Respiration
activity
tivum
C.
L.
rate
the
in
and
mitochondrial
cotyledons
during
of Pisum
sa-
germination
Kollöffel
Botanical
Laboratory,
Utrecht
SUMMARY
parallel
From
in
by
vitro
four
phases.
hydration
level
is
of the
a
certain
rate in
structure of the
the
in
fourth
respiration
of pea
and of the
in
only
to
a
in
rate
minor
transfer chain.
in the first
The
a
The increase
be
to
due
to
a
in
is
due to activation
fairly
constant
limited
in the
present
rate
whereas
enzymes,
supply but
oxygen
respiration
disorganization of
by
respiration
air-
in the third
the
decrease
the
subcellular
in
cotyledons.
INTRODUCTION
The
of seeds
germination
(1965),
Bain & Mercer
of
development
a
with the
starts
(1966) observed
complex
subcellular
of
uptake
that this
water.
uptake
is
Perner
in the
organization
due
to
(Opik
the activation
& Simon
The
by hydration
of enzymes
cotyledons
already present
continuing
rise
soluble oxidases
observed
(1963)
the
by
and axis
probably partly
in the
dry
seeds
1963).
in oxygen
an
for
account
increase
in
has been attributed
uptake during germination
several mechanisms. Mapson & Moustafa
that
(1965), Opik
accompanied
tissue of the seeds. The concomitant rise in metabolic activity is
to
activity
mediated
Its course may be divided
phase
due to
extent
of the mitochondrial
seems
and mitochondrial
of the electron transfer chain
potential activity.
activity
vivo
during swelling.
of the enzymes
phase
rate
cotyledons during germinationis
electron
respiration
two systems
is
hydration
attributed to a rise
respiration
1.
full
with
dry cotyledons
phase
rise
subsequent phase
to the
in
respiration
cycle
sharp
of the enzymes
in the
primarily
The
changes
on
that the
of the citric acid
enzymes
into
observations
it is concluded
this
(1956)
rise. Young
mitochondrial
and
et
al.
Öpik (1965)
suppose
and
Cherry
(1960)
which
activity,
suggests
an
im-
portant role of the enzymes of the electron transfer chain in this respiration
process.
söyr
et
Already
al.
Pfeffer
(1953)
anaerobiosis in the
The
object
and
(1885)
germination
more
with in vitro
tion
experiments,
to assess
2.
2.1
the
(1949),
Gok-
importance
the in
vivo
respiration
the role of the citric acid
respiration
find indications about the factors
to
Davison
stressed
is, by comparing
and of the electron transfer chain in the
and also
recently
(1959)
of
process of seeds.
of the present paper
measurements
and
Spragg & Yemm
process
controlling
the
of
pea
changes
rate
cycle
cotyledons,
in
respira-
rate.
MATERIALS AND
Seeds
Whole pea
and
seeds,
Acta Bot. Neerl.
METHODS
germination
var.
Rondo,
16(3), Sept.
1967
selected
on
equal air-dry weight
and
equal colour,
111
C. KOLLOFFEL
soaked under aeration for 20-22 hrs in tap
were
the
appropriate period
mination occurred in the dark
from the time the seeds first
for
cotyledons
both seed
or
the
at
23° C. The
in
came
In
experiment.
coat
and
with
water
to
as
naked
measured
was
the removal of the
to
soaking
to
and ger-
Swelling
the seed
dissected from the
carefully
were
for
germinated
next
germination
experiments prior
some
and axis tissue
of
period
contact
seeds. This will be referred
air-dry
water
moist filter paper in Petri dishes.
on
seeds
germinated
coat
cotyledons
of
excised
or
cotyledons.
