Enzyme Development and Glyoxysome Characterization in Cotyledons of Cotton Seeds

Abstract

Unimbibed, mature cotton seeds (cv. Deltapine 61) were found to possess activity for all gluconeogenesis-related enzymes examined, except for isocitrate lyase activity. This indicates that transcription and translation of most enzymes needed for postgerminative growth takes place during seed maturation. This is in contrast with the generalization that "germination" enzymes are synthesized de novo from previously untranslated mRNAs conserved in dry seeds. All enzyme activities increased 3-fold or greater following imbibition, and most remained constant after reaching their peak. Notable exceptions were activities for three beta oxidation enzymes and fructose bisphosphatase, which decreased precipitously after peaking with other enzyme activities.Standard sucrose gradient procedures with swing-out rotors were not useful for isolating cotton glyoxysomes. Satisfactory and reproducible results ultimately were obtained with sucrose gradients constructed in a Beckman JCF-Z zonal rotor. Specific activities of glyoxysomal enzymes were 2- to 3-fold lower than those reported for other oil seeds, except malate dehydrogenase which was 10-fold lower. Electron microscopy revealed that protein body fragments were the primary contaminant of glyoxysome fractions. Glyoxysomes were subfractionated by osmotic shock treatments to evaluate sub-organelle localization of constituent enzymes, several of which have not been examined in other oil seeds.

Full text

Plant
Physiol.
(1981)
68,
82-87
0032-0889/8
1/68/0082/06/$00.50/0
Enzyme
Development
and
Glyoxysome
Characterization
in
Cotyledons
of
Cotton
Seeds1
Received
for
publication
September
16,
1980
and
in
revised
form
January
31,
1981
STEPHEN
J.
BORTMAN2,
RICHARD
N.
TRELEASE3,
AND
JAN
A.
MIERNYK4
Department
of
Botany
and
Microbiology,
Arizona
State
University,
Tempe,
Arizona
85281
ABSTRACT
Unimbibed,
mature
cotton
seeds
(cv.
Deltapine
61)
were
found
to
possess
activity
for
all
gluconeogenesis-related
enzymes
examined,
except
for
isocitrate
lyase
activity.
This
indicates
that
transcription
and
translation
of
most
enzymes
needed
for
postgerminative
growth
takes
place
during
seed
maturation.
This
is
in
contrast
with
the
generalization
that
"germi-
nation"
enzymes
are
synthesized
de
novo
from
previously
untranslated
mRNAs
conserved
in
dry
seeds.
AB
enzyme
activities
increased
3-fold
or
greater
following
imbibition,
and
most
remained
constant
after
reaching
their
peak.
Notable
exceptions
were
activities
for
three
/t
oxidation
en-
zymes
and
fructose
bisphosphatase,
which
decreased
precipitously
after
peaking
with
other
enzyme
activities.
Standard
sucrose
gradient
procedures
with
swing-out
rotors
were
not
useful
for
isolating
cotton
glyoxysomes.
Satisfactory
and
reproducible
results
ultimately
were
obtained
with
sucrose
gradients
constructed
in
a
Beckman
JCF-Z
zonal
rotor.
Specific
activities
of
glyoxysomal
enzymes
were
2-
to
3-fold
lower
than
those
reported
for
other
oil
seeds,
except
malate
dehydrogenase
which
was
10-fold
lower.
Electron
microscopy
re-
vealed
that
protein
body
fragments
were
the
primary
contaminant
of
glyoxysome
fractions.
Glyoxysomes
were
subfractionated
by
osmotic
shock
treatments
to
evaluate
sub-organelle
localization
of
constituent
enzymes,
several
of
which
have
not
been
examined
in
other
oil
seeds.
A
major
metabolic
event
during
postgerminative
growth
of
oil
seeds
is
gluconeogenesis
from
storage
lipid.
Glyoxysomes,
spe-
cialized
organelles
containing
enzymes
catalyzing
,8
oxidation
of
fatty
acids
and
glyoxylate
cycle
reactions,
are
centrally
involved
in
this
process
(7).
They
have
been
well
characterized
in
endo-
sperm
of
germinated
castor
beans
(1,
3,
7,
9),
and
found
to
have
similar
enzyme
constituents
and
suborganellar
localizations
in
cotyledons
of
several
other
oil
seeds
(8).
Only
limited
data,
how-
ever,
are
available
on
enzyme
development
and
glyoxysome
char-
acterization
in
economically
important
cotton
seeds.
This
is
ap-
parent
when
one
examines
Table
8.2
in
Gerhardt's
recent
mono-
graph
(7)
on
plant
microbodies;
cotton
is
not
listed
among
the
angiosperm
species
from
which
glyoxysomes
have
been
isolated
and
characterized.
