Alcohol
the
dehydrogenase
cotyledons
maturation
C.
of peas
and
activity
in
during
germination
Kollöffel
Botanisch
Laboratorium,
Utrecht
SUMMARY
A
quantitative biochemical
pea
cotyledons
days
of
and histochemical
indicated that the
germination.
The
is formed
enzyme
of the
study
alcohol
decreased
activity
enzyme
during
seed
dehydrogenase activity
about
40
% during
and
development
the first
of
seven
is reactivated
by
hydration only during germination.
During homogenization
liberated
acids
which
are
-
absent
of seeds
genates
in
for
to avoid
are
also
To ascertain
day.
several
substances
and
cotyledons
one
germinated for
peas
These
enzyme.
germinated for
or
of
cotyledons
the
the intact
medium
extraction
of
germinated
of peas
cotyledons
the
inactivate
it is
days,
of
alcohol
the
substances
days,
are
probably long-chain fatty
absent in
the
the
tissue
and homo-
cotyledons
dehydrogenase activity
to add bovine
necessary
homogenization
several
very
-
by
albumin
serum
using
a
of
to
histochemical
method.
1.
INTRODUCTION
Germinating
caused
by
seeds
pea
the poor
Frietinger
and ethanol
1969).
ethanol
are
metabolized
The enzymes
onset
of
Cossins
high
an
Turner
in
et
1962;
the seeds
Faccioli
recently
activity
Acta
of
been
and
& Dupéron
1923;
of
period
Beevers
1963;
14
when the
ceases
largely depends
Leblovâ
1966;
conditions,
the
anaerobic
on
et
lactic acid
al.
and
C-labeled lactate and
Cossins
were
1959;
al.
1968).
The
from pea
Cossins
et
allowed
al.
to
described
&
Turner
1963;
1970
studied fre-
catalytic
cotyledons (Suzuki
1949;
a
few
days
Goksôyr
Kollöffel
germinate
been
and
1968)
et
pro-
1966;
after the
al.
1953;
but remains
under anaerobic conditions
1968).
(Kolloffel
was
1968;
have
physical
decreases
activity
Davison
1944;
Kollöffel
19(4), August
respiration
much known about the
7-day-old cotyledons
Bot. Neerl.
This
1967).
1963)
by experiments with
The histochemical localisation of ADH
has
1962;
under aerobic
&
(Fernandes
accumulation of lactic acid (Cossins
1959;
dehydrogenase (ADH)
1968; Cossins
when
(Maffei
shown
germination (Virtanen
&
1959; Kollöffel
anaerobiosis
partial
coat
1967).
relatively
of alcohol
of
The accumulation of ethanol
develops
as
involved
There is
Eriksson
coat.
1962; Cossins
Cameron & Cossins
perties
Yemm
period
a
of the intact seed
2
& Turner
(Cossins
seedling
(Cossins
quently.
0
during germination (Doireau
When the
ethanol
experience
accompanied by
radicle ruptures the seed
the conditions
&
Spragg
1927;
natural anaerobiosis is
1964)
always
permeability for
only
activity
1969).
in
It
germinating
was
pea
found that
cotyledons
the
somewhat lower than that of
ADH
1-day539
C.
old
in
cotyledons
with
contrast
earlier
In the present
(Kolloffel 1968).
assays
extended. It will be shown that the
results
obtained
extracts
from
MATERIALS
2.1.
23°
at
e.g. 7
will
days
of the
buffer and
tetrazolium
25° the reaction
at
0.1 N HC1. The
followed
A few ml
ature
of
a
not
may
mazan
ethanol
was
extract at
540
nm
for
washed with
phosphate
Ltd.).
buffer
(nitro-blue
After 30 min of incuba-
the sections
with
thoroughly
substrates
by endogenous
was
medium without ethanol. Details of this
a
(Kolloffel
1969).