2.2
of
Degree
swelling
In accordance with Kuhne & Kausch
as
the
water
Consequently,
the
[(fresh
minus
weight
is the
cotyledons
of
sample
of
degree
after
weight
can
kept
weight
of
through
was
to as
give
medium
the
8
centrifuged
containing
of
(Ig/ml).
again centrifuged
particles
at
particles
The
imbibe for 2 hrs
A
way
112
from
of tap
at
medium
formula
of the
dry weight
dry weight
means
g
as
was
for 15
pellet
air-dry
water
15 min. The
the fresh
in
of
a
of the
weight
of
(pH
pellet
of
in 20 ml
order
to
7.2),
cotyledons
a
per
were
added
pulverized
to
the
moment
and
grinding
supernatant
resuspended
was
buffer
grinding
a
(pH 7.6),
to
per ml of
medium and
washed mitochon-
give
ml
fraction from
pre-
equal
resulting slurry
medium
to
an
sucrose
cotyledons
obtain
in
extracts
The
thus obtained will be
pellet
phosphate
resuspended
buffer
seeds
was
The
1500 g for 10 min. Next the
min,
mitochondrial
0.4 M
containing
and 0.05 M
weight
with
mortar
a
cotyledons.
resuspended
was
phosphate
and the
use,
experiments.
diluted to 35 ml with the
from 1 gram fresh
0-5° C. From this
Fragmentation
suspension
in
ground
gauze,
at
sucrose
pellet
20,000
of the
cotyledons
same
aseptic
20,000 g for
from 2 grams of fresh
equal weight
in the
of
0.2 M
and 0.05 M
Isolation
Excised
defined
swollen cotyledons
of the
start
per gram of
centrifuged
at
were
grinding
(pH 7.2)
drial fraction. The washed
sucrose
is
cotyledons.
the
to
weight by
the unwashed mitochondrial fraction. The
equivalent
suspension
2.5
The
105° C for 20 hrs. The
at
weight)
ml of
layers
and the filtrate
fraction
a
1
buffer
phosphate
referred
An
100.
x
cooled to 0-5° C before
were
fresh
g
sand, adding
filtered
medium,
2.4
weight]
this temperature until the
at
cotyledons (10
was
of the
curve.
chilled
0.05 M
swelling
calculated according
was
drying
of
degree
dry weight
be calculated from the fresh
All apparatus and solutions
obtained
M
swelling
the
of the
Isolation ofthe mitochondrial fraction from
2.3
in
(1965)
cents
dry weight)/dry
cotyledons
time
swelling
in per
expressed
content
0.2
containing
the
equivalent
of
suspension (2 g/ml).
air-dry cotyledons
with
powder,
a
Braun “multimix”.
that
the imbibed
was
powder
allowed
was
to
treated
cotyledons.
of the
mitochondria
of unwashed mitochondria
(1 g/ml)
in 0.05 M
phosphate
Acta Bat. Neerl.
buffer
16(3), Sept.
1967
RESPIRATION
(pH 7.6)
RATE
AND
sonic
three
treatment
times. In
the sonicated
for
assays
suspension
fumarate
in
resuspended
a
some
of 800 KHz in
The supernatant obtained after
or
ACTIVITY
PISUM
IN
SATIVUM
DURING
CO
usually fragmented by freezing (ethanol-solid
was
thawing (0-5° C)
to
MITOCHONDRIAL
equal
and
min
aconitate
original
the
15
200-250 Watt.
at
The
hydratase.
exposed
was
suspension
used in the
was
and
mixture)
a
suspension
the frozen and thawed
centrifuging
to
the
Lehfeldt sonifier for 3 min
a
20,000 g for
at
hydratase
volume
experiments
GERMINATION
standard
sediment
was
unwashed mitochondrial suspens-
ion.
2.6
General remarks
All enzyme assays
c
performed
were
oxidoreductase which
always
proportional
calculated from
activities 15
(about
nation
a
linear
of the
process.
Respiration
The
respiration
technique.
Five
dry weight
of whole
the
ml
cotyledons
were
of reaction
were
-
rate
time
rate
activities
Enzyme
The enzyme
curve.
corrected for the decrease
not
cotyledons taking place during
in units
was
were
as
indicated in
the
the
germi-
figures.
cotyledons
were
was
placed
KOH
10%
of NADH- cyt.
The
added.
reaction
expressed
of whole cotyledons
flask with 0.2
ments
of the
are
exception
29-31°C.
enzyme
base
dry weight
with the
C,
at
of the
portion
cotyledons
25°
of
amount
Their activities
2.7
burg
the
to
at
performed
was
calculated for
%)
enzyme activities
on
in the
in
kept
measured
by
the
manometric
Warburg
in the main compartment of the
well.
centre
moist
the
During
ml
0.4
atmosphere,
War-
measure-
being
water
present in the side-arm.