In
our
ongoing
studies
on
cotton
seed
development
and
germi-
'This
work
was
supported
by
National
Science
Foundation
Grant
PCM-7823
156.
2
Present
address:
United
States
Department
of
Agriculture,
Science
and
Education
Administration,
Agriculture
Research,
Western
Cotton
Research
Laboratory,
4135
East
Broadway
Rd,
Phoenix,
AR
85040.
3
To
whom
reprint
requests
should
be
sent.
4Present
address:
Biology
Department,
Queens
University,
Kingston,
Ontario
K7L3N6,
Canada.
82
nation
(2,
17-19,
21,
26),
it
became
important
to
determine
which
gluconeogenesis-related
enzymes
were
already
present
in
unim-
bibed
seeds,
and
where
they
were
compartmentalized
within
cells
during
postgerminative
growth.
We
attempted
to
isolate
glyoxy-
somes
on
sucrose
gradients
in
swing-out
rotors
according
to
estab-
lished
procedures,
but
found
these
methods
were
unsuitable
for
separating
cotton
glyoxysomes
from
other
organelles.
In
this
paper,
we
show
the
activity
of
numerous
gluconeogen-
esis-related
enzymes
in
unimbibed
seeds,
and
tabulate
develop-
ment
of
these
activities
in
cotyledons
as
lipid
reserves
are
mobi-
lized.
A
reliable
procedure
is
described
for
isolating
and
purifying
cotton
glyoxysomes
via
isopycnic
centrifugation
in
a
zonal
rotor.
The
organelles
were
characterized
extensively
as
to
their
enzyme
content
and
suborganellar
localization.
Comparisons
are
made
with
similar
data
on
glyoxysomes
from
other
oil
seeds,
most
of
which
have
been
summarized
(7).
MATERIALS
AND
METHODS
Acid-delinted,
fungicide-treated
cotton
(Gossypium
hirsutum
L.
cv.
Deltapine
16
and
61)
seeds
were
presoaked
and
decoated
prior
to
plating
in
sterile
Petri
plates
(21).
Germination
and
growth
of
seeds
were
at
30
C
in
the
dark.
Homogenization
and
Centrifugation.
Total
enzyme
activity
in
cotyledons
of
unimbibed
and
germinated
seeds
was
assayed
in
French-pressed,
clarified
homogenates
similar
to
procedures
de-
scribed
by
Miernyk
et
al.
(17).
Decoated,
unimbibed
seeds
were
ground
to
a
coarse
meal
in
a
mortar
before
being
homogenized
in
a
medium
with
a
motorized
Teflon
pestle.
Although
most
organ-
elles
were
broken
during
this
procedure,
extracts
were
further
homogenized
in
a
French
pressure
cell
at
984
kg/cm2.
The
medium
used
for
most
enzyme
assays
contained
100
mm
K-phosphate
(pH
6.9),
12
mm
MgCl2,
and
3
mm
DTT.
DTT
in
media
did
not
interfere
with
assays
of
malate
synthase
or
citrate
synthase
activity
after
background
A
(412
nm)
was
recorded.
When
fructose
bis-
phosphatase,
NADP-isocitrate
DH,5
triosephosphate
isomerase,
and/or
aconitase
were
assayed,
cotyledons
were
homogenized
in
100
mm
Mops-KOH
(pH
6.9),
with
12
mm
MgC12.
For
preservation
of
organelles,
cotyledons
were
lightly
chopped
in
1.5
volumes
of
grinding
medium
at
4
C
with
razor
blades
attached
to
an
electric
knife
(26).
The
grinding
medium
contained
500
mm
sucrose,
2.5%
Ficoll,
10
mM
MgCl2,
and
3
mm
EDTA
in
50
mm
K-phosphate
(pH
6.9).
The
slurry
was
filtered
through
three
layers
of
buffer-moistened
Miracloth.
Minced
cotyledons
held
in
Miracloth
were
more
thoroughly
chopped
in
another
1.5
volumes
of
grinding
medium
until
only
small
(I
mm)
pieces
remained.
The
resultant
slurry
was
filtered
as
before.
To
prevent
breakage
of
starch-containing
plastids,
combined
fitrates
were
centrifuged
at
two
different
g
forces
without
removal
between
centrifugations:
at
240g
for
5
min
and
accelerated
to
522g
for
4
'Abbreviations:
DH,
dehydrogenase;
AT,
aminotransferase.
www.plant.org on February 17, 2015 - Published by www.plantphysiol.orgDownloaded from
Copyright © 1981 American Society of Plant Biologists. All rights reserved.

