Acta Bot. Need.
30(1/2), February 1981,
Glucose
and
in
sativum
Pisum
alcohol
metabolism
L.
and P.K. Wiersema
H. Winter
Centraal
131-138
p.
Isotopen-Laboratorium, Biologisch
9750 AA Haren
Centrum,
Universiteit
Groningen, postbus 14,
(Gn)
SUMMARY
Glucose absorbed
derivatives.
by
Lower
incubated, strongly
label of absorbed
ted
pea stem segments
alcohols when
of
1-
the
C
mainly
was
aliphatic alcohols
microchemical
and
alcohols.
of these
into
in
compounds.
present in
into
a
which the
number of
stem
In addition it
glucose-
segments
were
found that the
was
of these
Further
sugar-derivatives,
evidence
of the
which sugges-
glycosylation
of
could be obtained with the aid of enzymatical, chromatographical,
mass-spectrometrical
methods.
is present in
ethanol
one
ethyl-glucoside.
that in etiolated pea stem tissue,
If sufficient
metabolized
partly
to solutions
14
ethanol
absorbed lower
It is concluded
was
applied
stimulated the formation
ethanol
glycosylation
a
etiolated
aliphatic
the
glucose
will be
partly conjugated to
cells, ethyl-p-glucoside
becomes
lower
aliphatic
the main
glucose
metabolite.
1.
INTRODUCTION
Winter
(1967)
synthesis
reported
in etiolated
synthesized
the
formation of
could be stimulated
an
from absorbed
indole-3-acetic acid
by
2.
of related
synthesis
MATERIAL
Peas
AND
L.
80% relative humidity.
the
of 10
cm,
cv.
Alaska
After 7-8
mm stem
third internode. These
phosphate
buffer
After 24 hrs
segments
and
5-10
ground
in
D-Glucose
were
6.0)
of
at
successively
14
7)
were
days
segments
with
grown
were
placed
when the
segments
were cut
were
least 25
stem
plants
tate;
containing
trays
a
dark
room at
had reached
water
25 °C and
an
average
from the uppermost portion of
10
ml
aerated
20
mM
addition of other substances.
segments
harvested.
were
rinsed in deionized water, frozen in
C),
ethanol
(1-
14
C)
and
The
stem
liquid nitrogen
(England).
methyl (a)-D-glucoside
14
C
contents
prepared
acetic acid:
were
(U-
14
C)
measured with
a
counter.
Two directional chromatograms from the ethanol
were
in
in
floated in
without the
or
from Amersham
scintillation
segments
on
a mortar.
(U-
purchased
liquid
(pH
samples
were
aliphatic alcohols
compounds.
soaked autoclaved vermiculite. The trays
length
This
publi-
METHODS
sativum
(Pisum
glucose.
The present
(IAA).
cation reports its identification and also the effect of lower
the
glucose-derivative
unidentified
segments of peas
stem
water
on
extracts
Schleicher and Schiill 2043 b
(3:3:1)
and
of the
MgL
ground
with
pyridine: ammonium-hydroxide;
stem
butylace-
isobutanol
132
H.
(4:2:1)
solvents.
as
Kodak No-Sceen
300 g of
C
to a
floated in
were
for 1 min in
a
Virtis
Ionic
subjection
components
20000
at
mesh)
radioactivity detection
removed
Dowex
on
columns. Further fractionation
column
(0.23-1.93 m)
silicagel
with
purity
and
by
by shaking
was
x
added
performed
on
the fraction
water
by
in coldethanol
in
was
a rota
to
centri-
vapor
1
x
8
(100-200
Bio-Gel P
400 mesh
2
layer chromatography
The
(3:3:1).
on
silicagel impurities
mixture of Dowex 50 and Dowex
a
modification
a
at
extract
the supernatant.
Dowex
on a
determined with anthrone reagent after
was
prepared
suspension
8 and
one-dimensional thin
acetic acid;
butylacetate:
removed
WIERSEMA
purposes, part of an
was
50
rpm
evaporated
was
enzymatic hydrolysis
to
were
were
K.
ethanol solutions for 24 hrs and
0.4%
homogenizer
12000 g. The supernatant
at
small volume. For
intended for
The
segments
for 15 min
fuged
were
chromatograms
P.
film.
