Coniferyl alcohol metabolism in conifers - I. Glucosidic turnover of cinnamyl aldehydes by UDPG: Coniferyl alcohol glucosyltransferase from pine cambium

Article abstract of DOI:10.1016/S0031-9422(01)00107-8

UDPG: coniferyl alcohol glucosyltransferase (CAGT; EC 2.4.1.111) isolated from cambial tissues of Pinus strobus was able to convert cinnamyl aldehydes as well as dihydroconiferyl alcohol into their corresponding 4-O-β-D-glucosides in vitro, Cinnamyl aldeh

Full text of DOI:10.1016/S0031-9422(01)00107-8

Phytochemistry 57 (2001) 1085–1093
www.elsevier.com/locate/phytochem
Coniferyl alcohol metabolism in conifers — I. Glucosidic turnover
of cinnamyl aldehydes by UDPG: coniferyl alcohol
glucosyltransferase from pine cambium
b
a,
Valerie Steevesa,Hartmut Fo rsterb,1,Ulrich Pommer ,Rodney Savidge *
aFaculty of Forestry and Environmental Management, University of New Brunswick, Fredericton, NB, E3B 6C2, Canada
bFSU Jena, Institut fur Allgemeine Botanik, Bereich Pflanzenbiochemie, Kuhnhauser Str. 101, 99189 Erfurt-Kuhnhausen, Germany
¨
¨
¨
¨
¨
Received 25 July 2000; received in revised form 1 November 2000
Abstract
UDPG: coniferyl alcohol glucosyltransferase (CAGT; EC 2.4.1.111) isolated from cambial tissues of Pinus strobus was able to
convert cinnamyl aldehydes as well as dihydroconiferyl alcohol into their corresponding 4-O-b-d-glucosides in vitro. Cinnamyl
aldehydes were glucosylated with comparable efficiency to coniferyl alcohol,the physiological substrate for CAGT. Seasonal
patterns of CAGT activity for aldehydes were similar to those of coniferyl alcohol. Formation of cinnamyl aldehyde and additional
monolignol glucosides indicates that precursor flux and availability for lignification is likely greater than previously recognized.
# 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Pinus strobus; Coniferophyta; Pinaceae; Eastern white pine; Cinnamyl aldehyde; Cinnamyl alcohol; Dihydroconiferyl alcohol; Glucoside;
Glucosyltransferase; Seasonal cambial growth
1. Introduction
suspensions of Linum flavum (van Uden et al.,1990),
Paulownia tomentosa bark (Sticher and Lahloub,1982),
flue-cured tobacco leaves (Ito et al.,2000), Daphne
oleides stems (Ullah et al.,1999) and leaves and stems of
Viscum album ssp. (Deliorman et al.,1999). However,
its accumulation evidently is greatest in conifers,such as
Pinus banksiana (Leinhos and Savidge,1993; Leinhos et
al.,1994; Fo rster and Savidge,1995), Larix laricina
(Savidge,1991) and Picea abies (Schmid and Grisebach,
1982),where the contents of endogenous coniferin were
reported to be as high as 10 mmol/gfw. In isolated cam-
bial protoplasts the concentration of coniferin reached
1.6 mM and accounted fully for the content found in
tissue extracts (Leinhos and Savidge,1993; Leinhos et
al.,1994),indicating that very little if any was present in
walls of the primary-walled radially expanded cambial
derivatives from which the protoplasts were obtained.
In springtime,the cinnamyl alcohol glucosides accu-
mulate in cambial derivatives of conifers in the complete
absence of lignification (Savidge,1988,1989,1991;
Forster and Savidge,1995; Fukushima et al.,1997;
Rowland and Arora,1997; Savidge et al.,1998); how-
ever,there is ample evidence that once lignification is
The 4-O-b-d-glucopyranosides of trans-cinnamyl alco-
hols,i.e. E-coniferin and E-syringin,frequently accu-
mulate in the cambial region of woody plants,and they
have long been considered as participants in lignifica-
tion,conceivably as either storage reserves (see,for
example,Terazawa and Miyake,1984) or transported
forms of monolignols (Freudenberg and Torres-Serres,
1967). While coniferin is known to accumulate in con-
ifer cambial tissues (earlier examples include: Freuden-
berg and Harkin,1963; Marcinowski and Grisebach,
1977; Schmid and Grisebach,1982; Terazawa and
Miyake,1984; Terazawa et al.,1984ab, ; Savidge,1988,
1989,1991),it has also been isolated from cambial tis-
sues of various angiosperms (Terazawa et al.,1984ab, ),
needles of Douglas fir (Morris and Morris,1990) and
Norway spruce (Slimestad and Hostettmann,1996),cell
* Corresponding author. Tel.: +1-506-453-4919; fax: +1-506-453-
3538.
