
Protein was routinely extracted from the plant material at
4 °C using phosphate-buffered saline, pH 7.3. After centrifugation at 10,000 × g, the supernatant was desalted on a PD10
column before further use. Proteins were quantified by the method of
Bradford using the Bio-Rad reagents and bovine serum albumin as the
protein standard.
Enzyme Assay and Product Identification
The ScOMT1 assay was performed as previously described (21)
using a final concentration of 200 µM substrate (in 1%
dimethyl sulfoxide), 2.5 µM
[14CH3]AdoMet (containing 25 nCi), and up to
100 µg of protein in 1× phosphate-buffered saline in a total volume
of 100 µl. The reaction was started by the addition of the enzyme,
incubated at 30 °C for 30 min, and stopped by the addition of 10 µl of 6 M HCl. The methylated product was extracted in a
mixture of benzene-ethyl acetate (1:1, v/v) and an aliquot of the
organic phase was counted for radioactivity using a toluene-based
scintillation fluid. The remaining sample was chromatographed on either
a cellulose TLC plate (20 × 20 cm) using ethyl acetate, acetic
acid, H2O (1:3:7, v/v/v) or an Agilent Eclipse C18 silica
column (4.6 × 250 mm; particle size, 5 µm; Waters, Milford, MA)
using 20% methanol in 1% acetic acid for 2 min followed by a linear
gradient to 40% methanol and 1% acetic acid for 30 min; maintained
for 5 min, then equilibrated to the original conditions for 15 min. The
developed TLC plate was then exposed in a Bio-Rad molecular imager
system and the data were analyzed with the software provided.
Sequence Analysis of ScOMT1--
The nucleotide sequence of
ScOMT1 (Secale cereale OMT1) comprises
1373 base pairs containing 71 nucleotides of 5'-untranslated and 237 nucleotides of 3'-untranslated sequences including the poly(A) tail.
The cDNA encodes a polypeptide of 355 amino acid residues with a
calculated molecular mass of 38 kDa and a predicted pI value of 7.4 (Fig. 1A).
Comparison of the deduced amino acid sequence with other plant OMTs
reveals sequence identities ranging from 26 to 40% (Fig.
1B). However, the highest identity (57%) is observed with
the maize herbicide Safener-binding protein, SafBP (22). A sequence
alignment of these proteins shows five regions in the C-terminal
portion (regions I to V, Fig. 1B) that have been reported to
be conserved among most plant OMTs (23) and proposed to be involved in
the AdoMet binding site for plant OMTs (23-25). Several amino acids
not included in the five conserved regions were found to be conserved
between ScOMT1 and other plant OMTs (Fig. 1B). They may
correspond to binding sites for phenolic substrates, given that these
compounds share some structural similarities.
The phylogenetic relationship of ScOMT1 to other plant OMTs was deduced
from the amino acid alignment presented in Fig. 1B. The
parsimony tree generated from 13 different OMTs shows a good alignment
of their sequences that is supported by high (72-100%) bootstrap
frequencies. It also illustrates that the ScOMT1 and maize
Safener-binding protein sequences occupy a distinct branch relative to
the other plant OMTs (Fig. 1C). The fact that Pinus radiata, Arabidopsis thaliana, and
Populus tremuloides OMTs are phylogenetically more distant
and highly divergent from ScOMT1 and the other OMTs suggests that they
may have evolved independently despite the conservation of their
consensus motifs (Fig. 1C).
Expression and Partial Purification of the ScOMT1
Protein--
Expression of the recombinant ScOMT1 gave rise to a
fusion product that possessed a six-histidine tag as part of a leader sequence at the N terminus of the protein. The molecular mass of this
protein is consistent with the addition of a 3.7-kDa His tag leader
sequence fused to the 38-kDa ScOMT1. The purified protein did not
exhibit any significant enzyme activity. This may be attributed to the
inhibiting effect of Ni2+ ions that may leach from the
column and remain in contact with the enzyme protein (26). However,
dialysis and the addition of both EDTA and Substrate Specificity of ScOMT1 and Product
Identification--
The partially purified recombinant ScOMT1
exhibited exclusive specificity for daphnetin as a substrate for
O-methylation. The enzyme did not accept either the
6,7-dihydroxy analog, esculetin, or caffeic acid, 5-hydroxyferulic
acid, luteolin (a 3',4'-dihyroxyflavone), quercetin (a
3',4'-dihyroxyflavonol), umbelliferone (7-hydroxycoumarin), naringenin
(a 4'-hydroxyflavanone), apigenin (a 4'-hydroxyflavone), or kaempferol
(a 4'-hydroxyflavonol) among other substrates tested. This result
indicates that this novel OMT exhibits a high degree of both substrate
and stereospecificity. Kinetic analysis of the partially purified
ScOMT1 gave an apparent Km of 152 µM
for daphnetin and 19 µM for AdoMet. The
Km value for daphnetin is comparable with
Km values reported for other OMTs (11). The ScOMT1
reaction product was unambiguously identified as
7-hydroxy-8-methoxycoumarin. It gave a single product when chromatographed in a nonpolar solvent system on cellulose TLC (Fig.