After extraction with hot ethanol for 30 min, the
80%.
50
stem
homogenized
then
of the
Autoradiograms
X-ray
WINTER AND
by
1.
Fales
(1951).
The
glucose-derivative
ethanol -1was
dissolved
in
water
The
Mannheim).
mixed (1:1
(pH
6.0)
with
a
and
activity
was
Elementary analysis
of 40
U/mg
and
compound
(pH 6.0).
stem
in
segments
layer chromatography,
enzymatic
to
specific activity
C
the
incubating
subjected
glucose-containing
and incubated at 30
v/v)
decrease in
by
C solutions. After two-dimensional thin
glucosidase (EC 3.2.1.21)
ger
labelled
was
14
it
hydrolysis by P-
at
pH
6.0
(Boehrin-
P-glucosidase
After evaporation
to
were
dryness
the
measured.
was
carried
out
with
a
C-H-N Perkin
analyser
model 240
with katarometer detection system.
Mass
spectra
temperature
were
were
hr
acetylated
at a
recorded
with
100 C and the
an
A.E.l.-MS 9
ionizing potential
in the conventional
solved in acetic
manner
under
prior
anhydride-pyridine (10:1)
solutions
temperature of 120°C. The
overnight
3.
were
was
vacuum
instrument. The
70 eV. The
to
They
mass-spectrometry.
and heated in
were
source
dried fractions
evaporated
sealed flask for 1
a
at
55 °C and stored
above KOH.
RESULTS
Table I
shows
the
metabolites after
hrs.
distribution
incubating
These metabolites
(Winter
1967).
stimulates the
are
the
of
stem
version pattern of
of the
glucose
C
over
the
segments
in
main
14
C
at
but above
the expense of the
It
causes
solutions for 24
glucose-derivative
all, of ethanol
glucose-derivative.
soluble
80% ethanol
glucose
amino acids, malic acid and the
Addition of IA A,
synthesis
14
a
to
the
solutions,
shift in
the
con-
synthesis of amino acids and
malic acid.
Besides ethanol also methanol,
stimulate the
length,
the
synthesis
less
radioautographs
of
propanol
and butanol
glucose-derivatives
stimulation
was
involved.
{table 2).
The
significantly (P<0.05)
The
longer
Rf-values of the
the
spots
chain
on
suggested the synthesis of four glucose-derivatives, their
the
com-
GLUCOSE
Table
AND
ALCOHOL
METABOLISM IN
1. The effect of 1AA and
133
PISUM
ethanol
on
glucose absorption
and conversion
Medium
Glucose
Glucose not
Glucose-
solution
absorbed/25
metabolized
derivative
Aminoacids
Malic
acid
segments
I4
0.005%
+
+
12%
gg
40%
18%
33
6
27
36
ethanol
56
14
52
28
C-glucose
820
52
18
15
0.03%
0.1%
42
C-glucose
gg/ml IAA
10
14
+
10
+
0.03%
gg/ml IAA
ethanol
16%
10
5
6
780
41
25
II
9
980
41
46
10
3
position corresponding
to
the alcohol
These results intimated the bio-
applied.
synthesis of alcohol-glucosides.
test
To
sent
in
a
depended
60%
at
14
C-ethanol
this,
directional
high
to
stem
Rf-values
After
segments (fig. I).
0.66-0.85. The
ethanol concentrations. The Rf-values
main
to
the
strongly suggests
glucose-derivative
stem
the
hardly effected
synthesis
its
segments and
of
synthesis
ethyl-glucoside.
up
to an
14
to a
subsequent chromatography
and
values identical with the
Rf-values of the
methanol was
the
effect of lower
Medium solution
to
90%
14
C-glucose
l4
C-glucose
stem
aliphatic
Glucose
addition
+
0.05%
methanol
+
0.05%
ethanol
80%
ethanol
extract
autoradiography
glucose-derivative
alcohols
on
the
synthesis
Glucose-
absorbed/25
derivatives
1050
925
of
to
+
+
+
0.05% propanol-1
0.05%
butanol-1
0,05% pentanol-1
0.05%
hexanol-1
900
910
825
750
the
me-
of the
stem
resulted in Rf-
synthesized
when
glucose-derivatives.