E-mail address: savidge@unb.ca (R. Savidge).
Joint first author
1
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00107-8

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V. Steeves et al. / Phytochemistry 57 (2001) 1085–1093
underway they can contribute to that process (Forster et
al.,1999,2000),as earlier surmised by Freudenberg and
Harkin (1963). In order for coniferin to accumulate to
high levels,it must either be compartmentally shielded
or produced many times more rapidly than it turns over
(Marcinowski and Grisebach (1977) report a half-life of
60–120 h for coniferin) or is utilised in support of lig-
nification. Freudenberg (1964) reported that when the
coniferyl alcohol moiety of coniferin was radiolabelled,
the radioactivity was readily incorporated into spruce
lignin. The incorporation of isotopically labelled mono-
lignol glucosides into lignin has been further investi-
gated in autoradiographic studies by Terashima et al.
(1986,1993,1995,1997),Terashima and Fukushima
(1988) and Eglinton et al. (2000). Monolignol glucosides
enriched with carbon-13 at specific side chain carbons
were administered to growing tree stems resulting in
newly formed lignin shown to be carbon-13 enriched at
the side chain carbons corresponding to those of the
glucosides (Terashima et al.,1995; Xie and Terashima,
1991; Xie et al.,1994).
content varied parallel to seasonal cambial activity
(Savidge,1991),and biochemical investigations revealed
that the presence or absence of UDPG: coniferyl alco-
hol glucosyltransferase (CAGT; EC 2.4.1.111) activity is
the explanation for the qualitative seasonal variation of
coniferin,indicating that seasonal variation in coniferin
has its explanation in seasonal change in gene expres-
sion (Forster and Savidge,1995; Savidge and Fo rster,
1998; Forster et al.,2000).
Ibrahim and Grisebach (1976) were first to report
purification and characterization of a CAGT,isolated
from suspensions of Paul’s scarlet rose. The enzyme cat-
alysed the transfer of glucose from UDPG to coniferyl
alcohol and had the highest substrate specificity reported
to date for a CAGT (Km=3.3ꢀ10ꢁ9 M for coniferyl
alcohol; Km=2ꢀ10ꢁ5 M for UDPG). In 1977,Ibrahim
confirmed the substrate specificity using CAGT isolated
from young lignifying stems of Forsythia ovata,demon-
strating it to be distinct from other glucosyltransferases,
which are capable of nonspecifically glucosylating
hydroxy phenols,flavonols,anthocyanidins and phe-
nolic acids.
In agreement with the concept that the activity of
coniferin-hydrolysing b-glucosidase may influence the
rate of lignification (Grima-Pettenati and Goffner,
1999),a b-glucosidase specific to coniferin and of much
lower abundance and/or activity than numerous other b-
glucosidases of wood and cambial tissues was isolated,
primarily on the basis of its unique ability to hydrolyse
authentic coniferin to E-coniferyl alcohol,as confirmed
by GC/MS (Leinhos et al.,1994). It remains unclear
whether that coniferin-specific b-glucosidase is identical
to the enzyme investigated by Dharmawardhana et al.
(1995,1999),the N-terminal sequences of the two being
notably distinct although both evidently have similar pI
values,or to the enzyme isolated from inner secondary
cell walls in young spruce seedlings by Marcinowski et
al. (1979),or that localized to xylem epi-,endo- and
exodermis in stems and roots of chick pea seedlings by
Burmeister and Hosel (1981). A problem may exist,
however,in using antibodies for localisation; suppo-
sedly specific antibodies have been repeatedly observed
to bind non-specifically with lignified secondary cell
walls (R.A. Savidge,unpublished data).