GLYOXYSOMAL
ENZYMES
IN
COTTON
SEEDS
more
min
(Beckman
JS-13
rotor
in
a
J-21
B
centrifuge).
Clarified
homogenates
were
either
differentially
centrifuged
at
19,720g
for
30
min
to
obtain
a
glyoxysome-enriched
pellet,
or
layered
onto
a
sucrose
gradient
for
organelle
isolation.
For
swing-out
rotor
experiments,
general
procedures
outlined
by
Huang
(8,
9)
were
followed.
Modifications
of
the
methods
are
explained
in
the
text.
For
zonal-rotor
sucrose
gradient
centrifugation,
50
to
80
ml
of
clarified
homogenate
were
applied
to
a
700-ml
continuous
sucrose
gradient
generated
by
adding
100
ml
each
of
30,
35,
40,
45,
50,
55,
and
60%
(w/w)
sucrose
solutions
(in
20
mm
K-phosphate
(pH
6.9))
to
a
Beckman
JCF-Z
zonal
rotor
spinning
at
2,000
rpm.
The
gradient
was
supported
by
a
200-ml
62%
(w/w)
sucrose
cushion
in
the
same
buffer.
After
sample
application,
approximately
950
ml
20
mm
K-phosphate
buffer
was
added
as
overlay.
The
contents
were
centrifuged
at
25,000g
(calculated
at
the
radius
of
the
rotor
correponding
to
the
microbody
band,
8.2
cm)
for
2.5
to
3
h
in
a
Beckman
J-21
B
centrifuge.
After
rotor
deceleration
to
2,000
rpm,
contents
were
displaced
through
the
core
by
pumping
62%
(w/w)
sucrose
into
the
edge
line.
Fractions
were
collected
by
hand
in
25-
ml
aliquots
and
used
for
glyoxysome
subfractionation,
enzyme
assays,
and
electron
microscopy.
For
glyoxysome
subfractionation
experiments,
50
ml
glyoxy-
some
peak
(1.25-1.26
g/mi)
were
mixed
and
divided
into
three
12-ml
fractions:
A,
B,
and
C.
Each
fraction
was
diluted
to
a
final
volume
of
36
ml
with
50
mm
K-phosphate
(pH
6.9).
Dilution
buffer
for
fraction
B
contained
0.15
M
KC1,
resulting
in
a
final
concentration
of
0.1
M
KCI,
and
buffer
for
fraction
C
contained
0.3
M
KCI,
resulting
in
a
final
concentration
of
0.2
M
KCI.
Each
suspension
was
briefly
blended
in
a
Vortex
mixer
and
incubated
for
30
min
at
4
C
according
to
a
modified
procedure
(8).
Mixtures
were
then
centrifuged
at
39,000g
for
30
min
(Beckman
JA-20
rotor
in
a
J-21
B
centrifuge)
to
separate
membranes
from
solubilized
enzymes.
Pellets
were
resuspended
in
50
mm
K-phosphate
(pH
6.9),
containing
500
mm
sucrose
and
appropriate
KCI
concentra-
tion.
Enzyme
Assays.
All
chemicals,
substrates,
and
coupling
en-
zymes
were
obtained
from
Sigma
unless
noted
otherwise.
Isocitrate
lyase
(EC
4.1.3.1)
and
citrate
synthase
(EC
4.1.3.7)
were
assayed
as
described
by
Cooper
and
Beevers
(3).
Malate
synthase
(EC
4.1.3.2)
was
assayed
as
described
by
Miernyk
et
al.
(17),
malate
DH
(EC
1.11.1.6)
as
reported
by
Choinski
and
Trelease
(2),
3-
hydroxyacyl-CoA
DH
(EC
1.1.1.35),
enoyl-CoA
hydratase
(EC
4.2.1.17),
and
3-oxoacyl-CoA
thiolase
(EC
2.3.1.16)
according
to
Miernyk
and
Trelease
(19),
triosephosphate
isomerase
(EC
5.3.1.1)
according
to
Linskens
et
al.
(14),
and
succinate
DH
(EC
1.3.99.1)
as
reported
by
Singer
et
al.
(24).
The
assay
for
aspartate:a-keto-
glutarate
AT
(EC
2.6.1.1)
was
modified
from
Liu
and
Huang
(15):
70
,Lmol
Mops-KOH
(pH
8.2),
3
,umol
aspartate,
60
nmol
pyridoxal
5'-phosphate,
1.25
,umol
NADH,
and
extract
in
a
final
volume
of
1.0
ml.
