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Developmental Studies on Microbodies in Wheat Leaves : II. Ontogeny of Particulate Enzyme Associations

Authors:

Abstract

Crude particulate fractions from wheat leaves (Triticum vulgare L.) were separated on continuous sucrose density gradients, resulting in: broken chloroplasts, a mitochondrial fraction (indicated by cytochrome c oxidase), and microbodies. The visible band of the microbody fraction from adult leaves appears at a buoyant density of 1.25 grams per cm(3) and contains most of the activities of catalase, glycolate oxidase, and hydroxypyruvate reductase on the gradient. In the shoots of freshly soaked seeds, catalase is already highly particulate. During further development in light or in darkness, 40 to 60% of the total activities of catalase and glycolate oxidase and 25 to 40% of the total activity of hydroxypyruvate reductase are always found in the particulate fractions of the leaves. In young developmental stages, the peaks of the activity profiles of the microbody enzymes appear on sucrose gradients at relatively low densities, first between 1.17 to 1.20 grams per cm(3). During development in light, the buoyant density of the microbody fraction shifts to the final value of 1.25 grams per cm(3). However, even after 1 week of growth in the dark, the microbody fraction from etiolated leaves was observed at buoyant densitites 1.17 to 1.24 grams per cm(3) and did not appear as a defined visible band. A characteristic visible microbody band at a buoyant density 1.24 grams per cm(3) was found when the dark-grown seedlings received only three separate 5-minute exposures to white light. A similar peak was also obtained from light-grown leaves in which chloroplast development had been blocked by 3-amino-1,2,4-triazole.
Plant
Physiol.
(1972)
49,
33-39
Developmental
Studies
on
Microbodies
in
Wheat
Leaves1
II.
ONTOGENY
OF
PARTICULATE
ENZYME
ASSOCIATIONS
Received
for
publication
June
2,
1971
J.
FEIERABEND2
AND
HARRY
BEEVERS
Division
of
Natural
Sciences,
University
of
California,
Santa
Cruz,
California
95060
ABSTRACT
Crude
particulate
fractions
from
wheat
leaves
(Triticum
vulgare
L.)
were
separated
on
continuous
sucrose
density
gradi-
ents,
resulting
in:
broken
chloroplasts,
a
mitochondrial
frac-
tion
(indicated
by
cytochrome
c
oxidase),
and
microbodies.
The
visible
band
of
the
microbody
fraction
from
adult
leaves
appears
at
a
buoyant
density
of
1.25
grams
per
cm'
and
con-
tains
most
of
the
activities
of
catalase,
glycolate
oxidase,
and
hydroxypyruvate
reductase
on
the
gradient.
In
the
shoots
of
freshly
soaked
seeds,
catalase
is
already
highly
particulate.
During
further
development
in
light
or
in
darkness,
40
to
60
%
of
the
total
activities
of
catalase
and
glycolate
oxidase
and
25
to
40%
of
the
total
activity
of
hydroxypyruvate
reductase
are
always
found
in
the
particulate
fractions
of
the
leaves.
In
young
developmental
stages,
the
peaks
of
the
activity
profiles
of
the
microbody
enzymes
appear
on
sucrose
gradients
at
rela-
tively
low
densities,
first
between
1.17
to
1.20
grams
per
cm3.
During
development
in
light,
the
buoyant
density
of
the
microbody
fraction
shifts
to
the
final
value
of
1.25
grams
per
cm'.
However,
even
after
1
week
of
growth
in
the
dark,
the
microbody
fraction
from
etiolated
leaves
was
observed
at
buoyant
densities
1.17
to
1.24
grams
per
cm'
and
did
not
ap-
pear
as
a
defined
visible
band.
A
characteristic
visible
microbody
band
at
a
buoyant
density
1.24
grams
per
cm'
was
found
when
the
dark-grown
seedlings
received
only
three
separate
5-minute
exposures
to
white
light.
A
similar
peak
was
also
obtained
from
light-grown
leaves
in
which
chloroplast
development
had
been
blocked
by
3-amino-1,2,4-triazole.
In
the
preceding
paper
(3),
the
development
of
several
marker
enzymes
of
leaf
microbodies
has
been
described.
The
investigation
presented
in
the
following
will
describe
the
de-
gree
and
kind
of
particulate
association
of
the
enzymes
in
the
various
developmental
stages
and
under
the
different
condi-
tions
described
previously
(3).