Coniferin has been found present in the cambial zone
and its derivatives throughout the period of cambial
growth,but it becomes undetectable at the onset of cam-
bial dormancy in autumn,before the last cambial deriva-
tives have completed their maturation into latewood
(Savidge,1988,1989,1991; Fo rster and Savidge,1995;
Savidge et al.,1998). Coniferin has never been detected
in stems during cambial dormancy (Freudenberg and
Harkin,1963; Terazawa and Miyake,1984; Savidge,
1988,1989,1991; Fo rster and Savidge,1995; Savidge et
al.,1998). Quantitative investigations of endogenous
indol-3-ylacetic acid,sucrose and coniferin in Larix lar-
icina cambium revealed that,of the three,only coniferin
Schmid and Grisebach (1982) purified and character-
ized CAGT from Picea abies,and found coniferin to
inhibit CAGT in a noncompetitive manner,with pro-
duct inhibition starting at approximately 10 mmol/g.
Schmid et al. (1982) also immunolocalized CAGT to the
epidermis,subepidermis and vascular bundles of young
P. abies seedlings. Intracellularly,it was located in the
parietal cytoplasmic layer,indicating that CAGT is a
soluble enzyme of the cytoplasm,in agreement with
Ibrahim (1977). Forster and Savidge (1995) reported that
coniferin below 10 mmol/gtissue showed no inhibitory
effect on activity of CAGT of Pinus banksiana,but it did
inhibit CAGT above 10 mmol/g. The catalytic activity of
the coniferin-specific b-glucosidase isolated from lignify-
ing tissues was not found to reside in CAGT obtained
from the same tissues,at the same developmental stage
(Savidge and Forster,1998). Recently,Fo rster et al.
(1999,2000) confirmed the pronounced specificity of
CAGT for UDPG; however,they unexpectedly found
that CAGT from P. strobus also expressed high activity
when UDP-galactose was supplied to the enzyme.
E-Coniferaldehyde has been detected in the cambial
sap of conifers (Freudenberg and Neish,1968),bamboo
(Tanaka et al.,1998) and oak (Ferna andez de Simon et
al.,1996). Despite the generally low endogenous con-
iferylaldehyde levels in plants and the efficient reduction
of cinnamyl aldehydes to cinnamyl alcohols,coniferyl
aldehyde glucosides have nevertheless been isolated
from bark of Syringa velutina (Park et al.,1999) and
Kolopanax pictus (Sano et al.,1991).
Cinnamyl aldehydes have also been reported as sui-
table substrates for CAGT (Ibrahim and Grisebach,
1976; Schmid and Grisebach,1982; Fo rster et al.,1999,
2000). Preliminary investigations indicated that CAGT

V. Steeves et al. / Phytochemistry 57 (2001) 1085–1093
1087
catalysed the conversion of cinnamyl aldehydes to their
corresponding 4-O-b-d-glucosides in vitro (Forster et
al.,1999,2000). CAGT from Pinus strobus appeared to
produce higher amounts of cinnamyl aldehyde gluco-
sides than monolignol glucosides; however,the specific
turnover rate (i.e. ratio between administered substrate
and obtained product) was approximately 8 times lower
(Forster et al.,2000). In seasonal investigations of
CAGT activity,Fo rster et al. (2000) presented evidence
for in vitro generation of coniferyl aldehyde glucoside to
parallel similar seasonal patterns to that observed for
coniferin. In the present study,we confirm,for the first
time,by proton NMR spectroscopy,the enzymatic glu-
cosidic turnover of cinnamyl aldehydes catalysed by
CAGT isolated from pine cambial tissue. Seasonal pat-
terns of CAGT activity for monolignols and cinnamyl
aldehydes are provided and their Km values reported for
the first time. This is the first report of in vitro bio-
synthesis of dihydroconiferyl alcohol glucoside.
and Grisebach’s (1982) findings for Picea abies.