formazan of Nitro-BT
N,N-dimethylformamide
was
and
0.1
heated
carefully
collected and the
extract was
determined
germinated
and Nitro-BT
(0.4 M)
Houses
Drug
sections in
were
of peas
containing;
be exceeded because otherwise the sections
extracts
water
moist filter
paper in
thoroughly
were
reduction of Nitro-BT
mixture of
few minutes the
combined
to
cotyledons
medium
a
the stained sections and this
to
soaked in aerated tap
cotyledons.
stopped by washing
was
Extraction of the
added
The
cotyledons
in
have been described elsewhere
procedure
from
sections and conditions of incubation
(1.35 mM),
unspecific
by incubating
were
transferred
“7-day-old”
as
The British
chloride;
”
Rondo
next
germination.
incubated
NAD
(0.05 M, pH 6.4),
tion
“
cv.
and
freehand sections of the
cut
phosphate
2.3.
in darkness
be mentioned
Preparation
Freshly
germinated
extracts
conditions
Petri dishes for further
large
absent in
are
METHODS
seeds of Pisum sativum L.
Air-dry
2.2.
AND
Germination
for 20-22 hrs
of seeds
cotyledons
seeds.
newly germinated
2.
biochemical
these studies have been
investigation,
for several days contain inhibitors of ADH, which
with
KOLLOFFEL
centrifuged
to
the
optical
destroyed.
were
amount
of the
After
a
twice. The
repeated
was
density
standard solution of
a
are
(1:1)
80°. This temper-
cell debris. The
remove
by comparing
with that of
procedure
N HC1
to
of for-
centrifuged
reduced Nitro-
chemically
BT.
Because of its
high affinity
for cellular structures Nitro-BT
washed from the sections after incubation and,
cally
at alkaline
After
pH, it
incubation,
was
necessary
the sections
were
all
perform
to
it is
as
dried for
was
easily
operations
about 20
not
completely
reduced
hrs
at an
at
unspecifiacid
pH.
60° and
next
weighed.
ADH
weight)
activity
per
was
expressed
30 min. The
reduction of Nitro-BT
Although
the
occurring
formazan
method described is yet
a
was
a
also
of tissue
540
the
amount
linear
formazan
always
produced
corrected for
per
a
g
tissue
(dry
slight unspecific
in the control sections.
measure
response
-
mg
was
extracted
direct
because it recorded
to
as
activity
with
only partially
of the alcohol
respect
to
from the
sections, the
dehydrogenase
activity
the time of incubation and
and hence enzyme-present.
Acta Bot.
Need.
19(4), August
1970
ALCOHOL
2.4.
DEHYDROGENASE ACTIVITY
cotyledons
medium containing:
bovine
(40)
from
The
(BSA).
double cheesecloth and the volume
The
homogenate
fraction
then
was
dry cotyledons
furt
a.
next
M.).
An
allowed
2.5.
way
as
of alcohol
Assay
of tap
imbibe for 2 hrs
same
3.0 ml
with
NADH.
different dilutions of
M
-1
cm
-1
3.
was
stored
RESULTS
Extracts
from
Table
was
at
from
Effect
extract.
to
(Braun A.G.,
the
Air-
dehydrogenase.
powder,
Frank-
and this
was
powder
was
imbibed
cotyledons.
phosphate
a
buffer,
in
preparation
NAD
blank
at
25° in
containing
with
0.01
a
M
acetalde-
total volume of
addition of acetaldehyde,
followed
linearly
time for
initial reaction
a
Unicam S.P.
to
3
velocities of
the
800
buffer and
enzyme,
1
min.
Enzyme
least
at
A molar extinction coefficient of 6.22
distilled before
cotyledons
bovine
or
the
serum
cotyledons
three
3
X
10
use
and diluted with
water.
A 0.2 M solu-
days.
showed
presence
oxidized
activity
albumin
of
a
was
(BSA)
on
sizable ADH
very
the
germinating peas.
(+BSA)
per
of BSA
cotyledon
per
low
alcohol
activity
(table
1,
but in those
left
column).
dehydrogenase activity
Extracts were
prepared
of
in the absence
(2%). Enzyme activity expressed
as
ixmoles
of
min.