After
recording
Awo
for
"NADH
oxidase"
activity,
reaction
was
initiated
with
0.1
ml
5
mM
a-ketoglutarate.
Addition
of
exogenous
malate
DH
was
unnecessary.
Alanine:a-ketoglutarate
AT
(EC
2.6.1.2)
was
assayed
in
the
same
manner,
but
was
coupled
with
33
units
pig
heart
lactate
DH.
The
fructose-bisphosphatase
(3.1.3.11)
assay
was
modified
from
Youle
and
Huang
(29):
120
,umol
Mops-KOH
(pH
7.0),
4
,umol
MgCl2,
2
,umol
EDTA,
0.5
,umol
NADP,
2.5
units
hexosephosphate
isomerase,
and
2
units
glucose-6-P
DH
in
a
final
volume
of
1.0
ml.
Background
Am0
was
recorded
and
reaction
was
started
by
addition
of
50
,tl
80
mim
fructose
1,6-bisphosphate.
NADP-isocitrate
DH
(EC
1.1.1.41)
as-
say
was
modified
from
Curry
and
Ting
(4):
54
,mol
Hepes-KOH
(pH
7.4),
62.5
nmol
NADP,
and
1
,umol
MnCI2
in
a
final
volume
of
1.0
ml.
After
recording
Am0
background,
reaction
was
initiated
with
20
pd
10
mm
isocitrate.
Aconitase
(EC
4.2.1.2)
activity
was
measured
similarly
except
4
,umol
DTT
was
included,
reaction
was
coupled
with
0.44
unit
pig
heart
NADP-isocitrate
DH,
and
initi-
ated
with
20
,il
10
mm
cis-aconitate.
The
hydroxypyruvate
reduc-
tase
(EC
1.1.1.81)
assay
mixture
contained
90
,imol
K-phosphate
(pH
6.0),
62.5
nmol
NAD,
and
extract
in
a
final
volume
of
10
ml.
Reaction
was
initiated
with
20
pl
30
mm
hydroxypyruvate.
Hy-
droxypyruvate
was
30
times
more
active
as
substrate
than
glyox-
ylate.
Units
for
enzyme
activities
in
homogenates
are
nmol
min-1
cotyledon
pair-',
except
for
catalase
activity
which
is
expressed
as
Luck
units
per
cotyledon
pair.
Protein
was
measured
as
described
by
Lowry
et
al.
(16).
Electron
Microscopy.
Organelles
from
gradient
fractions
were
fixed
for
20
min
by
adding
an
equal
volume
of
6%
glutaraldehyde
and
54%
sucrose
in
20
mm
K-phosphate
(pH
6.9),
at
room
tem-
perature.
Preserved
organelles
were
collected
by
centrifugation
for
20
min
at
20,000g
(JA-20
rotor).
Pellets
were
resuspended
in
20
mm
K-phosphate
(pH
6.9),
and
concentrated
on
0.22-,um
Gelman
cellulose
triacetate
filters
using
a
Swinney
adaptor
attached
to
a
10-ml
syringe.
Filters
were
folded
once,
encased
in
warm
2%
agar,
postfixed
for
1
h
with
2%
OS04
in
50
mm
K-phosphate
(pH
6.9),
dehydrated
in
a
graded
acetone
series,
and
embedded
in
Spurr's
resin.
RESULTS
The
morphology
of
cotton
seedlings
germinated
and
grown
in
moist
Petri
dishes
is
shown
in
Figure
1.
Embryos
were
fully
imbibed
by
5
h;
radicle
protrusion
-1
cm
(germination)
was
complete
by
12
h.
Cotyledon
unfolding
and
expansion
were
apparent
at
30
h
when
fresh
weight
of
seedlings
was
increasing
linearly.
Enzyme
Development.
Development
of
enzyme
activity
from
levels
in
dry
seeds
through
72
h
is
shown
in
Table
I.
Except
for
isocitrate
lyase,
unimbibed
seeds
possessed
activity
for
all
enzymes
examined.
Following
imbibition,
activities
of
all
enzymes
in-
creased
to
a
peak
between
36
and
50
h.
Except
for
malate
DH,
triosephosphate
isomerase,
and
alanine
AT,
the
increases
were
3-
fold
or
greater.
Once
peak
activity
was
attained,
activities
of
most
enzymes
leveled
off
or
decreased
slowly.
Exceptions
were
activities
for
three
f8
oxidation
enzymes
(hydroxyacyl-CoA
DH,
enoyl
hydratase,
and
thiolase)
and
fructose
bisphosphatase.