Microbodies
of
adult
green
leaves,
like
those
from
other
tissues
(2,
10),
are
characterized
by
a
buoyant
density
of
1.25
g
per
cm'
after
centrifugation
on
continuous
sucrose
density
gradients.
Therefore
equilibrium
density
centrifugation
on
sucrose
gradients
was
used
to
sep-
arate
organelle
fractions
from
developing
wheat
leaves
and
'This
work
was
supported
by
National
Science
Foundation
Grant
GB24961.
2Permanent
address:
Pflanzenphysiologisches
Institut,
34
Got-
tingen.
Unterf,
Karspille
2,
Germany.
the
occurrence
of
marker
enzymes
in
these
fractions
was
ex-
amined.
The
development
of
glyoxysomes,
the
specialized
micro-
bodies
of
fatty
seedlings,
has
been
described
(4, 7).
Develop-
mental
studies
on
leaf
microbodies
have
already
been
reported
in
the
special
instance
of
greening
cotyledons
where
they
re-
place
a
population
of
glyoxysomes
after
the
breakdown
of
the
reserve
fat
(5,
6,
9).
No
such
problem
of
a
changing
micro-
body
population
arises
in
normal
leaves,
since,
as
in
wheat,
they
do
not
originate
directly
from
fatty
organs.
MATERIAL
AND
METHODS
Plant
material
and
growing
conditions
have
been
described
in
the
preceding
paper
(3).
Sylvania
fluorescent
tubes
(No.
F72T12-CW-VHO)
were
used
as
a source
of
white
light.
The
intensity
was
approximately
14.8
X
10'
ergs
cm-2
sec'.
Preparation
of
Cellular
Organelies.
For
the
preparation
of
the
cell
organelles,
the
coleoptiles
were
removed
and
only
the
leaves
were
used.
In
more
advanced
stages
(5
and
6
days),
only
the
upper
three-quarters
of
the
leaves
were
used.
The
grinding
medium,
modified
from
that
described
by
Gerhardt
and
Bee-
vers
(4),
consisted
of
0.451
M
sucrose,
2.5%
Ficoll,
0.15
M
tricine
buffer,
pH
7.5,
1
mm
EDTA,
pH
7.5,
10
mm
KCl,
1
mM
MgCl1,
2
mm
sodium
ascorbate.
In
ice-cold
grinding
medium
a
fine
mince
of
the
leaf
material
was
prepared
with
razor
blades.
This
mince
was
further
briefly
and
gently
ground
in
a
mortar,
with
the
addition
of
a
small
quantity
of
sand.
Two
to
3
ml
of
grinding
medium
were
used
per
gram
fresh
weight
of
tissue.
The
enzyme
recoveries
showed
that
less
than
40%
of
the
cells
were
broken
under
these
conditions.
The
homogenate
was
passed
through
four
layers
of
cheesecloth
and
centrifuged
for
5
min
at
120g.
The
supernatant
solution
was
recentrifuged
for
30
min
at
7,700g.
The
resulting
crude
particulate
pellet
was
resuspended
(1
ml
solution
per
2
g
fresh
weight
of
the
tis-
sue
originally
used)
in
a
modified
grinding
medium
containing
twice
the
concentration
of
sucrose
and
no
ascorbate.
Usually
1.5
or
2
ml
of
this
suspension
were
layered
on
top
of
a
con-
tinuous
nonlinear
gradient
of
sucrose
contained
in
1
mM
EDTA,
pH
7.5.
The
tubes
were
centrifuged
for
4
hr
at
64,300g
and
2
C
in
a
Spinco
rotor
SW
25.2.
The
gradients
had
a
total
volume
of
approximately
40 ml
layered
on
a
cushion
of
8
ml
60%
sucrose
(w/w).
The
con-
cave
gradients
were
prepared
by
an
ISCO
Dialagrad
gradient
pump,
Model
380.
Slightly
modified
gradients
were
used
for
better
separation
of
the
subcellular
particles
from
leaves
grown
under
different
conditions.
In
the
standard
program
used
for
normal
light-grown
materials,
the
ratios
of
the
flow
rate
of
1
mM
EDTA,
pH
7.5,
to
that
of
60%
(w/w)
sucrose
(in
1
mm
EDTA,
pH
7.5)
set
at
the
11
dials
were:
0,
9,
17,
24.5,
30.5,
34.5,
38,
40,
43,
48,
and
53%.