-BSA
+BSA
0
40
39
1
46
58
3
35
45
5
12
34
7
4
25
germinationperiod
in
medium.
activity (Alcohol:
after the
sec
1-day-old cotyledons
of
NADH
grinding
DISCUSSION
extracts from
(-BSA)
added
from fresh
was
against
-8° for several
AND
7-day-old
1.
nm
proceeded
one
through
used.
was
Acetaldehyde
tion
340
calculated from the
was
2%
squeezed
1.1.1.1)
About 15
at
10 ml
and
and the supernatant
min,
of alcohol
Thereafter the
dehydrogenase
spectrophotometer
The reaction
activity
was
and 0.1 ml enzyme
of 7.2.
pH
a
decrease in absorbance
recording
water
at 0°-5°.
(pH 7.2)
for 10 min. The supernatant
g
“multimix”
a
mixture contained: 0.05 M
0.225 mM NADH
hyde,
with
was
35 ml with
to
x
g for 15
X
10 g sand and
buffer
slurry
source
preparations
oxidoreductase E. C.
The reaction
the
as
pulverized
equal weight
to
treated in the
used
1500
air-dry cotyledons
with
phosphate
made up
at
and
mortar
a
resulting
20,000
at
now
was
first
were
was
centrifuged
recentrifuged,
was
fraction thus obtained
in
0.05 M
sucrose,
PEAS
OF
swollen
ground
were
0.4 M
albumine
serum
COTYLEDONS
of extracts
Preparation
Prechilled
IN
days
Acta Bot. Neerl.
19(4), August
1970
541
C.
Table
2.
Inactivation
of
cotyledons by
and
kept
oxidized
in
the
an
alcohol
ice for the
per
time.
from
extract
an
The extracts
7-day-old cotyledons.
appropriate
cotyledon
of
dehydrogenase activity
from
extract
Enzyme activity expressed
were
as
1-day-old
mixed
(1:1)
(xmoles NADH
min.
per
time
after
activity
%
of calculated
mixing
treatment
KOLLOFFEL
activity
min
46
1-day-old
4
7-day-old
mixture
+2%
BSA
M
+0.01
glutathione
When
these
lower
than that
{table 2).
two
extracts
which
albumin
100
60
14
56
120
10
40
180
8
32
300
7
28
300
24
96
300
8
32
(BSA)
to
from
tracts
cotyledons
the
the
put in buffer
It is
able
to
put
were
of the ADH
or
in
preparation
mixed.
were
sections
air-dry
much
was
individual values
extracts
increased
from
cotyledons
but
question
was
that
two
ex-
1-day-old
for about 1
hr
contain substances which
cotyledons
The effect of BSA
used. It
of
at
much lower than that of controls
arose
resulted from
cotyledons
when
occur
sections
1-day-old cotyledons.
from older
cotyledons
not
When
serum
Glutathione had
(table I).
7-day-old cotyledons
was
of the extracts. Therefore BSA
the
period of
activity
added
of the combined
extracts
two
from
extract
extract
an
{table I, right column).
from
an
of these
extracts
from older
from the
of the ADH activity did
cotyledons
inactivate ADH. Now the
extracts
ADH
resulting
activity
mixture of the
in
activity
clear that
the
expected
be
prevented completely, however, by adding bovine
1-day-old
ADH
mixed
were
little effect. Inactivation
only
of
25
could
depression
The
with time. It could be
4°,
0
(1:1)
was
some
kind of inactivation
during
added to the extraction medium
largely depended
without effect
from
are
activity
whether the low ADH
7-day-old
on
the
on
the
germination
of
activity
cotyledons
was
extracts
greatly
en-
hanced.