2.8
Succinate oxidase system
Oxygen
uptake by
the
Warburg apparatus.
volume of 2.4 ml with
HCL buffer;
MgCl2 ;
a
0.0125 M
final
pH
of 7.2: 0.02 M sodium
phosphate
buffer;
0.2 M
reaction
suspension
mixture
was
intended
originally
2.9 Succinate
a
conventional
contained,
succinate;
sucrose;
0.01
M
cytochromec;
of mitochondria (2
succinate and other intermediates of the citric acid
EC
measured in
was
0.3 mM NAD; 0.3 mM NADP; 0.01 mM
and 0.6 ml of a washed
This
isolated mitochondria
The main compartment of the vessel
in
0.01
a
total
M tris-
NaF; 5mM
ADP
1 mM
g/ml).
for
the
oxidation
of
both
cycle.
dehydrogenase (succinate: (acceptor)oxidoreductase,
1.3.99.1.)
Succinate
dehydrogenase
was
measured
tion of 2,6-dichlorophenol-indophenol
phate (Pier pont
1963).
tion: 0.04 M sodium
KCN;
phenazine
indophenol
The reaction
succinate;
0.05 M
methosulphate (0.3
and 0.1 ml unwashed
Acta Bot. Neerl.
16(3), Sept.
1967
spectrophotometrically by
in the
presence
mixture
of
contained in
phosphate buffer;
mg/ml);
suspension
phenazine
0.03
a
0.2 M
mM
2,6
of mitochondria
final
the reducmethosulconcentra-
sucrose;
0.01 M
dichlorophenol(1 g/ml)
in
a
final
113
C.
volume of 3
ml
with
omitted from the
the
partly
Enzyme
using
rate
reduction
that
non-enzymic
activity
calculated
was
Nadh:cytochrome
Enzyme activity
M
NADH
and 0.1
final volume of 2 ml and
added
ml
a
7.6. At
the cuvette, and within
to
for 6 min in
a
Unicam S.P. 800
without NADH
as
15
zero
Fumarate
aconitate
Both enzymes
for
fumarate
measured
a
buffer;
0.6 mM
final volume of 3 ml and
The
3
reaction mixture
for
volume of 3 ml and
In both assays
a
pH
use
Zeiss M 4 Q III
as
an
in
M -1
E
24flnm
(0.15%);
5000nm
a
solution was
measured
was
calculated
a cuvette
by using
a
cmA
EC
EC
4.2.1.2.)and
4.2.1.3.)
by
a
slight
modification
(Pierpoint
1960)
0.04
and 0.3 ml of
an
M
sodium malate;
enzyme
preparation
aconitate
hydratase
of
an
contained 0.027 M
enzyme
preparation
sodium
in
final
a
of 8.0.
was
made of the
supernatant fraction of
means
of
a
24onm
three times
a
was
measured in
with the reaction mixtures without substrate
activities
Enzyme
by
c
of 8.0.
spectrophotometer,
blank.
optical
changes
modification
slight
cytochrome
frozen and thawed mitochondrialsuspension. Increase in E
a
1962).
In final concentrations the reaction mixture
0.045 M tris-HCl buffer and 0.3 ml
citrate,
4
10 M
cysteine
pH
a
& Singer
a
was
spectrophotometrically
(1950).
contained:
hydratase
0.045 M tris-HCl
in
by
hydratase (L-malate hydro-lyase
were
by
1.6.99.3.)
increase in E
Enzyme activity
x
60onm
. Enzyme activity
of unwashed mitochondria in
hydratase (citrate hydro-lyase
of the method of Racker
succinate.
E
recording spectrophotometer against
molecularextinction coefficient of 18.5
2.11
(EC
in
time the reduced NAD
the
sec
blank.
optical
an
(Arrigoni
5mM KCN,
suspension
a
1
lO^M’cm
x
added
decrease
The reaction mixture contained:
(1963).
sucrose;
of
pH
without
observed
was
corrected for
always
was
spectrophotometrically
0.2 M
phosphate buffer;
0.3 mM
occurred
the
oxidoreductase
c
of the method of Nason & Vasington
0.05
dichlorophenol-indophenol
concentrations
dye
measured
was
from
The
of reduction
coefficient of 20.5
corrected for infinite
was not
of 7.6
pH
blank. The
molecular extinction
a
2.10
final
a
optical
KOLLOFFEL
were
calculated
molecular extinction
from
the
observed
coefficient of 2.4
x
3
10
1
cm^
.