This
group
of
enzymes
showed
a
precipitous
drop
in
activity
after
reaching
a
peak
between
36
and
40
h.
Differential
Centrifugation.
A
major
consequence
of
chopping
cotyledons
(Table
II),
rather
than
homogenizing
with
a
Teflon
pestle
(and
French-pressing)
(Table
I),
was
a
substantial
reduction
(57-90%)
of
enzyme
activities
in
the
clarified,
chopped
homoge-
nates.
Of
several
different
conditions
tested,
centrifugation
of
clarified
homogenates
at
19,720g
for
30
min
(Table
II)
yielded
the
[l--0|20-30|4
Q
45|50|~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~---
FIG.
1.
Morphology
of
cotton
seedlings
removed
from
Petri
plates
at
successive
times
(h)
after
decoating.
Seed
coats
were
removed
by
hand
4
h
after
initial
soaking.
Entire
length
of
60-h
seedling
is
7
cm.
Plant
Physiol.
Vol.
68,
1981
83
www.plant.org on February 17, 2015 - Published by www.plantphysiol.orgDownloaded from
Copyright © 1981 American Society of Plant Biologists. All rights reserved.

BORTMAN,
TRELEASE,
AND
MIERNYK
highest
enzyme
activity
in
glyoxysome-enriched
pellets.
Per
cent
recovery
in
pellets
ranged
from
1%
for
aconitase
to
53%
for
citrate
synthase.
Purification
of
enzyme
activity
in
19,720g
pellets
ranged
from
0-
to
1.6-fold,
which
was
low
compared
to
other
oil
seeds
(1.5-
to
3-fold).
Except
for
isocitrate
lyase,
Triton
X-100
(0.05%
final
concentration)
enhanced
activities
of
all
enzymes
examined
in
the
glyoxysome
pellet.
Malate
DH
activity
was
increased
3-fold
over
that
measured
in
the
absence
of
detergent,
while
from
7
to
25%
increases
were
evident
for
the
other
enzymes.
Effects
of
added
KCI
(0.2
M)
also
were
determined.
Except
for
malate
DH,
activities
of
the
enzymes
were
inhibited
or
unaffected.
Data
in
Table
II
are
from
experiments
in
which
the
per
cent
recovery
in
pellets
and
activity
for
each
enzyme
were
optimized.
Sucrose
Gradient
Centrifugation.
Numerous
attempts,
with
Table
I.
Development
of
Enzyme
Activity
in
Cotyledons
of
G.
hirsutum
(cv.
Deltapine
61,
1977
harvest)
Seeds
were
germinated
and
grown
in
the
dark
on
moist
filter
paper
at
30
C.
Values
are
means
from
at
least
four
separate
experiments
in
each
of
which
12
to
16
cotyledon
pairs
were
homogenized.
Hours
after
Initial
Soaking
EnzymeI
a
I
E0
10-12
122-26
36-40
45-50
70-75
nmol
min
cotyledon
pair-'
X
I0-1
Isocitrate
lyase
0
0
12
41
42
38
Malate
synthase
32
43
71
200
200
160
Catalase'
4
11
22
59
55
56
Malate
DH
840
1,190
1,650
1,840
1,970
1,850
Citrate
synthase
28
42
45
52
84
51
Aconitase
6
8
24
55
51
29
Enoyl
hydratase
110
350
490
620
187
23
Hydroxyacyl-CoA
DH
38
78
116
208
147
42
Oxoacyl
thiolase
3
4
17
26
16
6
Aspartate
AT
38
33
110
137
160
112
Alanine
AT
32
28
41
42
36
21
Triosephosphate
isomerase
1,840
1,900
1,950
1,950
2,160
1,880
Fructose-bisphosphatase
7
7
15
122
96
32
NADP-isocitrate
DH
6
6
14
23
27
20
a
Luck
units.
modification
of
many
parameters,
failed
to
yield
satisfactory
isolation
of
glyoxysomes
from
Deltapine
cotton
cultivars
in
swing-
out
rotors.
Typical
results
were
a
single
protein
plateau
between
1.18
and
1.25
g/ml
where
overlapping
broad
peaks
of
catalase,
isocitrate
lyase,
malate
DH,
and
fumarase
activities
persistently
equilibrated.
Results
were
the
same
when
samples
(either
resus-
pended
19,720g
pellets
or
520g
clarified
supernatants)
were
cen-
trifuged
4
or
17
h
at
53,000gav
in
either
a
Beckman
SW
25.2
or
SW
25.1
rotor.