A similar artifact of isolation has been described
They
showed that the decrease in the
istics
in
pea
chondria
the
themselves,
from
pea
were
bound
cotyledons
low ADH
A crude
542
during germination
but
mitochondria. These
tissue and
was
cotyledons
activity
activity
to
an
fatty
by
not
was
by
some
due
acids
were
formed
fatty
acids it
7-day-old cotyledons
uncentrifuged homogenate
a
was
kept
is
et
al.
to
changes
fatty
(Eriksson 1968)
was
investigated
caused
by
acids
on
of the
that ADH
whether the
these substances.
in ice for 1 hr. The
chloroform-methanolmixture
Acta
(1968).
in the mito-
during homogenization
BSA. As it is also known
extracted from this with
Zeevaart
mitochondrial character-
increased concentration of free
is inhibited by
from
of
by
Bot. Neerl.
lipid
material
the method of
19(4), August
1970
ALCOHOL DEHYDROGENASE ACTIVITY IN
Table
3.
The
alcohol
tissue
in
dehydrogenase activity
with
assayed
COTYLEDONS OF
nitro-blue
(dry weight)
tetrazolium and
Dyer
(1959).
(TLC)
in
plates
and the
A
TLC of
of
comparison
a
in the
exclusively
0.73
5
0.53
7
0.45
extracted
of
lipids
Kieselgel
nol. The
acids
fatty
spots
were
extract
an
extract
The
weight).
main
whereas smaller
10%)
earlier
were
(Eriksson
of the
amounts
1967;
shown in table
4.
Methanol
et
are
al.
activity
only
in
acid
an
(about
acid
palmitic
had
they
(95:5, by
60%)
(about
linolenic acid and short-chain
The
extract
the solvent
-
and
were
in metha-
after
good agreement with
1968).
of
linoleic
was
10%)
of stearic acid,
Zeevaart
the ADH
(about
present
were
homogenized
were
vapour.
These substances
with methanol-sulfuric acid
mixture
of oleic acid
The
acid
with that of
monoglycerides
7-day-old cotyledons.
methylated
component
on
iodine
to
1-day-old cotyledons
acids and
fatty
of
present. These results
leic acid and oleic
is
and
amounts
and much smaller
acids
fatty
gel
g
thin-layer
40-60°)-ether-formic
visualized by exposure
from
by
(E. Merck, Darmstadt).
analyzed by gasliquid chromatography
were
produced per
per
(b.p.
absent, however, when pea cotyledons themselves
been eluted from the
cotyledons
pea
formazan
fractionated
were
HF
ether
petroleum
showed that
7-day-old cotyledons
almost
germinating
mg
30 min
0.59
plates
(60:40:1.5, by vol.)
g per
1
with
developed
were
days
3
The
on
of
as
mg formazan
germination
Bligh &
sections
expressed
30 min.
per
period
chromatography
PEAS
inhibitory
from
those reported
action
of
1-day-old cotyledons
inhibited the reaction
-
lino-
only
weakly.
These results thus
cotyledons fatty
also in
Table
clearly
acids
are
indicate that
and their
1-day-old cotyledons
4.
Effect of oleic
of
tract
acid
acid
(0.3 M)
pipetted
zyme
to
addition
and linoleic
1-day-old cotyledons.
in methanol
tion. The mixture
into
a
was
cell
during homogenization
liberated which
were
stirred
and 0.15
activity expressed
as
acid
Ten
absent in intact
As
homogenates.
on
pi
added
are
the
of
to
a
alcohol
solution
10 ml
of
a
these
dehydrogenase activity
of oleic
strongly
acid
(0.3 M)
diluted
pmoles
the
NADH
ml
oxidized
preincubation
cotyledon
were
per
min.
time in min
none
56
57
54
methanol
44
45
42
42
15
4
42
7
1
oleic
acid
+
linoleic
acid
Acta Bot. Need.