Rf-values
Relative
amounts
17%
0.55-0.80
55
0.66-0.85
45
64
0.55-0.80
100
40
0.55-0.80
0.66-0.85
+
to
0.4%.
segments.
Glucose
775 pg
low
and ethanol
stem segments
0.1%
at
identical with the Rf-
ethanol concentration of
C)
applied
two
pre-
percentage metabolized
were
formed when
mainly
was
This reversed labelling experiment
segments (table 2).
Adding methyl (ot)-D-glucoside (U-
Table 2. The
the
the ethanol concentration applied and varied from
on
applied
dium
with
compound
applied
the label of the absorbed ethanol
chromatography
values of the
were
was
34
31
19
21
0.55-0.80
14
86
15
0.74-0.87
85
0.66-0.85
55
0.94-0.93
45
0.55-0.80
35
0.66-0.85
65
0.55-0.80
52
0.66-0,85
48
%
134
Fig.
H.
1. Ethanol
metabolite, isolated
from pea stem
segments
WINTER
after incubation
AND
P.
K.
WIERSEMA
for 24 hrs in
medium
14
solutions
o
Fig.
pea
o
2.
containing
various
0.5% glucose.
•
concentrations
•
0.1% glucose.
of
A
C-ethanol
A
segments
after incubation
in
l4
a
C-ethanol
glucose.
A
A
I
% glucose.
0.01% glucose.
Enzymatic hydrolysis by P-glucosidase (pFl 6.0)
stem
and
of the
14
solution.
C-glucose
derivative obtained from
GLUCOSE AND
Fig.
A:
3.
ALCOHOL METABOLISM IN
Mass-spectra
0.5% ethanol,
B:
of the
0.4%
glucose-derivatives synthesized in
methanol.
135
PISUM
pea stem segments
during incubation
in
136
H.
The labelled
14
glucose-derivative, prepared by incubating
C-ethanol solutions,
subjected
was
derivative yielded
a
the
AND
P.
K.
WIERSEMA
segments in
stem
enzymatic hydrolysis by p-glucosidase.
to
2 shows that the greater part is broken down.
Fig.
WINTER
Micro-analysis
of this
glucose
ratio of 2, which is in agreement with
hydrogen/carbon
ethyl-
glucoside.
extracted from the
Highly purified compounds
by
electron
solutions
acetyl
(Biemann
glucoside’
corresponded
to
base
mann
1963).
originated
molecular
However
from the
the
could
signals
glucose-derivative
very
4.
+
at
The
3.
sugars with
on
145 and
m/z
peracetylated
original
the
molecular
ion
‘ethyl-
record.
M
3
157
+
It
of the
detected, which is quite usual for
m/z
331
impact (Heyns
present and
was
by
362
the various
much
fig.
of about 10 times the
of the
of electron
m/z
at
analysed
incubated in
shown in
are
peracetylated
the
m/z
same
& Schar-
likely
very
loss of the OCH
a
group.
3
the observation that the
structure was
purchased
M
be
not
were
were
m/z 103, m/z
at
376.
means
fragment
and relative intensities of
glucopyranose
ion
of
molecular ion with
were
of
clearly present
weight
ionization by
upon
Conclusive for the
segments
intensity
an
fragments
nevertheless
peracetate of ‘methyl-glucose’
sugar-derivatives
with
peak
the
were
molecular
a
segments,
present in the spectra of all per-
was
fragments. Typical
The
1963).
small but
was
which
3
in succession
al.
et
+
CO
a
gave
of the other
peaks
groups
CH
was
and
sugars
stem
stem
either methanol or ethanol. The results
fragment
acetylated
highest
impact mass-spectrometry.
containing
The main
The
fragmentation pattern
values of this
those
as
of
plant-made
methyl-P-D-
from Baker Chemicals B.V.
DISCUSSION
The
and
experiments
analyses
show that
glucose externally applied
for the
conjugation
segments is partly used
centration
glucoside synthesized.
The internal sugar
I). Ethanol absorbed by the
also
converted
to
stem
synthesized, though
Evidently
of
the
strongly
at
Raising
stimulates the
now
The
absence
of
a
the
availability
concentration
(Winter
etal.