3.
RESULTS
3.1
The
of whole
Respiration
course
of
respiration
nience, into four phases
I. A
II
rapid
rise in
Hereupon
114
a
a
fairly
cotyledons {fig. I)
follows
respiration
follows
maintained at
as
cotyledons
of the
rate
phase
be
analysed,
for
conve-
for the first 8-10 hrs.
of
constant
can
:
about
level,
15
hrs
or even
during
tends
to
which
drop
respiration
is
somewhat.
Acta 801. Need.
16(3), Sept.
1967
RESPIRATION
AND
RATE
MITOCHONDRIAL
III. A second slower rise
of
onset
IV. A slow
Further
The
Fig. 1.
no
of
The
Fig.
3.
The
Fig.
4.
to
with
which
of the
of the
of
The relation
respiration
It is
up
to
by
now seen
a
about 60 hrs from the
to
plotting
that
in
of about
degree
cotyledons
coat
respiration
rate
phase
I
100%.
There exists,
respiration
cotyledons
without
of excised
from intact
seed
coat
the
during
germinated
respiration
seeds
(•)
(naked cotyledons O).
phases (I, II,
III and
and
The
IV).
cotyledons.
from intact seeds
(•)
and
of
cotyledons
from seeds
germi-
(O).
the
degree
of
swelling
may have been limited
the effect of high
intact and naked
high
and the
oxygen
germinated
cotyledons,
tion
cotyledons
16(3), Sept.
of the
seeds. This
does
not
during
1967
by
the
tension
oxygen tension
constant, rise
tion time of the
Acta Bot. Neerl.
cotyledons
is divided into four
respiration rate
between
slight, fairly
of the
rate of
of the
germinated
seeds
As appears from table 1
rate
GERMINATION
respiration
rate
of
cotyledons
germinatedseeds.
measurements
a
made easier
respiration
respiration rate
without seed
by investigating
only
is
swelling
the
from
of the
swelling
from intact
rate
DURING
after about 60 hrs.
rate
{fig. 4).
content
linearly
course
nated
respiration
water
course
course
2.
SATIVUM
from about 25 hrs
extending
of these results
cotyledons
Fig.
PISUM
linear relation between swelling and respiration in phase II and III.
extent
The
in
drop
study
increases
however,
IN
germination.
directly against
rate
ACTIVITY
of oxygen
respiration
during
respiration
the
course
measurements
rate
of
caused
cotyledons
of the
from
germina-
for the differences in the
of the
studied
was
rate.
rise, being independent
account
the
supply
on
respira-
germination.
115
C. KOLLOFFEL
Table
1.
The effect
of high
seeds
from
and
tion.
hours
the rate
without
measured
cotyledons
of
coat
moles 0
p
first
in
in
and
air,
from intact
of
phases
germinain
next
from seeds
cotyledons
germinatedwithout
in air
oxygen
in oxygen
16.2
19.1
20.0
22.6
14
16.8
20.8
20.1
24.9
18
15.4
18.8
19.2
23.3
19
18.0
21.0
15.6
23.2
24
18.1
23.8
23.3
29.0
40
30.0
34.0
41.2
46.2
the
respiration
and
during swelling
the rise in
rate at
in time
the
and
seedling
coat
table 2 it
to
have been influenced
This
indicated
was
the transition from
therefore
was
by
the
supply
observation that
III
phase
to
by
supply.
coat
the oxygen
the
correspondradicle
elongating
The influence of the
From
investigated.
be concluded that the intact seed
may
by
II
coat
with better oxygen
consequently
rate
phase
of the seed
perforation
respiration
on
may
germination.
respiration
closely
rate
coat
seed
11
However,
100%
/hr/g dry weight.
2
from intact
air
cotyledons
at different
manometrically
as
of
respiration
seed
germinated seeds
in
seed
was
Oxygen uptake expressed
germination
of the
on
germinated
Oxygen uptake
oxygen.
ed
tension
oxygen
seeds
the
data of
limited the gaseous diffu-
sion.