Although Ibrahim (1977) obtained small amounts of
iso-coniferin (i.e. trans-coniferyl alcohol-1-O-b-d-gluco-
side) from Forsythia ovata,this isomer of coniferin to
our knowledge has not yet been reported in conifers.
1H NMR data for the enzymatic products,along with
that of coniferyl alcohol,are presented in Table 1,while
corresponding molecular structures are found in Fig. 1.
Carbon 9 (Table 1) refers to either the primary alcoholic
carbon atom (i.e. as in coniferin (1),syringin ( 2),dihy-
droconiferyl alcohol glucoside (5) and coniferyl alcohol)
or the aldehydic carbon atom (i.e. as in coniferyl alde-
hyde glucoside (3) and sinapyl aldehyde glucoside (4)).
As evidenced by large coupling constants for the 7-H/8-
H of the double bond (i.e. J=15.8,15.8,16.6 and 14.8
Hz for 1–4,respectively),products
1–4 maintained a
trans orientation. Chemical shifts for glucosyl hydro-
gens are not reported,but were observed ranging from
ꢀ=3.1–3.8 ppm.
Chemical shifts of 9.61 and 9.63 ppm,corresponding to
the aldehyde hydrogens for 3 and 4,respectively,clearly
demonstrate that the glucosyl moiety must have attached
at the 40-position of the aromatic ring,as opposed to the
primary alcohol of the phenylpropane side chain.
When comparing the 1H NMR spectra of coniferin and
coniferyl alcohol,a larger up-field shift (i.e. up to 0.38
ppm) is observed for the aromatic hydrogens compared
to the aliphatic ones. This suggests the glucose attached
to the phenol oxygen,and not that of the primary alco-
hol. Since the structures and chemical shifts for 2 and 5
are similar to 1,the same conclusions were obtained.
As previously mentioned, E-coniferin formation has
been reported in various plants throughout the plant
kingdom; however,by way of contrast,American beech
exceptionally accumulates cis-monolignols. Yamamoto
et al. (1990) reported a glucosyltransferase in bark of
Fagus grandifolia,which showed an unusual preference
for Z-coniferyl alcohol catalysing the formation of Z-
coniferin. The glucosyltransferase isolated from cambial
tissues of Pinus strobus did not accept cis-coniferyl
alcohol and a mixture of cis and trans substrates always
resulted in formation of E-coniferin,as evidenced by
HPLC separation (results not shown). Plausibly,the
maintenance of a trans orientation during catalysis is an
essential requirement for converting suitable substrates to
4-O-b-d-glucosides in the cambium of Pinus strobus.
The catalytic activity of UDPG: CAGT in the for-
mation of monolignol glucosides increased more than 3-
fold in early June compared to CAGT isolated in mid-
May (Fig. 2). The data agree with results obtained from
cambial tissue of Pinus banksiana (Savidge and Forster,
1998); however,considerable differences in CAGT
activity observed between cambium and developing
xylem of Pinus banksiana did not appear in correspond-
ing tissues of Pinus strobus (data not shown). Seasonal
fluctuations in CAGT activity and levels of monolignol
2. Results and discussion
Molecular structures of the predicted enzymatic pro-
1
ducts were confirmed by H NMR spectroscopic ana-
lyses. Two-dimensional COSY and HMQC NMR
spectra were obtained to aid in structure elucidation. For
both semi-preparative and analytical HPLC,an assay
was developed to effectively separate the cofactor uri-
dine 50-diphosophoglucose (UDPG),the monolignol
substrates and the corresponding product glucosides,
allowing exact quantitation of the catalysed reaction.
Based on this assay,enzymatic products were separated
using semi-preparative HPLC techniques,collected and
lyophilized,resulting in mg quantities of the desired
compounds.
The monolignol-specific UDPG was extracted and
partially purified (Savidge and Forster,1998) from
approximately 400 g of differentiating xylem from Pinus
strobus harvested in late May. IEF and native PAGE
indicated purification to homogeneity,however,SDS–
PAGE and N-terminal sequencing of the resulting major
band revealed the presence of two proteins,one without
any evident homology to known proteins and the other
yielding perfect homology to the internal sequence of a
glucosyltransferase precursor. Because of the almost
identical physico-chemical features (pI 5.0 and mole-
cular weight 43 kDa) of both proteins,complete pur-
ification could not yet be achieved (Forster et al.,2000).