%
of ethanol
range
the
1970),
least up
an
to
amount
of
are
ethanol
as
is the
absorption
of
rate
reserves.
mobilized for the
not
the
high
rate
as
limiting
4%,
which
amount
of
synthesis of ethyl-P-glucoside.
(0.5%)
synthesis
stimulates the
at
limiting step
glucose
is
0.4%,
ethyl-P-glucoside
that the total
seems
for the
sugar
con-
ethyl-(3-
external
synthesis.
glucose
indicates that somewhere in this concentration
again
stem
applied glucose
glucose
reserves
0.4%
of ethanol, it
difference in
of
ethanol concentration of
ethanol concentration
limiting step
the
also combine with ethanol (fig.
internal sugar
externally applied
significant
centrations of 0.5 and 1
at
absorption
the rate
Under these conditions
is
the
amount
in the absence of
effects the total
which the
ethyl-P-glucoside
available sugar is
to
the total
it does compete with the internal
rate
step in the process.
Up
not
reserves
segments
ethyl-P-glucoside.
externally applied glucose hardly
synthesis
this is
dependent (table I). However,
to
with ethanol. This process is
in the process.
is still concentration
con-
range
In
this
dependent
GLUCOSE AND
A considerable
methanol
the
possible
two
might
source
(Betz
1960).
the
stimulating
Avery
these
the
relatively
respiration
small but
conditions it
will be
ethanol and
because,
Besides
as
it
difficult
in
part
the
Liu
et
to
glucose.
acetaldehyde
al.
experiments
either
lower
to
as
of alcohol in
amounts
glucose
the
a
role of
a
crude
enzyme
a
reactive
highly
toxic substances
be
might
this
tissue,
or
nature.
important (3is
not
clear. It
by conjugation
to
the
increasing
the
advantageous
ratio without
stem
to
be drawn
segments of peas is that
absorbed from the medium
In the
of
presence
to
appears
from
from
that ISG, detected in
physiologically
metabolism in
in the cells
cells. Under
energy-rich compound
ethyl-(3-glucoside
that it
alcohols.
the
sufficient oxygen
toxic levels. The main conclusion
storage
on
that IAA
ethyl-(3-glucoside
an
of
was
high NAD/NADH
to
aliphatic
be
of other
(1966) suggested
on
present
aglycon
physiological
concentration
isolating
(ISG). They suggested
synthesis
maintain a
rapidly growing
synthesize
to
render ethanol into less
to
meristematic cells
the
place
respiration
effect of IAA
maintain
to
(1964), might
the
they demonstrated,
takes
conjugated
able
was
Vitek
seedlings by
be necessary
glucose
more
isosuccinimide-glucose
glucosides. However,
from the
which
seedlings
of pea
extracts
with
significant
in
This
1938, Bonner 1949, and Kelly &
(Reinders
even
anaerobic
explained by assuming
Liu & Castelfranco (1970) succeeded in
supply.
etiolated pea
might
could be
to
our
starts
of 24 hrs.
experimental period
effects fermentation in the
1951), indirectly
growth
in the incubation medium. In the second
synthesis
alcohol-P-glucoside synthesis {table I)
by
In
(table 2).
bacterial
place
be internal for meristematic cells tend
Likewise the
with ethanol or
conjugated
externally applied
In the first
during
is
glucose
not
are
sources.
medium
result in alcohol
might
the
are
containing
sugar
of the absorbed
amount
if these alcohols
even
there
opinion
137
ALCOHOL METABOLISM IN PISUM
become
the
main
be
can
sufficiently
large
glucose
metabolite.
ACKNOWLEDGEMENTS
The
authors wish
A.
to thank
mass-spectrometrical analysis
Kiewiet
and
Dr.
(org.
chem. lab.
RU
Groningen)
J. Bakker for the correction
for this
of the
English
assistance
the
on
text.
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A.
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aerobe
Biemann,
K., D.
C.
De
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Bonner,
J.
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W.
Physiology (Edited by
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chem. Soc. 85:
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and
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Springer-Verlag
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1763-1771.
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Pales,
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und
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K.
Wiersema.
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by growing
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(1970):
The
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in pea stem
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between
Acta. Bot.
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Neerl.
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