The
gradients
were
fractionated
33
FEIERABEND
AND
BEEVERS
50
Glycolate
OxidaseI
0E
E6
40
0
20
30
Fraction
number
In@
:un
,
O
FIG.
1.
Separation
of
the
crude
particulate
fraction
from
adult
wheat
leaves
(plants
grown
for
2-3
months
in
the
greenhouse)
on
a
continuous
sucrose
density
gradient.
The
material
on
the
gradi-
ent
was
derived
from
3
g
of
leaf
tissue.
The
dashed
lines
mark
the
locations
of
densities
1.165
(left)
and
1.25
(right)
g/cm3.
by
collecting
successive
1-ml
samples
from
an
ISCO
density
gradient
fractionator,
Model
D.
Analytical
Methods.
The
methods
for
the
determination
of
the
enzyme
activities
and
of
the
protein
content
have
been
re-
ported
in
the
previous
paper
(3).
The
chlorophyll
content
of
the
gradient
fractions
was
determined
spectrophotometrically
(14).
Sucrose
concentrations
were
determined
on
each
of
the
gradient
fractions
with
a
Bausch
and
Lomb
Abbe-3L
refrac-
tometer.
RESULTS
Separation
of
Subceliular
Particles.
Figure
1
shows
a
typical
distribution
of
subcellular
particles
after
equilibrium
centrifu-
gation
of
the
crude
particulate
fraction
obtained
from
adult
Plant
Physiol.
Vol.
49,
1972
wheat
leaves
on
a
continuous
sucrose
density
gradient.
The
crude
particles
used
for
this
gradient
contained
80%
of
the
chlorophyll,
70%
of
the
cytochrome
oxidase,
37%
of
the
cata-
lase,
35%
of
the
glycolate
oxidase,
and
22%
of
the
hydroxy-
pyruvate
reductase
present
in
the
leaf
material.
As
has
already
been
shown
by
Tolbert
and
others
for
preparations
from
leaves
of
other
species
(10-12),
three
main
fractions
of
subcellular
particles
can
be
discriminated
on
the
gradient.
At
a
buoyant
density
around
1.165
g/cm3,
there
is
a
strong
band
containing
most
of
the
chlorophyll
and
protein
present
on
the
gradient.
As
for
the
gradients
from
spinach
(10,
11)
this
fraction
repre-
sents
broken
chloroplasts.
Cytochrome
oxidase
was
used
as
a
marker
for
mitochon-
drial
material.
Its
activity
shows
a
major
peak
at
buoyant
densities
between
1.20
to
1.21
g
per
cm3
but
spreads
broadly
over
the
gradient
into
the
chloroplast
material
(Fig.
1).
The
range
between
densities
1.20
to
1.21
is
regarded
as
the
true
mitochondrial
fraction.
In
comparative
experiments
with
spin-
ach
leaves,
a
clear
peak
of
cytochrome
oxidase
appeared
only
between
1.20
to
1.22
g
per
cm3,
which
is
also
reported
as
typi-
cal
for
the
mitochondrial
fraction
by
Rocha
and
Ting
(10).
In
the
wheat
material,
mitochondrial
particles
appear
to
be
trapped
by
the
broken
chloroplasts
(Fig.
2).
The
mitochondrial
fractions
from
younger
stages,
all
etiolated
leaves,
or
even
light-grown
plants
in
which
greening
had
been
artificially
pre-
vented
by
herbicide
treatment,
appear
at
considerably
lower
densities
on
sucrose
gradients
than
the
mitochondrial
fraction
from
green
leaves
(see
Figs.
3-6).
A
sharp
peak
of
cytochrome
oxidase
activity
is
observed
at
buoyant
densities
of
1.17
to
1.18
g
per
cm3,
close
to
the
value
typical
of
mitochondria
from
many
nongreen
plant
tissues
(2).
When
a
crude
particulate
fraction
from
etiolated
leaves
of
6-day-old
seedlings
(see
Fig.
4)
is
mixed
with
a
chloroplast
fraction
before
being
centrifuged
on
a
sucrose
density
gradient,
the
activity
profile
of
cytochrome
oxidase
is
drastically
modified
(Fig.
2).
A
major
portion
of
the
activity
is
now
found
trapped
in
the
broken
chloroplasts
coin-
CL
0.2
9-c
0
0
0.1
E
5-
E
zv
10
0.
01)
(I)
0
o
0-0
3l
0
20
30
Fraction
number
FIG.