2A) with a higher
RF value (0.6) than that of daphnetin (0.25),
suggesting the presence of a methyl group and, consequently, the
reaction product elutes later (Rt 26.0 min) than daphnetin (Rt 19.0 min) after HPLC on a
C18 Agilent column (Fig. 2B). The enzyme
reaction product coeluted with an authentic sample of
7-hydroxy-8-methoxycoumarin (Rt 25.8 ± 0.5 min) on HPLC, and was well separated from the 7-methoxy-8-hydroxy isomer (Rt 27.5 ± 0.4 min) (Fig.
2C). Moreover, the UV absorption maxima of the enzyme
reaction product, which were identical to that of an authentic sample
of 7-hydroxy-8-methoxycoumarin, is lower than that of daphnetin
( Effect of Growth Temperature and Growth Irradiance on PSII
Excitation Pressure--
Table I
summarizes the estimated 1-qP values for PSII excitation pressure for
rye plants that were grown at either 20 or 5 °C under increasing
irradiance. The data demonstrate that increasing irradiance results in
an increased 1-qP at both 20 and 5 °C. It is significant to note
that plants grown at either 20/800 or 5/250 not only exhibited
comparable 1-qP values but also displayed the highest values, thus,
considered to be grown under high excitation pressure. In contrast,
plants grown at either 20/250 or 5/50 exhibited a comparable low
excitation pressure relative to those grown at either 20/800 or 5/250.
As expected, plants grown at 20/50 exhibit the lowest 1-qP value and
are considered to be grown at a very low PSII excitation pressure
relative to those grown at 20/250 or 5/50. Thus, by employing an
experimental design that includes plants grown at 20/50, 20/250,
20/800, 5/50, and 5/250, it may be possible to discern the individual
effects of low temperature, light, and excitation pressure.
Accumulation of ScOMT1 mRNA--
Northern blot analysis (Fig.
3A) shows that the
ScOMT1 transcript exhibits a high level of expression in
plants grown at 5/250 or 20/800 (°C temperature/light intensity
µmol m OMT Activities of Cold-acclimated Rye Leaves--
The endogenous
ScOMT1 activity of both cold-acclimated and high-light grown rye leaves
show that ScOMT1 exhibits the highest enzyme activity when rye plants
are cold acclimated (5/250), as compared with those exposed to high
light (20/800) or the control plants (20/250) (Fig.
4A). These results are
consistent with the higher accumulation of ScOMT1 mRNA
at 5/250 as compared with 20/800 (Fig. 3A).
Moreover, using various phenylpropanoid and flavonoid substrates we
show that cold acclimation increases not only ScOMT1 activity against
daphnetin, but also for other OMTs that utilize the lignin precursors,
caffeic acid and 5-OH-ferulic acid as substrates (Fig. 4B)
(27). It is well known that lignin contributes to the strength of plant
cell walls, facilitates water transport, and impedes the degradation of
wall polysaccharides (28). Therefore, an increase in the activity of
OMTs involved in lignin biosynthesis suggests a participation in the
plant defense response not only against pathogens, insects, and other
herbivores but also against cold stress.
We report here the molecular and biochemical characterization of a
cDNA clone (ScOMT1) that encodes a novel OMT in rye.