Table!.
The
effect
peeled
of
of
uptake
off
peeling
whole
-
but
the
seeds
seed
coat from
left in the flasks
Oxygen uptake expressed
as
p
moles
2
uptake
22.0
13.5
21.2
been
and
cotyledons
germinated
was
during
then
naked
germination
from intact
rate
in
phase
the
rate
the seed
studied
coat.
As
respiration
I is
rate
by
by
seeds
rate
The
that of
from
time
probably partly
{fig. S).
the
fig. I,
curves.
due
to
there is
Acta Bot.
cotyle-
uptake
from
only
sharper
more
barrier
supply during
oxygen
rate in
a
coat
closed
cotyledons.
cotyledons
The
the
higher respiration
as
of whole
from the
comparing
appears
the seed
tightly
the limited oxygen
with
off
have acted
measurements
artificially
off
seedling
remains
coat can not
is influenced
was
then it
even
respiration
germinated
without seed
cotyledons
the radicle of the
always stripped
respiration
difference between both
respiration
peeled
diffusion because
cotyledons. However,
How far the
swelling
perforated by
were
was resumed.
seed coat
10.9
having
coats
/hr/g dry weight.
12
limit the oxygen
Oxygen
rate.
seed
20
dons, because it
116
0
the
whole
diffusion of oxygen
the
respiration
next
seeds
around the
to
on
germination
Also after
may
seeds
and measurement of oxygen
-
hours
whole
manometrically,
measured
was
a
rise
slight
of the
rapid swelling
phase
of
seeds
II and
Need. 16(3),Sept.
of
III
1967
RESPIRATION
tion
coat
rate
AND
be induced
seems to
seed
RATE
prior
MITOCHONDRIAL
oxygen
uptake
naked
between the
probably
phase
and
whether this
investigate
3.2
radicle
seedling
from
their remains
is much lower than in the
The transition from
the
the better oxygen
by
soaking
to
II
with
of excised
As
spite
DURING
GERMINATION
of the removal of the
in which the rise in
to
of the
the
respira-
from
respiration
In
of the
with
is
order
to
cotyledons,
that of
2 there
fig.
of
emergence
tissue.
respiration
and excised
cotyledons
on
axis
compared
was
with the
corresponds
growth
appears
influenceof the axis tissue
no
SATIVUM
In
phase (II)
III
phase
faster
cotyledons
of naked
PISUM
supply.
a
is related
seeds.
IN
preceeding (I) and following (III) phase.
to
a
growth
germinated
respiration
ACTIVITY
cotyledons
difference
no
and there is
cotyledons
rate.
Mitochondrial activity
3.2.1
Distribution
of
the
mitochondrial enzymes
after
of the
fragmentation
mitochondria
From
preliminary
fumarate
and
hydratase
substrates it seemed
that
emerged
aconitate
Because of the
suspensions.
it
experiments
it
hydratase
necessary
them
disrupt
obtain
to
with
and
by freezing
high
mitochondrial
of the mitochondria
possible inaccessibility
to
difficult
was
activities
the
to
thawing
by
or
sonication.
Table
3.
Distribution
of the
and sedimental
of several
activity
fraction
obtained
chondrial suspension.
The reaction
section
methods.
materials
and
chondrial
suspensions
natant
sedimental
or
at different
ties
are
phases
expressed
of
freezing
whereas
were
as
the
the
indicated in the
succinate
composed
contained
sucrose
same
figs. 6,
described
as
M
was
supernatant
sonication
or
sucrose
omitted
obtained
distribution.
mito-
a
under
when
when
from
the
mitosuper-
cotyledons
The enzyme
activi-
7 and 8.