All substrates tested in the assay were successfully
converted to their 4-O-b-d-glucosides,as evidenced by
the chemical shifts of the glucosyl moieties (not repor-
1
ted) in the H NMR spectra. The glucosyl moiety did
not bond to the alcoholic hydroxyl group of the phe-
nylpropane side chain of the monolignol substrates (i.e.
as in iso-coniferin),which is in agreement with Schmid

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V. Steeves et al. / Phytochemistry 57 (2001) 1085–1093
Table 1
1H NMR spectral data for the monolignol glucosides and coniferyl alcohol
ꢀ 1H (ppm) [int,mult, J (Hz)]
C
Coniferyl aldehyde
glucoside
Sinapyl aldehyde
glucoside
Dihydroconiferyl Coniferin
alcohol glucoside
Coniferyl
alcohol
Syringin
20
7.32
7.03
[1 of 2H, s]
6.86
7.06
[1H, d,1.9]
7.00
[1H, d,1.9]
6.74
[1 of 2H, s]
[1H, d,1.6]
[1H,d,2.2]
50
7.21
–
7.07
[1H, d,8.2]
7.10
[1H, d,8.3]
6.72
[1H, d,8.0]
–
a
a
[1H, d,8.3]
60
7.25
7.03
[1 of 2H, s]
6.74
6.94
[1H, dd,8.3,1.9]
6.84
[1H, dd,8.1,1.6]
6.74
[1 of 2H, s]
[1H, d,8.3]
[1H, dd,8.2,2.1]
7
7.61
7.61
[1H, d,16.0]
2.63
[2H, t,7.6]
6.54
[1H, br d,15.8]
6.50
[1H, br d,15.9]
6.54
[1H, br d,15.9]
[1H, d,16.0]
8
6.71
6.78
[1H, dd,14.8,7.0]
1.82
[2H, tt,7.7,6.3]
6.27
[1H, dt,15.8,5.6]
6.19
[1H, dt,15.6,5.9]
6.32
[1H, dt,15.8,5.6]
[1H, dd,16.6,7.7]
9
9.61
9.63
[1H, d,7.7]
3.55
[2H, t,6.5]
4.20
[2H, dd,5.8,1.4]
4.19
[2H, dd,6.0,1.1]
4.21
[2H, dd,5.6,1.0]
[1H, d,8.0]
OCH3
3.91
3.86
3.85
3.87
3.85
3.85
[3H, s]
[6H, s]
[3H, s]
[3H, s]
[3H, s]
[6H, s]
Both C30 and C50 methoxyls yield equivalent proton shifts.
a
Fig. 1. Structure of monolignol glucosides.
glucosides in cambial tissue parallel each other (Fig. 2,
Savidge et al.,1998) with comparable efficiencies in the
formation of aldehydic and alcoholic glucosides.
The effective turnover of both cinnamyl alcohols and
cinnamyl aldehydes by CAGT during the growing sea-
son was confirmed by the determination of the apparent
Km values of the various substrates (Table 2). The appar-
ent Km values were obtained from Lineweaver–Burk plots
at saturating concentrations of UDPG. For coniferyl
alcohol,when assayed in phosphate buffer at pH 7.6,a
Km of 120 mM was obtained. Sinapyl alcohol,although
not synthesized in pines,also served as a substrate with an
apparent Km of 154 mM. The data confirm former results
for Pinus banksiana,where sinapyl alcohol reached 96%
of the relative activity of coniferyl alcohol (Savidge and
Forster,1998). A comparable Km value (250 mM) was
reported for spruce (Schmid and Grisebach,1982).
In addition,dihydroconiferyl alcohol (DHCA) was
accepted as a suitable substrate for the pine CAGT and
converted to its corresponding 4-O-b-d-glucoside (Fig. 1,
Table 1). The 4-O-b-d-glucoside has been reported in
needles of Pinus contorta (Higuchi et al.,1977). Ralph et
al. (1997) recently reported dihydroconiferyl alcohol as a
main constituent of lignin in a mutant loblolly pine. The
involvement of dihydrophenylpropanoids in the bio-
synthesis of plant pigments (Schmitt and Schneider,
1999) suggests,in general,that dihydro-compounds
play a role in secondary metabolism.