This novel gene exhibits 26 to 40% amino acid sequence identity to a
number of plant OMTs and is most closely related to SafBP, a Safener-binding protein that may protect maize against injury from
chloroacetanilide and thiocarbamate herbicides (22), although it did
not exhibit any enzymatic activity. This is not surprising because
ScOMT1, but not SafBP, possesses in the first motif, the conserved
aspartic acid residue proposed to be involved in the binding to AdoMet
(29). ScOMT1 exhibited an exclusive specificity for the methylation of
the 7,8-dihydroxycoumarin, daphnetin. The fact that it did not accept
the 6,7-dihydroxycoumarin analog, esculetin, implies that
meta-directed methylation of esculetin to
7-hydroxy-6-methoxycoumarin (scopoletin), an ubiquitous coumarin derivative, is catalyzed by another distinct OMT (Fig.
5), which has yet to be isolated and
characterized at the molecular level. The ScOMT1 reaction product was
unambiguously characterized as 7-hydroxy-8-methoxycoumarin by
chromatographic and spectroscopic methods in comparison with chemically
synthesized compounds. 7-Hydroxy-8-methoxycoumarin was first isolated
and partially characterized in 1961 from Hydrangea macrophylla and given the trivial name, hydrangetin (30). Since then both the 7-hydroxy-8-methoxy and 7-methoxy-8-hydroxy isomers have
been identified by spectroscopic methods from Daphne
tangutica (31) and Daphne giraldii (32), respectively,
as the main constituents used as analgesic herbal medicines. This also
suggests the existence of a position-specific daphnetin 7-OMT that has
not been yet isolated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
2 s
1 (5/250)
(°C temperature/light intensity µmol m
2
s
1) exhibit a similar tolerance to photoinhibition as
plants grown at high light (20/800) because both cold-acclimated and
high-light plants are exposed to a comparable high excitation pressure
measured as 1-qP (10). Similarly, nonacclimated rye and wheat grown at 20/250 exhibit a similar sensitivity to photoinhibition as plants grown
at low temperature but low light (5/50) because both nonacclimated plants and plants grown at 5/50 are exposed to comparable low excitation pressure. Our previous studies have shown that in addition to the traditional role of photosynthesis in energy transduction, the
redox state acts as a signal that initiates a transduction pathway
coordinating genetic and biochemical responses in wheat and rye plants
(8, 9). Genetic analysis revealed that several genes are associated
with increased photosystem II excitation pressure (9). Of these genes,
one exhibited homology to several plant O-methyltransferases (OMTs).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 s
1 at 5 °C (5/50 and
5/250, respectively) and 50, 250, or 800 µmol m
2
s
1 at 20 °C (20/50, 20/250, or 20/800, respectively).
maxMeOH nm 265, 330; EIMS
m/z (relative intensity (rel. int.) %)
178 [M+] (53), 150 (42), 58 (35), 43 (100);
1H NMR (CD3OD, solvent peak at
3.30 as
internal standard (int. std.)) 6.16 (1H, d,
J = 9.6), 6.79 (1H, d, J = 8.4), 6.97 (1H, d, J = 8.4), and 7.80 (1H, d, J = 9.6).
max nm. 1H NMR spectra
were recorded on a 270 MHz JEOL JHM-EX270 spectrometer in
acetone-d6 or in CDCl3; NOESY by a
500 MHz Bruker AMX500 in acetone-d6 and coupling
constants (J values) in Hz.
maxMeOH nm 254, 324; EIMS
m/z (rel. int.) 192 [M+] (100), 177 (19), 164 (15), 149 (13), 121 (10); 1H NMR
(acetone-d6, 270 MHz, solvent peak at
2.04 as int. std.): 3.93 (3H, s), 6.17 (1H,
d, J = 9.6), 6.87 (1H, d,
J = 8.4), 7.26 (1H, d, J = 8.4), 7.85 (1H, d, J = 9.6), 8.93 (1H,
s, OH). 1H NMR (CDCl3, 270 MHz,
TMS): 4.13 (3H, s), 6.24 (1H, d,
J = 8.4), 6.24 (1H, d, J = 9.6), 6.90 (1H, d, J = 8.4), 7.11 (1H,
d, J = 8.4), and 7.62 (1H, d,
J = 9.6).
maxMeOH nm 259, 319; EIMS
m/z (rel. int.) 192 [M+] (100), 177 (10), 164 (11), 149 (25), 164 (10), 121 (9); 1H NMR
(acetone-d6, 270 MHz, solvent peak at
2.04 as int. std.): 3.94 (3H, s), 6.19 (1H, d,
J = 9.6), 7.01 (1H, d, J = 8.6), 7.12 (1H, d, J = 8.6), 7.84 (1H,
d, J = 9.6), 8.25 (1H, s, OH).