NADH-cyt.c
dehydrogenase
0,2
the
among
thawing
Mitochondria
tested.
germination showed
in units
and
mixtures were
They always
tested,
were
fractions
mitochondrial enzymes
after
oxidoreduc-
fumarate
aconitate
hydratase
hydratase
tase
original
mitochondria
supernatant
sediment
freezing/thawing
supernatant
sediment
After
te
0.21
freezing/thawing
sonication
sonication
the mitochondria had
hydratase activity
been
these
results
freezing
rations with
The
activity
high
highest
was
and
was
0.00
0.34
0.32
0.14
0.96
0.08
0.08
0.05
0.31
0.42
0.31
0.11
0.63
0.03
0.06
disrupted
are
thawing,
used
as
a
likely
succinate
16(3),Sept.
the
highest
being
dehydrogenase
suspensions
1967
to
a
be
fumarate and aconita-
{table 3)
readily
and
hydratase
and
hardly
any
solubilized. Because of
milder method and
routine method for
fumarate- and aconitate
found in the
Aclaßot. Neerl.
0.16
0.03
detected in the supernatants
was
in the sediments. Both
enzymes
reproducible results,
1.14
0.00
obtaining
giving
more
enzyme prepa-
activities.
NADH-cyt.
c
oxidoreductase
with intact mitochondria. After sonica-
117
C. KOLLOFFEL
tion little
activity
was
recovered in the supernatant, and after
ing scarcely anything.
ments.
Both enzymes
brane.
In the
NADH-cyt.
was
applied
Fig. 5,
6,
7
c
Most of the
are
likely
be
determination of the
oxidoreductase the
as
was
firmly
activity
original
freezing
recovered, however,
bound
to
and thaw-
in the sedi-
the mitochondrial
of succinate
mem-
and
dehydrogenase
unwashed mitochondrial
suspension
usual.
and 8. The
course
of the
dehydrogenase (fig. 6.);
(fig. 7.)
to
activity
and
aconitate
activity
NADH
of the succinate oxidase
cyt.
c
oxidoreductase
system (fig. 5.);
(fig. 6);
fumarate
succinate
hydratase
hydratase (fig. 8.).
3.2.2 Succinate oxidase system
Mitochondrial
seeds
at
(phase
suspensions
I and
II)
obtained
showed
an
from
oxygen
least 60 minutes. The succinate oxidase
IV)
declined with time and therefore the
portion
of the
correcting
118
for
curve
the
(15-30
min).
endogenous
The
which
activity
activity
mean
respiration
of
cotyledons
uptake
was
from older
was
germinated
cotyledons (phase
calculated from the linear
values of
have
newly
linear with time for
been
triplicate
plotted
Acta Bot. Neerl.
in
flasks
fig.
after
5.
16(3), Sept.
The
1967
RATE
RESPIRATION
succinate
AND
oxidase
it rises much
MITOCHONDRIAL
increases
activity
till it reaches
more
ACTIVITY
a
IN
PISUM
SATIVUM
DURING
little between 0 and 20
a
peak
but after that
hrs,
about 60 hrs after the
at
GERMINATION
beginning
of the
germination.
the
During germination
sions
showed
weight
slight
a
about 2.4
to
3.2.3 Succinate
When in the
suspensions
endogenous respiration
constant
moles 0
p,
2
NADH-cyt.
c
nadh-cyt.
oxidoreductase
the
used,
activity
c
plotted,
of both enzymes remains
activity
it increases till it reaches
a
dry
fairly
added enzym.
to
constant
2 min. The
to
in
dehydrogenase,
for about
about 60 hrs after the
peak
/hr/g
diluted mitochondrial
slightly
with that of succinate
together
2
much diluted mitochondrial
proportional
sions. The reaction proceeded then linearly with time for 1
has been
moles 0
\x
oxidoreductase
assay
not
was
estimated therefore with undiluted or
was
of the mitochondrial suspen-
from about 0.6
/hr/g dry weight.
and
dehydrogenase
were
increase
20
activity
fig. 6.
The
after that
hrs,
of the
beginning
It
suspen-
germina-
tion.
3.2.4 Fumarate
A
solubilized enzyme
Their
activity
after the
4.
onset
and aconitate
hydratase
preparation
was
hydratase
used
of the
7 and
germination (fig.
in
the
of
assays
till it reaches
increases during germination
both enzymes.
peak
a
about 60 hrs
fig. 8).
DISCUSSION
When the
is
{fig. I)
enzymes, such
{fig.
with
a
This difference is
of several
activity
{fig. 6)
{fig. 5),
succinate
fumarate
or
appear that
changes
of these enzymes.
activity
of whole
during germination
of the
difference between the
system. The respiration
activity
mitochondrial enzymes
ate
it will
in
is there
whereas the
level.
rate
course
oxidoreductase
and of the in vitro
sharply,
same
c
changes
germination
in vivo
the
hydratase {fig. 8),
parallel
hrs of
respiration
with
the succinate oxidase system
as
NADH-cyt.