V. Steeves et al. / Phytochemistry 57 (2001) 1085–1093
1089
Fig. 2. Seasonal activity pattern of UDPG:CAGT in developing xylem of Pinus strobus as determined by in vitro experiments.
Table 2
Enzymatic parameters of the partially purified UDPG:CAGT from
cambium of Pinus strobus
Scarlet Rose (Ibrahim and Grisebach,1976) was
expressed as relative activity in comparison to coniferyl
alcohol. Because of the lack of authentic compounds for
co-chromatography,the structure of the presumed glu-
cosidic products could not be unequivocally elucidated.
Hence,the data in this report based on HPLC separa-
tion and NMR investigation (Table 2) confirm,to our
knowledge for the first time the real structure of the
enzymatic products (Fig. 1). In contrast to the gluco-
syltransferase purified from Forsythia ovata (Ibrahim,
1977) and in agreement with the glucosyltransferase
purified from Picea abies (Schmid and Grisebach,1982)
the CAGT from Pinus strobus expressed almost no
activity with cinnamic acids (Forster et al.,2000). The
glucosylation of cinnamic acids (Nurmann and Strack,
1980; Lorenzen et al.,1996; Fons et al.,1998; Wang and
Ellis,1998) has been demonstrated previously,with the
glucosyl moiety attached to the carboxyl group of the
side-chain.
The observed seasonal fluctuations of both cinnamyl
alcohol and aldehyde glucoside formation (Fig. 2) coin-
cide with events of wood formation,i.e. formation of pri-
mary walled radially enlarged cells and the first
appearence of differentiated tracheids undergoing sec-
ondary-wall formation and lignification (Savidge and
Forster,1998). Preliminary examination revealed both
high activities of the glucosyltransferase and develop-
mental processes during June and July (Fig. 2) in the
cambial zone,thus concentrating the characterization
and purification of the CAGT to this time of the season.
Lignification is not developmentally correlated to the
formation of monolignol glucosides as shown previously
Substrate
Enzymatic product
Km (mM) Vmax
ꢁ1
(nMꢀmg
protein
ꢀminꢁ1
)
Coniferyl alcohol Coniferin
Sinapyl alcohol Syringin
Coniferaldehyde Coniferaldehyde-4-
119.6
153.5
317.4
1.17
1.54
1.85
O-b-d-glucopyranoside
Sinapaldehyde-4-
Sinapaldehyde
241.7
1.38
O-b-d-glucopyranoside
Coniferyl aldehyde and sinapyl aldehyde glucosyla-
tion resulted in Km values of 317 and 242 mM,respec-
tively,thus working in close proximity to the order of
cinnamyl alcohols. Nevertheless,existing differences in
the enzymatic turnover of cinnamyl alcohols and alde-
hydes,evident by the reported Km values,underline that
the hydroxyl group of the phenylpropane side-chain
constitutes a more efficient substrate configuration. As
reported before (Forster et al.,2000),cinnamyl alcohols
and cinnamyl aldehydes compete with each other for
CAGT when provided as dual substrates,with alcohols
being more efficient in inhibiting the formation of cin-
namyl aldehyde glucosides.
Cinnamyl aldehydes have been previously reported as
appropriate substrates; however,structure and physi-
ological relevance was not investigated. The amount of
aldehydic glucosides obtained in investigations of the
substrate specificity of CAGT in Picea abies (Schmid
and Grisebach,1982) and suspension cultures of Paul’s

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V. Steeves et al. / Phytochemistry 57 (2001) 1085–1093
(Leinhos and Savidge,1994; Savidge and Fo rster,1998).
However,some observed metabolic connections to
wood formation and lignification once the glucosides
are produced in abundance needs further clarification.