1H NMR (CDCl3, 270 MHz, TMS): 3.99 (3H,
s), 5.71 (1H, s, OH), 6.26 (1H, d,
J = 9.6), 6.86 (1H, d, J = 8.6), 7.01 (1H, d, J = 8.6), and 7.62 (1H,
d, J = 9.6).
3.93 (s) and 7-OH at
8.93 (s) (weak), 7-OH and H-6 at
6.87
(d, J = 8.4 Hz) (weak), H-6 and H-5 at
7.26 (d, J = 8.4 Hz) (medium), H-5 and H-4
at
7.85 (d, J = 9.6 Hz) (medium), and
between H-4 and H-3 at
6.17 (d, J = 9.6 Hz) (medium), whereas in that of 8-hydroxy-7-methoxycoumarin NOE strong
correlation signals were observed between 7-OCH3 at
3.94 (s) and H-6 at
7.01 (d,
J = 8.6 Hz) and medium correlation signals between H-6 and
H-5 at
7.12 (d, J = 8.6 Hz), H-5 and H4
at
7.84 (d, J = 9.6 Hz), and H-4 and H-3
at
6.19 (d, J = 9.6 Hz). These results
indicate unequivocally the substituted patterns of the methoxy protons,
a hydroxy proton, two aromatic protons, and two olefinic protons in
both compounds.
2 s
1 was used to
synthesize double stranded cDNA (Amersham kit) and ligated to
XhoI-EcoRI adaptors as previously described (9).
The library was screened with 32P-labeled cDNA probes
prepared from poly(A)+ RNA isolated from plants grown at
20 °C/800 µmol m
2 s
1 and at
20 °C/250 µmol m
2 s
1 (control plants).
The plaques showing a differential hybridization signal with these
probes were selected and purified using standard molecular biology
techniques (20).
80 °C.
-thiogalactopyranoside
at a final concentration of 1 mM, then harvested 3 h
later. The cells were collected by centrifugation at 8,000 × g for 10 min, and resuspended in 8 mM Tris-HCl,
pH 8.0, 200 mM NaCl. The mixture was sonicated using a
sonic dismembrator 550 (Fisher Scientific) for 1 min 40 s and then
centrifuged at 10,000 × g for 10 min at 4 °C. The
clear supernatant was collected for further purification and enzyme assays.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ScOMT1 sequence analysis. A,
nucleotide and deduced amino acid sequences of ScOMT1.
Shaded amino acids represent conserved residues proposed for
plants OMTs. B, amino acid alignment of ScOMT1 with
various plant OMTs. CjnOMT, Coptis japonica
norcoclaurine 6-OMT (D29811); CjmOMT, Coptis
japonica 3'-hydroxy-N-methylcoclaurine 4'-OMT (D29812);
PshOMT, Pisum sativum 6a-hydroxymaackiain 3-OMT
(U69554); Ms7-OMT, Medicago sativa isoflavone
7-OMT (AF023481); PdOMT, Prunus dulcis OMT
(U82011); ZmOMT, Zea mays OMT (P47917);
TaOMT, Triticum aestivum OMT (U76384);
ZmSafener, Zea mays Safener-binding protein 1 (T01354); AtOMT, A. thaliana flavonol 3'-OMT
(U70424); PtCOMT, P. treuloides caffeic acid
3-OMT (X62096); HvFOMT, Hordeum vulgare flavonoid
7-OMT (X77467); PrCOMT, P. radiata caffeic acid
OMT (X70873). Sequence alignment was done using the Biology Workbench
website version 3.2 at www.workbench.sdsc.edu and Canadian
Bioinformatics Resource (CBR) at www.cbr.nrc.ca. Percentages of amino
acid sequence identity of the various OMTs against ScOMT1 are indicated
at the end of the alignment. Dashes represent
inserted spaces for maximum sequence alignment. I to
V correspond to the conserved regions proposed for plants
OMTs (23) and the shaded areas indicate highly conserved
sequences. C, phylogenetic analysis of various plant
OMT amino acid sequences (substrate preferences in brackets) using
Phylip (Phylogeny Interference Package version 3.57). ScOMT1 is
highlighted. Bootstrap confidence values (n = 100) are shown at the forks. OMT sequences used in this analysis are
the ones listed in A.