Modifying
sample
protein
content
(20
or
60
mg),
homogenizing
buffer
(K-phosphate,
Tricine,
Tris-HCl),
pH,
ad-
dition
of
PVP
or
BSA,
or
altering
type
and/or
concentration
of
osmoticum
(0.25-0.6
M
sucrose,
including
mixtures
of
sucrose
and
Ficoll)
did
not
appreciably
alter
distribution
of
overlapping
marker
enzyme
activities.
In
one
experiment,
a
clarified
homoge-
nate
of
germinated
cucumber
seedlings
was
prepared
and
centri-
fuged
in
the
same
manner
as
a
cotton
homogenate.
Marker
activities
for
the
cucumber
enzymes
separated
as
expected,
whereas
the
same
anomalous
results
were
obtained
with
the
cotton
preparation.
Satisfactory
isolation
of
cotton
glyoxysomes
ultimately
was
obtained
with
a
Beckman
JCF-Z
zonal
rotor
at
25,000gav
for
3
h
(Fig.
2).
These
results
were
reproducible.
More
than
25
zonal-
rotor
experiments
were
done
on
cotton
embryos
of
various
ages
and
germinated
seedlings
without
encountering
problems
similar
to
those
with
swing-out
rotors.
A
major
protein
peak
typically
banded
at
1.19
g/ml
coincident
with
peak
activities
of
mitochon-
drial
marker
enzymes
(cyto-oxidase
and
fumarase
profiles
not
shown).
These
enzyme
peaks
were
clearly
separated
from
glyox-
ysomal
enzyme
activities
peaking
at
1.26
g/ml.
Perceptible
activ-
ities
of
the
plastid
marker
enzymes,
triosephosphate
isomerase
and
acetyl-CoA
carboxylase,
were
not
detected
in
the
gradients.
Assays
for
carotenoids
and
flavonoids
also
failed
to
resolve
a
particulate
plastid
region.
Fractions
12
through
16
(1.21-1.23
g/ml)
were
noticeably
yellow,
therefore
may
be
the
region
of
broken
plastids.
Electron
microscopic
examinations
of
the
1.26
g/ml
region
of
gradients
revealed
that
the
glyoxysomes
were
intact
with
a
ho-
mogeneous
matrices
(Fig.
3).
Virtually
absent
from
these
fractions
were
mitochondria
and
plastids.
The
main
and
persistent
contam-
inant
was
protein
body
fragments.
Although
a
large
protein
peak
was
not
apparent
in
the
microbody
region
of
gradients
(Fig.
2),
Table
II.
Per
Cent
Recovery
and
Puriflcation
of
Selected
Enzymes
in
Rate-Sedimented
Pellets
and
Zonal-Rotor
Gradient
Fractions
Homogenates
of
cotyledons
from
48-h-old
cotton
seedlings
were
prepared
with
razor
blades,
clarified
at
520g,
then
either
centrifuged
at
19,720g,
30
min,
or
applied
to
a
Beckman
JCF-Z
zonal
rotor
and
centrifuged
at
25,000g
for
3
h.
Enzyme
units
are
nmol
min-'
cotyledon
pair-'.
Specific
activity
is
units
mg-'
protein;
protein
concentration
in
the
clarified
homogenate
is
3.36
mg
cotyledon
pair-'.
Per
cent
(%)
values
are
per
cent
of
activity
in
the
clarified
homogenate.
Fold
purification
is
computed
from
ratios
of
specific
activities
in
the
fractions
and
clarified
homogenate.
Clarified
Chopped
Pellet
l9,720g,
30
Zonal
Gradient
1.235-1.265
Homogenate
min
g/ml
Enzyme
Units
Specific
%
Pfi-
%
Pf
Specific
activity
cation
caio
activity
catlon
~~cation
Isocitrate
lyase
72
21
26
0.8
19
5.7
120
Malate
synthase
260
77
48
1.5
30
9.6
742
Catalase
93
27
46
1.4
30
9.6
259
Malate
DH
4500
1339
10
0.3
9
2.9
3857
Citrate
synthase
77
23
53
1.6
32
10.4
239
Aconitase
217
64
1
0.1
NDa
ND
ND
Hydroxyacyl-CoA
DH
472
141
37
1.1
27
8.6
1213
Aspartate
AT
386
115
ND
ND
44
6.4
735
Alanine
AT
35
10
ND
ND
38
5.7
57
Hydroxypyruvate
reductase
11
4
ND
ND
36
9.4
38
a
ND,
not
determined.
84
Plant
Physiol.
Vol.
68,
1981
www.plant.org on February 17, 2015 - Published by www.plantphysiol.orgDownloaded from
Copyright © 1981 American Society of Plant Biologists. All rights reserved.

Plant
Physiol.