19(4), August
1970
was
added. En-
30
+
ex-
linoleic
sample
15
preparation
of an
or
0
enzyme
are
enzyme prepara4°. A
acetaldehyde
per
and
acids
fatty
thoroughly in nitrogen atmosphere at
ml NADH and 0.15
of 7-day-old
cotyledons
543
C.
able
inactivate
to
ADH
inactivation of ADH
fatty
acids
are
bound
sibility, however,
it is
from
that
probable
7-day-old
BSA. The present
by
they
that other substances
involved
are
It is
cotyledons.
in the
generally
do
experiments
in
vitro
known
that
exclude the pos-
not
also involved in the
are
KOLLOFFEL
inactivation of
ADH.
When the ADH
that
appeared
only
about
consistent
ADH
cotyledons
7-day-old
of older
medium
to
or
extracts
showed
1-day-old
which BSA
to
it is thus
cotyledons,
was
an
histochemically
activity
ones.
add
to
tissue
of the
of pea
7
decreased about 40
cotyledons
%
over
a
sizable
ascertain
BSA
by
a
it
was
are
the
ex-
histo-
that the ADH
germination
a
the
to
using
chemical method. The results of both methods indicate anyway
activity
which
These results
added. To
necessary
homogenization
avoid
determined
was
cotyledons
lower than that of sections of
40%
with those of
activity
traction
of the
activity
sections of
period
of
days.
Extracts
the seed.
activity
(water
water
from
reached
was
from 50
accompanied by
the enzyme is
relative
a
water
of the
content
13% (air-dry,
to
mature
cotyledons
seeds)
Kollöffel & Sluys
In
during germination.
which
highest
of
ADH
of about 47
this
dehydrogenase
maturation
during
activity.
%
was
So it is clear that
of the seed
reversibly inactivated during maturation
mitochondrial malate and succinate
activity
development
the
The decrease of the relative
decrease of about 15 % of the ADH
by hydration only
ADH
during
pea seeds indicated that the
maturing
the fresh weight).
percentage of
as
content
vated
at a
synthesized
have been
must
with
Experiments
content
showed
air-dry cotyledons
that the enzyme
implies
and reacti-
respect it resembles the
activity (Kollöffel
1970;
1970).
ACKNOWLEDGEMENTS
The
of Dr. G.
help
and identification
M.
A.
L.
Scherphof of
of the
T. Gebbink
the
acids is
fatty
and Miss
A. A.
Biochemisch
Laboratorium
gratefully acknowledged.
van
Schaik for their
skilful
(Utrecht)
The
in the isolation
author thanks
technical
also
Miss
assistance.
REFERENCES
Bligh, E.
Can.
G. & W. J.
J.
Cameron,
pea
Biochem.
D. S.
& E.
cotyledons.
Cossins,
E.
A.
Dyer
(1959);
Physiol.
A.
Cossins
Biochem.
(1962):
37:
J.
A
(1967):
105:
Utilization
rapid
method of total
lipid
extraction
and
purification.
911.
Studies
of
intermediary metabolism
in
germinating
323-331.
14
of ethanol-2-
C
by
pea
slices.
Nature
(Land.)
194: 1095-
1096.
—
—
—
(1964):
Formation
Nature
(Land.)
& H. Beevers
&
E.
R.
(Land.)
—
&
—
(1962):
Ann. Bot.
544
metabolism
of lactic
acid
during germination
of pea
seedlings.
989-990.
(1963):
Turner
183:
and
203;
Ethanol
(1959);
metabolism
Utilization
in
plant
of alcohol
tissues. Plant
in
Physiol.
germinating
pea
38: 375-380.
seedlings.
Nature
1599-1600.
Losses
of alcohol and
alcohol
dehydrogenase activity
in
germinatingseeds.
26: 591-597.
Acta Bot. Need.
19(4), August
1970
ALCOHOL DEHYDROGENASE ACTIVITY IN
&
—
of ethanol
The metabolism
(1963):
—
COTYLEDONS OF PEAS
in
germinating pea seedlings.
J.
Bol.
Expt.
14:
290-298.
L. C.
B. Blawacky
Kopala,
—,
alcohol
D. C.
Davison,
& A. M. Spronk
dehydrogenase. Phytochem.
The
(1949);
distribution
plants, with particular reference
Proc. Linn.