2.
Trapping
of
mitochondria
by
chloroplasts.
Distribution
of
cytochrome
oxidase
when
the
crude
particulate
fraction
from
etiolated
leaves
(6
days
old)
was
centrifuged
on
a
sucrose
density
gradient
(A).
Distribution
of
cytochrome
oxidase
(A)
and
chloro-
phyll
(0)
on
the
gradient
when
the
crude
particulate
fraction
from
the
etiolated
leaves
was
mixed
with
a
chloroplast
fraction
(pellet
obtained
after
5
min
centrifugation
at
l,OOOg
from
a
5
min
120g
supernatant)
from
light-grown
wheat
leaves
of
the
same
age
be-
fore
centrifugation.
34
0.3~
5[
DEVELOPMENT
OF
MICROBODIES
IN
LEAVES.
II
ciding
with
the
chlorophyll
peak
so
that
the
original
location
of
the
mitochondria
can
hardly
be
recognized.
This
trapping
effect
is
probably
the
reason
for
the
broad
tails
of
the
cyto-
chrome
oxidase
profiles
seen
on
all
gradients
obtained
from
more
advanced
green
leaves.
At
a
buoyant
density
of
1.25
g
per
cm3
a
sharp white
band
with
a
very
small
percentage
of
the
total
particulate
protein
is
found.
This
contains
most
of
the
activities
of
several
enzymes
which
have
been
reported
to
be
housed
in
leaf
microbodies
(10-13):
catalase,
glycolate
oxidase,
and
hydroxypyruvate
re-
ductase
(see
Figs.
1
and
3,
6-day
stage).
Occasionally
some
ac-
tivity
of
these
enzymes
also
appears
at
the
top
of
the
gradients
due
to
a
changing
degree
of
leakage
or
breakage
of
the
micro-
body
particles
during
the
isolation
procedure.
Behavior
of
the
Microbody
Fraction
during
Normal
Devel-
opment
of
the
Leaves
in
Light
and
Darkness.
A
large
percent-
age
of
the
catalase
activity
present
in
the
tiny
shoots
of
freshly
soaked
wheat
seeds
is
found
in
the
crude
particulate
fraction,
whereas
the
latter
contains
only
a
small
portion
of
the
total
activity
of
hydroxypyruvate
reductase
(Table
I).
During
fur-
ther
development
in
light,
as
well
as
in
the
dark,
between
40
to
60%
of
the
total
activities
of
catalase
and
glycolate
oxidase
in
the
leaves
are
always
found
in
the
particulate
fraction.
The
particulate
portion
of
the
activity
of
hydroxypyruvate
reduc-
tase
is
always
smaller,
representing
between
25
to
40%
in
leaves
of
plants
more
than
2
days
old.
The
portion
of
the
ac-
tivities
found
in
the
particulate
fraction
decreases
slightly
with
increasing
age
of
the
plants
(Table
I).
Figure
3
illustrates
the
development
of
the
microbody
frac-
tion
during
germination
in
light.
The
youngest
developmental
stages
pose
certain
problems
because
of
the
low
enzyme
ac-
tivities
(3)
and
the
difficulty
of
obtaining
enough
leaf
material.
The
small
leaves
(5-10
mm)
after
2
days
of
germination
in
light
do
not
contain
glycolate
oxidase
(3).
Their
particulate
catalase
fraction
appears
on
sucrose
gradients
at
buoyant
den-
sities
1.17
to
1.19
g
per
cm3,
overlapping
but
not
truly
coin-
ciding
with
the
mitochondrial
fraction
(Fig.
3,
Table
II).
On
gradients
prepared
from
green
leaflets
of
3-day-old
plants,
no
typical
microbody
band
can
yet
be
found.
Catalase
appears
at
similar
densities
as
in
2-day-old
plants
but
spreads
much
fur-
ther
into
higher
densities.
Only
at
higher
densities
is
it
accom-
panied
by
glycolate
oxidase
(Fig.
3).
At
the
4th
day
of
germi-
nation
a
clear
peak
of
catalase
activity
appears
at
a
buoyant
density
of
1.24
g
per
cm3.
Finally,
after
6
days
of
germination,
a
sharp
(and
now
also
visible)
typical
microbody
band
can
be
isolated
from
the
leaves
on
sucrose
density
gradients.
It
con-
tains
most
of
the
activities
of
catalase,
glycolate
oxidase,
and
hydroxypyruvate
reductase
on
the
gradient.