-mercaptoethanol to the
protein extract following affinity chromatography did not prevent the
loss of enzyme activity. On the other hand, ammonium sulfate
precipitation followed by DEAE-cellulose chromatography resulted in an
enzymatically active ScOMT1.
max 265 and 330), whereas its mass is increased by 15 mass units, indicating the introduction of a methyl group into
daphnetin. In addition, the chemical structure of the enzyme reaction
product was verified by NOESY in comparison with the other methylated
isomer, which indicates unequivocally the substitution pattern of the
methoxy protons, a hydroxy proton, two aromatic protons, and two
olefinic protons in both 7-hydroxy-8-methoxycoumarin and
8-hydroxy-7-methoxycoumarin as described under "Experimental Procedures."
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Fig. 2.
Analysis of ScOMT1 reaction product.
A, computer enhanced image of the cellulose-TLC
separation of the enzyme reaction product generated by the recombinant
gene product of ScOMt1 using 7,8-dihydroxycoumarin
(daphnetin) as substrate and [14C]AdoMet as cosubstrate.
The conditions used for the enzyme assay and chromatography of the
reaction product are described under "Experimental Procedures."
B, HPLC elution profile of the substrate daphnetin
(Rt = 19.0 min) and the ScOMT1 monomethylated
reaction product (Rt = 26.0 min). C, HPLC
elution profile of the co-chromatography of the ScOMT1 reaction product
(peak 2, Rt = 26.0 min) and
7-methoxy-8-hydroxycoumarin (peak 3, Rt = 27.5 min) co-chromatographed.
Effect of growth temperature and irradiance on 1-qP
2 s
1) as compared with those grown
at 20/250 or 5/50. However, the transcript level is higher in
cold-acclimated plants (5/250) than in 20/800. This could possibly be
because of an increase in mRNA stability at low temperature. The
levels of expression exhibited by ScOMT1 under the various
growth conditions cannot be explained as responses to
either growth temperature or growth irradiance. For example, if we
compare plants grown at either 20/250 or 5/250 we may conclude that the
expression of ScOMT1 is regulated by low temperature. This
is clearly not the case for two reasons. First, rye plants grown at
20/800 and 5/250 exhibited a higher level of ScOMT1 mRNA
than plants grown at 20/250 and 5/50. Second, plants grown at 5/50
exhibited a lower expression of ScOMT1 mRNA than plants
grown at 5/250, indicating that the ScOMT1 transcript is
regulated by PSII excitation pressure. The ScOMT1 cDNA
insert detects a transcript of ~1000 bases, which is within the
expected range for a cDNA clone of that length. This is
corroborated by the kinetics of ScOMT1 transcript
accumulation during exposure to low temperature (5/250) and high light
(20/800) (Fig. 3B). When rye plants grown at 20/250 are
transferred to high excitation conditions, the ScOMT1
transcript gradually accumulates and was highest at 12 days exposure to
20/800 and at 40 days to 5/250 (Fig. 3B). Other treatments
such as wounding, ABA application, heat shock, or salt stress did not
reveal any effect on the expression of ScOMT1 (result not
shown). To our knowledge, these results represent the first report of a
plant OMT transcript that is regulated by PSII excitation pressure and
not by either low temperature or high light per se.
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Fig. 3.
Expression analysis of
ScOMT1. A, Northern blot of
ScOMT1 regulated by PSII excitation pressure. 20/800,
20/250, and 20/50 represent rye plants grown at 20 °C and 800, 250, or 50 µmol m 2 s
1, respectively. 5/250 and
5/50 represent rye plants grown at 5 °C and 250 or 50 µmol
m
2 s
1, respectively. B, kinetics
of ScOMT1 mRNA accumulation during high-light and
cold-temperature exposure: plants grown at 20/250 were transferred to
either 20/800 or 5/250 for the indicated times. Equal amounts of total
RNA (10 µg) were separated by agarose gel electrophoresis in the
presence of formaldehyde and transferred to nitrocellulose membranes.
*, represents an ethidium-bromide stained 28 S ribosomal band as a load
control.
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Fig. 4.
OMT activities of cold-acclimated rye leaves.