CAGT,in catalyzing the formation of cinnamyl alco-
hol glucosides,evidently competes with coniferyl alco-
hol dehydrogenase (CAD) and monolignol oxidising
enzymes for coniferyl alcohol. Our data (Fig. 2,Table 2)
indicate that the glucosyltransferase must also compete
with CAD for available cinnamyl aldehydes by forming
the corresponding aldehydic glucosides (Fig. 1).
Results obtained in the past few years have prompted
a reconsideration of the established lignin biosynthetic
pathway. It is clear now that inter-conversions of lignin
precursors may occur at varied points along the lignifica-
tion pathway,and not necessarily solely at the phenyl-
propanoid acid stage. In addition,the ratio of guaiacyl to
syringyl units can be modified at the level of CoA esters
(Grimmig et al.,1999),cinnamyl aldehydes (Osakabe et
al.,1999),cinnamyl alcohols (Chen et al.,1999ab, ) and
monolignol glucosides (Matsui et al.,1996).
cryogenic vials (Savidge et al.,1982). The tissue was
then stored in liquid nitrogen until preparation for
CAGT analysis.
3.3. Enzyme isolation
Buffer systems used include: extraction buffer (0.2 M
phosphate (Na2HPO4,NaH PO4),10% polyvinylpyrroli
2
done,7.5% glycerol,15 mM aminocaproic acid,10 mM
dithiothreitol,14 mM b-mercaptoethanol,pH 7.6),equi-
libration buffer (0.1 M phosphate (Na2HPO4,NaH 2PO4),
14 mM b-mercaptoethanol,pH 7.6),sodium phosphate
buffer (Na2HPO4,NaH PO4,100 mM,pH 7.6). Cam-
2
bial tissue was pulverized to a homogeneous powder
and extracted for 1 h in extraction buffer. The suspen-
sion was filtered and the filtrate centrifuged for 20 min
(4ꢂC) at 15,500ꢀg. The supernatant was centrifuged (1
h,4 ꢂC) at 100,000ꢀg to remove microsomes. Protein
in the supernatant was precipitated with (NH4)2SO4.
Protein precipitated between 40 and 85% (NH4)2SO4
was collected by centrifugation (23,300ꢀg,20 min,4 ꢂC).
The resulting pellet was resuspended in phosphate
buffer (2.5 ml),then applied to Sephadex G-25 (20 mm
i.d.ꢀ100 mm,previously equilibrated in equilibration
buffer). One ml fractions of soluble protein were
collected. Fractions showing transferase activity were
pooled and concentrated by centrifugation at 2455ꢀg
using a Centricon YM-10 concentrator. All subsequent
isolation and purification of UDPG: coniferyl alcohol
glucosyltransferase (CAGT) was carried out according
to Forster and Savidge (1995) and Savidge and Forster
(1998).
Taken together,the questions of if and how CAGT
(i.e. alone or in a regulatory manner with the coniferin-
specific b-glucosidase) may participate in controlling
availability of cinnamyl alcohols and/or cinnamyl alde-
hydes for lignin formation deserves further efforts.
3. Experimental
3.1. General experimental
Coniferyl alcohol,sinapyl alcohol,coniferyl aldehyde
and sinapyl aldehyde were purchased from Aldrich. E-
coniferin was isolated from P. strobus (Savidge,1989)
and dihydroconiferyl alcohol was obtained as pre-
viously described by Savidge (1987). Before processing
and HPLC analysis,handcut sections of cambium and
developing xylem were examined by microscopy as pre-
viously described by Savidge (1988).
3.4. Protein determination
Protein concentrations were determined using the
method developed by Lowry et al. (1951),modifying final
volumes to 40% of the original assay. The UDPG: CAGT
was then applied in a 40-fold dilution,using bovine serum
albumin as a standard,and measured at 578 nm.
3.2. Trees
3.5. Semi-preparative HPLC and product separation
Healthy Pinus strobus between 15 and 18 years old
growing in the University of New Brunswick Woodlot,
Fredericton,NB,Canada,were selected. Sufficient
amounts of protein for this study were isolated for pur-
ification from a number of trees harvested in late May
1997. Individual trees were harvested during the growing
season (April–September) for investigations into seaso-
nal CAGT activity patterns. On each date,stem seg-
ments from the main axis were removed with a handsaw
and brought directly to the laboratory for processing.