6),
aconitate
run
of the
course
compared
and
respiration
rate
in
the
during
to
the
artificially
of the
in this
accelerated
their isolation, which
the first
Only during
of the isolated enzymes remains
due
dehydrogenase
hydratase (fig. 7)
course
rate
cotyledons
mitochondrial
brings
activity
phase
10
of the
increases
practically
hydration
at
the
of the
about their immedi-
activation.
The succinate
transfer
oxidase system
particle),
oxidoreductase
succinate
dehydrogenase
(complex II) represent
transfer chain. The fumarate
which
enzymes
enzymes
are
a
catalyse
the
acid
most
changes
in
fully,
of the
in
16(3), Sept.
1967
of
an
electron
NADH-cyt.
hydratase represent
Assuming
enzymic
activity
I)
IV
and
c
the enzymes of the electron
and aconitate
cycle.
in vivo, it may be concluded from these
Acta Bot. Need.
III and
(complex
almost
hydratase
citric
reliable index for
chondrion and that their
activity
(the complexes I,
that
the
the
investigated
activities within the mito-
vitro represent real
experiments
that the
changes
in
respiration
119
C. KOLLOFFEL
cotyledons during germination
of the pea
acid
is mediated
The enzymes of the electron transfer chain
thawing
by
sonicating
or
recovered after
were
mitochondrial suspension
a
supernatant fraction. These results
also
transfer
in
mitochondria from
chain
localized
are
whereas the citric acid
The
respiration
cycle
tissues
The
cotyledons changes
rise in
sharp
electron
It
whole
possible
was
and
of whole
citric
acid
It is
to
NADPH
the
on
by
at
that
Öpik
following
0-5°C,
enzyme
Bevilaqua
Riggio
synthesis
structures were
mechanical
{fig. I)
10 hrs of
the
(1)
respira-
of
swelling
cotyledons.
powder
powder
has occurred. This
ascertained
the
air-dry
The
degree
imbibed
of
the
of
air-dry
with water,
with
agrees
NADH
and
-
2
of
precipitates
dry
and
germi-
dons of
(1966)
germinating
mitochondrial
arisen de
cells
as
novo
as
PHASE
In this
cell
organells
of considerable
“ghosts”
became
authors
of
embry-
and
cyto-
there is
shrinkage
most
water
content
increasing
suppose
that
in the
(pro-mitochondria);
the
became
an
hydrated.
origin
or
cotyle-
development
of
mitochondria
of mitochondria which had lost their
dormant
cotyledons
increasing
associated with
These
structure.
the
was
present study indicate, however, that
the
spite
observed that
pea seeds
could have arisen from
machinery
In
preserved.
microscopic investigations
during dormancy
damage.
Bain & Mercer
they
enzymic
have
may
The biochemical data of the
from
pre-existent
mitochondria
likely.
II
period, lasting
germination,
reorganization
Probably
120
an
who
acetone
concluded from electron
(1965)
cells of air-dried seeds that
root
plasmatic
of
evidence:
with
in
& Simon (1963) for bean
(1964),
in
the first
present
the imbibition of this
during
oxidoreductase activities
Ferner
seems
already
linearly
2
the
time
pea seeds.
nating
no
mitochondrion,
of enzymes
hydration
isolate active enzymes from
likely
not
of
results
onic
electron
membrane.
germination
cotyledons during
cycle
increases
cotyledons
maximal 2 hrs
lasting
outer
the
distinguished.
the activation
This fits in with the results of
cotyledons.
the
chain
rate
to
This conclusion is based
of the
{fig. 4).
respiration
attributed
is
transfer
cotyledons.