A, specific activity of endogenous ScOMt1 protein in rye
leaves grown at the indicated temperature/light (°C/µmol
m 2 s
1) conditions using daphnetin as
substrate. B, specific activities of various OMTs in rye
leaves grown at low temperature. 20/250 represents rye plants grown at
20 °C and 250 µmol m
2 s
1; 5/250,
represents rye plants grown at 5 °C and 250 µmol m
2
s
1. The phenolic substrates used were: Daph,
daphnetin; Esc, esculetin; Api, apigenin;
5-OHF, 5-hydroxyferulic acid; Nar, naringenin;
CA, caffeic acid; Quer, quercetin;
Lut, luteolin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
A proposed pathway for the biosynthesis of
hydroxycoumarins demonstrating the role of the new ScOMT1.
OH, hydroxylase; PAL, phenylalanine ammonia
lyase
Several substituted coumarins have been shown to effectively remove superoxide anions (33). This result was confirmed with fraxetin, a 6,7,8-trisubstituted coumarin that is capable of scavenging superoxide anion radicals, presumably to protect sites of human cytokine activation during the inflammation process (34). These results suggest that coumarins are potent scavengers of peroxyl radicals and are potential candidates for evaluation as protective agents against disorders in which oxidative reactions are implicated. We have demonstrated that the ScOMT1 transcript level is up-regulated in response to PSII excitation pressure created by either low temperature or high light. Maximum activity of ScOMT1 was reached after ~40 days of low temperature acclimation or 12 days at high light exposure. In plants, these two conditions result in increased oxidative stress during prolonged exposure (35, 36). Thus, the enhancement of the active oxygen scavenging system that was induced by low temperature or high light could result in a combined increase of ScOMT1 enzyme activity and accumulation of coumarin derivatives. This suggests a possible role for ScOMT1 methylation of coumarins as a general defense response against oxidative stress.
The exclusive O-methylation of daphnetin by ScOMT1 is quite significant considering the fact that this coumarin has recently been reported as a protein kinase inhibitor (37). It is well known that the process of protein phosphorylation is governed by the complementary activities of protein kinases and phosphatases, both being important for the regulation of cell functions (37, 38). Low temperature treatment has been shown to increase kinase activity and stimulate protein phosphorylation in wheat (39). Therefore, the methylation of daphnetin by ScOMT1 may be considered as a means of modulating the effect of daphnetin on protein kinases, allowing them to function during exposure to high PSII excitation pressure and cold acclimation. It may be possible that daphnetin O-methylation could shift the phosphorylation state of the protein kinase, thus activating the expression of specific genes involved in the modulation of PSII excitation pressure and cold acclimation.
It has recently been reported that daphnetin exhibits a potent inhibitory activity on inflammatory cytokines, because it can be used to treat rheumatoid arthritis, lumbago, and reduce fever in Turkish folk medicine (40). Daphnetin is also being used in China for the treatment of coagulation disorders (41), and may block the action of the various protein kinases involved in the development of these diseases. Taken together, these observations support the potential role of daphnetin methylation in the modulation of protein kinases.
In summary, we have cloned and characterized a novel OMT that catalyzes
the exclusive O-methylation of daphnetin to
7-hydroxy-8-methoxycoumarin. ScOMT1 expression is up-regulated by PSII
excitation pressure and low temperature. Because daphnetin is known to
act as a protein kinase inhibitor, we suggest that its methylation may
be involved in low-temperature signaling. Further studies are required
to confirm the importance of daphnetin methylation in signal
transduction involving protein kinases and/or in the defense mechanism
of the plant against oxidative stress.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ingrid Muzac for generating the phylogenetic tree and Drs. Y. Fukushi and S. Tahara for the synthesis of 7-hydroxy-8-methoxycoumarin and 7-methoxy-8-hydroxycoumarin samples.
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FOOTNOTES |
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* This work was supported by research grants from the Natural Sciences and Engineering Research Council of Canada, Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (to F. S., N. P. A. H., and R. K. I.), and Genome Canada and Génome Québec.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.
To whom correspondence should be addressed: Dépt. des
Sciences biologiques, Université du Québec à
Montréal, C.P. 8888 Succursale Centre-ville, Montréal,
Québec H3C 3P8, Canada. Tel.: 514-987-3000 (ext. 3363); Fax:
514-987-4647; E-mail: sarhan.fathey@uqam.ca.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M209439200
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ABBREVIATIONS |
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The abbreviations used are: PSII, photosystem II; OMT, O-methyltransferase; HPLC, high performance liquid chromatography; ScOMT1, rye cDNA encoding O-methyltransferase; TLC, thin layer chromatography; qP, photochemical quenching.
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