Doireau,
de la
Soc.
des
germination
C. E.
Eriksson,
J. Food Sci.
—
N.S.W. 74:
& R. Dupéron
P.
of formic
to their
(1923):
G.
de
J.,
alcohol
dehydrogenases
in the pea
distribution
lipoxidase,
du
aspects
Légumineuses.
Aerobe
und
C.
higher plant
in the
higher
plant during its life cycle.
‘métabolisme
R. Soc.
of
Biol.
fermentaire’
160:
au cours
1926-1932.
and substrate
enzyme
E.
in
green peas.
the
&
Atmung
bei
J. Food Sei.
peas.
525-532.
33:
Pisum sativum.
Keimlingen von
107-256.
20:
Samen. Flora
Boeri
activity during
from
(E.C. 1.1.1.1.)
anaerobe
über
(1927): Untersuchungen
nahme bei keimenden
Goksöyr,
of a
26-36.
graines
Bot. Néerl.
Trav.
properties
32: 438—441.
D. S.
Frietinger,
and
variation
(1966): Quelques
Pea
Some
(1968):
1125-1134.
Alcohol: NAD Oxidoreductase
(1968):
Fernandes,
Ree.
(1967):
7:
R. K.
122:
Bonnichsen
germination of
the
die
Kohlensäureabgabe
und
Sauerstoffauf-
167-201.
The
(1953):
green pea
of ADH
variation
(Pisum sativum).
Acta
and
Chem.
catalase
Scand. 7:
657-662.
Kollöffel,
C.
sativum L.
—
The
(1969):
—
& J.
V.
Sluys
Bot. Need.
LeblovA, S.,
plants
and
maturation
during
Acta Bot. Neeri.
dehydrogenase
conditions.
histochemical
and mitochondrial
rate
I.
localization
of
seed.
cotyledons
Planta
Mitochondrial
17 :
activity
of peas
germinated
under
in
the
of Pisum
cotyledons
18: 406-419.
of mitochondria
91;
(Berl.)
of Pisum
70-77.
dehydrogenases
Neeri.
Bot.
cotyledons
from
cotyledons
pea
321-328.
in pea
cotyledons during germination. Acta
503-508.
ZimAkovA,
in the
the
phosphorylative activity
of the
(1970):
19;
in
in the
activity
16: 111-122.
Acta Bot. Neeri.
during germination. Acta
Oxidative
(1970):
alcohol
environmental
sativum L.
—
of
(1968): Activity
different
—
(1967): Respiration
during germination.
course
of
D, SofrovA &
growth
J. BarthovA
under normal
(1969);
Occurrence of ethanol
and anaerobic conditions.
Biol. Plant.
in
pea
(Praha)
11: 417-423.
Maffei
A.
Faccioli,
germinazione
di
Variazioni
(1959):
Pisum sativum
ed
di alcuni
effetti della
enzimi
di alcuni
e
anaerobiosi.
Boll.
coenzimi
Soc.
liai.
durante
Biol.
Sper.
la
35:
2166-2171.
Spragg,
and
S. P. & E.
ascorbic
Suzuki,
Y.
W. Yemm
acid
(1966);
in
Alcohol
seedling. Phytochem.
Virtanen,
als
A.
I.,
A.
J.,
mitochondria
Acta Bot.
Neerl.
J.
mechanisms and the
Expt.
Bot.
dehydrogenase (alcohol:
(1944):
& N. Rautanen
Geschehen.
M. Gruber
from
peas.
10 :
NAD
changes
of
glutathione
409-425.
oxido-reductase)
from
the pea
5: 761-765.
A. Kärkelä
biochemisches
Zeevaart,
(1959): Respiratory
germinating
&
Suomen
M.
H. van
germinating peas.
19(4), August
1970
Kemislilehti
Raalte
Acta
Bot.
Die
B17:
Keimung
(1968):
Neeri.
und
Reifung
der Samen
21-23.
17:
Oxidative
phosphorylation
in
349-356.
545