The bark was peeled from the wood and the exposed
inner bark surface (i.e. cambial zone) and wood surface
(i.e. developing xylem) were scraped individually into
Reversed-phase HPLC of the enzyme assay aliquots
was performed according to Savidge (1989) on a Milton
Roy system with a semi-preparative C18 ODS Ultra-
sphere column (Beckman,5 mm spheres,150 ꢀ10 mm
i.d.). Assays were carried out on a larger scale (i.e. total
volume 2 ml) in order to obtain sufficient product for
analytical investigations. The enzyme incubation mixture
consisted of 100 mM phosphate buffer (pH 7.6),2 mM
UDPG,1mM of the substrates in question and
approximately 800 mg protein. Enzyme assay incubation
time was 60 min at 36ꢂC. Product separation was
achieved with a linear gradient over 30 min (5–100%
MeOH,adjusted to pH 3.0 with acetic acid) with a flow

V. Steeves et al. / Phytochemistry 57 (2001) 1085–1093
1091
rate of 5 ml/min and UV detection at 260 nm: Rt con-
iferin 8.0 min,266 nm: Rt syringin 8.7 min,330 nm: Rt
coniferyl aldehyde glucoside 9.0 min,320 nm: Rt sinapyl
aldehyde glucoside 9.8 min,262 nm: Rt p-coumaryl
alcohol glucoside 8.7 min,276 nm: Rt dihydroconiferyl
alcohol glucoside 8.3 min.,266 nm. Enzymatic products
were collected from semi-preparative HPLC eluant and
freeze dried. The compounds were characterised by
with a flow rate of 0.5 ml/min. Column temperature was
adjusted to 26ꢂC. Data analysis was accomplished using
Borwin PDA (version 1.0) and chromatography (ver-
sion 1.22.03 B) software.
The enzyme incubation mixture (i.e. total volume 200
ml) contained 200 nM UDPG in 10 ml H2O,various
amounts of substrate in 10 ml MeOH, 80 ml sodium phos-
phate buffer and approximately 100 mg protein dissolved
in sodium phosphate buffer. The various substrates were
incubated for 10 min at 36ꢂC,then terminated using 20
ml HOAc (50%). Km values were calculated at fixed
concentration of UDPG (1 mM) by linear regression of
1/v vs. 1/s (Lineweaver-Burk plot) with Grafit (version
4.0.1). All experiments were performed in triplicate
and compared to a control containing all constituents
but terminated prior to catalytic activity by HOAc
precipitation of the enzyme.
1
GC–MS and H NMR and used as standards in analy-
tical HPLC.
3.6. Ultraviolet-visible spectra
Ultraviolet-visible spectra of the purified enzymatic
products were obtained using a computer-controlled
UV/vis spectrophotometer (Shimazu,UV-2102-PC).
Spectra were recorded from 195 to 450 nm and com-
pared to spectra obtained in analytical HPLC work-up.
Coniferin (1) lmax (MeOH) nm (log "): 260 (1,3), 214,
lmin (MeOH) nm: 238, lshoulder (MeOH) nm: 296; syr-
ingin (2) lmax (MeOH) nm (log "): 266,(12, 3),221, lmin
(MeOH) nm: 240; coniferyl aldehyde glucoside (3) lmax
(MeOH) nm (log "): 330 (1,16), 236, lmin (MeOH) nm:
266, lshoulder (MeOH) nm: 306; sinapyl aldehyde gluco-
side (4) lmax (MeOH) nm (log "): 320 (0,55), 236, lmin
(MeOH) nm: 268; dihydroconiferyl alcohol glucoside
(5) lmax (MeOH) nm: 276,224, lmin (MeOH) nm: 248.
Acknowledgements
We would like to thank Dr. L. Calhoun for support
on compilation of the NMR data for the cinnamyl glu-
cosides. V.J.S. and R.A.S. thank NSERC for research
funding. HF was supported by a Grant-in-Aid (FO 271/
4-1) of the Deutsche Forschungsgemeinschaft (Bonn,
Germany).
3.7. Nuclear magnetic resonance
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