Its
buoyant
den-
sity
approaches
1.25
g
per
cm3,
but
is
still
significantly
lighter
than
that
from
older
leaves
(Table
II).
The
specific
activity
of
the
microbody
enzymes
in
the
peaks
of
activity
increases
in
a
striking
manner
during
development.
Two
features
contribute
to
this
increase:
(a)
the
increasing
total
amounts
of
the
en-
zymes,
and
(b)
the
fact
that
at
younger
stages
the
microbodies
overlap
with
other
fractions
of
higher
protein
content.
On
the
gradients
obtained
with
material
from
4-
or
6-day-old
plants
(Fig.
3),
the
profile
of
the
glycolate
oxidase
activity
shows
a
remarkable
shoulder
or
extra
peak
at
lower
densities
which
does
not
coincide
with
the
rather
uniform
behavior
of
catalase
and
hydroxypyruvate
reductase.
This
phenomenon
is
not
yet
completely
understood.
However,
it
is
most
probable
that
this
fraction
of
glycolate
oxidase
of
slightly
lower
density
represents
broken
microbody
particles.
The
other
two
marker
enzymes
obviously
leak
out
of
the
particles
much
more
easily;
thus
more
catalase
than
glycolate
oxidase
may
appear
in
the
supernatant
fraction
(Table
I,
especially
the
6-day
light
stage)
or
on
the
top
of
the
gradient.
When
the
crude
particulate
frac-
tion
(from
green
leaves
of
6-day-old
seedlings)
was
deliberately
Table
I.
Percenztages
of
Enizyme
Activities,
Chlorophyll,
anid
Protei,z
Fountd
in
the
Crude
Particulate
Fractioni
of
Wheat
Leaves
The
data
shows
the
sum
of
the
values
recovered
in
the
pellets
after
centrifugation
at
120g
and
7,700g
as
percentage
of
total
in
the
homogenates.
The
120g
pellets
alone
contained
only
between
3
and
7%
of
the
total
activities
of
the
microbody
marker
enzymes.
For
the
preparations
from
ungerminated
seeds,
the
whole
shoots
were
used
after
soaking
the
seeds
for
6
to
7
hr
in
distilled
water.
The
120g
pellet
obtained
from
this
material
contained
17%
of
the
total
catalase
activity.
In
the
final
entry
the
seedlings
were
exposed
to
5
min
light
at
the
ages
of
3,
4,
and
5
days.
Hiy-
Cata-
pyco
rou-
Cyto-
Chloro-
Treatment
lase
late
vate
chrome
phyllProtein
Oxidase
Reduc_
Oxidase
tase
7%
of
total
Shoots
of
seeds
47.3
...
13.1
88.9
...
31.2
4
Days
dark
68.6
67.3
...
13.3
6
Days
dark
58.9
69.6
42.8
87.3
31.0
2
Days
light
61.4
...
8.7
85.8
...
3
Days
light
65.8
62.9
30.2
84.6
85.6
33.8
4
Days
light
56.4
52.6
31.6
90.5
92.6
38.1
6
Days
light
44.5
50.6
36.9
75.9
82.3
43.1
Adult
leaves
35.6
34.2
24.9
76.2
89.8
36.4
6
Days
light,
80
yM
42.7
39.9
35.9
82.6
...
27.5
aminotriazole
6
Days
light,
0.1
mm
56.0
58.9
...
85.3
...
29.5
aminotriazole
6Daysdark+3treat-
55.7
67.9
44.1
84.4
.
...
ments
5
min
light
broken
by
resuspending
it
in
distilled
water
and
thereafter
centrifuged
on
a
sucrose
gradient,
almost
all
of
the
catalase
activity
stayed
at
the
top
of
the
gradient.
However,
a
large
por-
tion
of
glycolate
oxidase
accompanied
by
only
very
little
of
the
catalase
still
appears
on
the
gradient
at
a
density
range
slightly
lower
than
that
of
the
intact
microbody
fraction.
The
behavior
of
the
microbody
fraction
during
development
in
the
dark
is
demonstrated
in
Figure
4.
As
indicated
by
the
yellow
color
of
carotenoids,
etioplast
material
seems
to
over-
lap
with
the
mitochondrial
fraction
on
the
gradient
or
goes
into
the
pellet
at
the
bottom
of
the
tube.
After
4
days
of
growth
in
the
dark,
a
broad
fraction
with
catalase
activity