Vol.
68,
1981
GLYOXYSOMAL
ENZYMES
IN
COTTON
SEEDS
$
*-wi
r
dMh.
t
.,.
;.
z
;
A
iim
-gz§
R:
*
i
*
*
5
t
4
*
:
9
f
*
w
t\;
*
8;
t
.
<.:
:
f
>-.4i.
*
jw
*
.f
Si_
{s<
'
s
h
>
ttEra
*
x
\
vet
,*
8S
sa
x
*
eB.Fv
tZ
f
.2.
..
e':
s
>
h
i
*:
:'
s:
s:6.
...
{
G
{
tX2W
S
W
X
a
61..
,o.#
i
.:
|>8)
+S:
-
*2
/''<
..
t
B
o4E
#
j
n
M.:S
tF
if
^
.
2.;
XX s
^t
';
0d
#
S
u
s
si
&
tC
#
.}.
*
...
w
ts.
a
4
2s
v
2
G
v8;*emv
FIG.
3.
Representative
electron
micrograph
of
organelles
in
the
1.26
g/ml
region
of
a
sucrose
gradient.
Glyoxysomes
(G)
are
scattered
among
the
numerous
protein
body
fragments
(arrows).
Notably,
mitochondria
and
plastids
are
absent.
x
9,000.
protein
levels
were
well
above
base
line
in
all
fractions
nearby
the
peak.
Electron
microscopy
of
fractions
17
and
21
revealed
protein
body
fragment
as
the
main
component.
Attempts
were
made
to
purify
glyoxysomes
from
gradient
frac-
tions
further
by
differentially
centrifuging
them
from
protein
body
fragments.
Glyoxysomes
in
approximately
55%
(w/w)
sucrose
were
diluted
to
50,
45,
40,
or
35%
sucrose
prior
to
centrifugation.
Electron
microscopy
revealed
that
dilutions
to
40
and
35%
were
deleterious,
causing
excessive
loss
of
matrix
material.
Greater
than
70%o
recovery
of
glyoxysomes
could
be
achieved
by
dilutions
to
45%
sucrose,
but
enzyme
specific
activities
were
not
increased
over
those
given
in
Table
II.
Protein
body
fragments
continued
to
contaminate
glyoxysomes
under
all
centrifugation
schemes
tried.
Glyoxysome
Subfractionation.
Table
III
shows
per
cent
enzyme
activity
released
from
isolated
glyoxysomes
following
osmotic
breakage
in
buffer,
or
buffer
containing
KC1.
Treatment
with
buffer
alone
released
more
than
90%o
the
activities
of
isocitrate
lyase
and
the
two
aminotransferases,
indicating
these
enzymes
to
Fraction
Number
FIG.
2.
Distribution
of
cotyledon
proteins
and
enzyme
activities
on
a
continuous,
sucrose
density
gradient.
A
clarified
homogenate
of
cotyledons
from
48-h-old
cotton
seedlings
was
centrifuged
in
a
Beckman
JCF-Z
zonal
rotor
for
3
h
at
16,000
rpm
(25,000g
at
rotor
radius
where
glyoxysomes
banded).
Activity
at
the
top
of
the
gradient
is
not
shown
since
it
was
diluted
by
900
ml
overlay.
Sixty
ml
homogenate
from
165
cotyledon
pairs
were
layered
on
a
linear
gradient
(30-60o,
w/w)
through
the
rotor
core.
For
actual
activity
(nmol
min-'
fraction-1),
multiply
ordinate
values
by
factors
given
for
each
enzyme:
succinate
DH
(SDH),
50;
catalase
(Cat),
190;
citrate
synthase
(CS),
130;
isocitrate
lyase
(ICL),
60;
aspartate
AT
(Asp
AT),
45;
malate
synthase
(MS),
600;
malate
DH
(MDH),
2,500;
hydroxyacyl-CoA
DH
(HAcCoA
DH),
620;
alanine
AT
(Ala
AT),
45;
hydroxypyruvate
reductase
(HPR),
30.
(I,
cn
U1)
C)
ca
Cu3
0
0
CI
c
10
0)
0-
3
2
3
2
>)
0)
E
N
c
w
0
ll
._
z
a:
85
www.plant.org on February 17, 2015 - Published by www.plantphysiol.orgDownloaded from
Copyright © 1981 American Society of Plant Biologists. All rights reserved.

BORTMAN,
TRELEASE,
AND
MIERNYK
be
in
the
glyoxysomal
matrix.
Buffer
treatment
released
approxi-
mately
80%o
catalase
and
malate
DH,
but
salt
treatment
did
not
release
malate
DH
as
it
did
catalase
activity.