(2)
of
enzymes
with
indicate
1966). They
I
germination
tion
al,
et
the
localized on the
and for convenience four phases have been
PHASE
sedi-
found in the
were
inner membrane of the
are
enzymes
of pea
rate
(Bachmann
plant
the
on
in the
in accordance with those obtained pre-
are
mammalian tissues
with
dominantly
cycle
and
freezing
predominantly
mental fraction, whereas the enzymes of the citric acid
that
enzymes of the citric
and the electron transfer chain.
cycle
all
the
of
between about
hydration
the
of the
10 hrs
ultrastructure
pre-existent
enzymes
and about 25
cotyledonary
is
hrs from the
tissue is
continued
completed
(Bain
have been activated
now
Acta Bot.
onset
and the
& Mercer
1966).
by hydration
NeerL
16(3),Sept.
and
1967
RESPIRATION
thus
both
This lag
RATE
extent to
Yemm
(1959).
to
PHASE
The
in
and
the
did result in
oxygen, reduces the
increase in
a
to
Neither
raise of this
fairly
a
the
is
an
GERMINATION
(Breidenbach
activity (Akazawa
activity
cell content,
was
PHASE
activity.
may be the
mitochondrial
to
a
of Spragg &
increase of the oxygen
from the
coat
The testa,
cotyledons
cotyle-
acting
as
a
in situ.
result
al,
et
as
& Beevers
case
in
actual increase in
an
whether it
or
content
is
rate
and
It is
1957).
respiration
the
of
phase is associated
yet investigated
not
was
1966),
and
in this
cotyledons
It
a
to
further
a
the result of
intrinsic mitochonthat
unlikely
very
due
be
may
higher
whether this
the number of
this
rise in
hydration
of the
I.
phase
IV
Respiration
of
and mitochondrial
rate
Cherry
germination.
activity
after about 60 hrs from the
drop
(1963), Öpik (1965)
and
Bain & Mercer
observed that the mitochondrial ultrastructure in
bean and
peanut,
pea
dons, respectively,
tion
level.
constant
attributed
opinion
lag phase.
of the
rate
of whole
rate
increase in the mitochondrial
an
onset
at
cotyledons
the removal of the seed
respiration
mitochondrial
activity
mitochondria
drial
remain
(1)
DURING
SATIVUM
III
increasing
both
nor
PISUM
supply, contrary
The evidence for this is:
swelling
IN
of whole
rate
the limited oxygen
increasing respiration
with
ACTIVITY
enzymic activity
respiration
during respiration (2)
dons before
barrier
MITOCHONDRIAL
respiration
phase
minor
tension
AND
in
the
succinate oxidase
after several
désintégrâtes
present
study
the
assays
nated seeds remains
supporting
oxygen
these
uptake by
days
of
is,
that
mitochondria from
whereas that of older
constant
An
germination.
observations
cotyledons
(1966)
cotyleindicathe
during
newly germi-
decreases with
time.
Histochemical observations
significance
of the observed
with vital redox
particle
indicators,
bound succinate
cambium and less
drogenase activity
During
the
from the
procambium
bundles
in
of these
investigated
activity.
In
found
was
potential xylem,
are
the
16(3),Sept.
only
and Öpik
the
to
together
and
in
chloride,
in the
Succinate
pro-
dehy-
parenchyma.
within 3
to
but remained in the
tissue which
into
(1965),
of pea
respiration partly
to
about the
experiments
mainly
subepidermal layers.
undergoes
4
days,
adjacent
a
differen-
correlation between this
in mitochondrial activity
(1956)
soluble oxidases
in relation
Actaßot. Need.
more
procambium, taking place
respiration
vivo
attributed the rise in
and
Investigations
changes
Mapson & Moustafa
the
know
Janus Green and blue tétrazolium
epidermal
during germination.
with
to
also localized in certain cells of the storage
was
differentiationand the
role
as
the differentiation of the
cells. These
ments
changes
such
in order
in mitochondrial
dehydrogenase activity
in the
activity disappeared
tiation
made
were
are
in progress.
comparing
bean
in vitro
cotyledons,
experi-
respectively,
the increase in soluble oxidases. The
with
the
role
of anaerobiosis will
be
the present results.
1967
121
C. KOLLOFFEL
ACKNOWLEDGEMENTS
The author wishes
to
J.
constant
van
Die for their
Thanks
tions
also
are
due
the
regarding
to
his thanks
express
interest and
Prof.
R. van
stimulating
der Veen and
to
Prof.
discussions.
Dr. H. P. Bottelier for his interest and critical sugges-
to
manuscript.
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