Thirty
to
forty
%
malate
synthase,
hydroxyacyl-CoA
DH
and
citrate
synthase
activ-
ities
remained
associated
with
pellets
following
osmotic
shock
in
buffer.
Malate
synthase
was
readily
removed
from
membranes
with
the
use.
of
increased
ionic
strength,
whereas
pellet
activity
of
both
the
other
enzymes
was
not
released
in
salt
solutions.
A
major
portion
of
hydroxypyruvate
reductase
activity
was
bound
to
glyox-
ysomal
membranes.
Nearly
35%
remained
in
pellets
even
after
0.2
M
KC1
treatment.
DISCUSSION
The
central
role
of
glyoxysomes
in
the
mobilization
of
reserve
lipids
is
well
documented
(1, 7).
Recent
evidence
summarized
by
Beevers
(1)
indicates
that
glyoxysomes
and
their
constituent
en-
zymes
in
castor
bean
endosperm
are
synthesized
de
novo
from
RER
following
seed
imbibition.
This
is
consistent
with
Ihle
and
Dure's
proposal
(11)
that
mRNAs
for
these
"germination"
en-
zymes
in
cotton
seeds
are
transcribed
during
embryogenesis,
but
are
not
translated
until
needed
for
postgerminative
growth.
Studies
in
our
laboratory
(2,
18,
19,
26)
and
by
others
(6,
12,
13)
indicate
that
many
of
the
enzymes
involved
in
gluconeogenesis
from
reserve
lipid
already
are
active
in
maturing
embryos
and
are
incorporated
into
microbodies
prior
to
desiccation.
Table
I
shows
that
mature,
dry
cotton
seeds
possess
activities
for
all
the
gluco-
neogenesis-related
enzymes
examined
(except
isocitrate
lyase).
Additionally,
activities
for
numerous
enzymes
related
to
reserve
protein
(5,
21,
28)
and
raffmose
(23)
mobilization
have
been
found
in
unimbibed
cotton
seeds.
These
data
indicate
that
activities
of
embryo-synthesized
enzymes
persist
in
dry
seeds,
presumably
as
part
of
the
developmental
program
to
equip
seeds
for
germination
and
subsequent
mobilization
of
reserves.
Thus,
Ihle
and
Dure's
original
concept
(11)
that
events
occur
during
embryogenesis
to
prepare
the
seed
for
germination
and
growth
still
holds,
but
must
include
translation
of
mRNAs
coding
for
many
of
the
enzymes
required
for
postgerminative
growth.
Furthermore,
the
relation-
ship
between
glyoxysomal
enzyme
activities
in
dry
seeds
and
de
novo
synthesis
of
glyoxysomes
(1)
needs
resolution.
The
apparent
absence
of
isocitrate
lyase
activity
in
cotton
and
18
other
species
of
oil
seeds
(17)
is
a
curious
phenomenon.
Neitfier
we
(2,
18,
26)
nor
Ihle
and
Dure
(1
1)
detected
catalytic
activity
in
developing
cotton
embryos,
whereas
Frevert
et
al.
(6)
measured
low
levels
in
late-stage
cucumber
embryos
when
the
enzyme
was
centrifuged
from
crude
albumen
extracts.
The
latter
authors
sug-
Table
III.
Per
Cent
Solubilization
of
Enzymes
from
Glyoxysomes
Osmotically
Disrupted
with
or
without
KCI
in
buffer
Twelve-ml
portions
of
a
glyoxysome
fraction
(1.26
g/ml)
from
zonal
gradients
were
diluted
to
36
ml
with
K-phosphate
(pH
6.9),
or
buffer
plus
KCI.
Following
a
30-min
incubation,
the
mixtures
were
centrifuged
and
activities
assayed
in
the
supernatants
and
membrane
pellets.
Values
are
per
cent
of
activity
in
supernatants.
Enzyme
Buffer
0.1
M
0.2
M
%
released
Isocitrate
lyase
95
98
Aspartate
AT
91
93
94
Alanine
AT
93
99
99
Catalase
84
95
95
Malate
synthase
62
96
98
Malate
DH
81
82
87
Hydroxyacyl-CoA
DH
69
82
91
Citrate
synthase
68
74
85
Hydroxypyruvate
reductase
33
56
66
gested
that
the
activity
was
masked
by
action
of
an
inhibitor,
but
we
could
not
confirm
this
in
cotton
(17,
18).
In
either
case,
it
appears
that
development
of
isocitrate
lyase
activity
in
embryos
and
its
presence
in
dry
seeds
are
different
than
for
its
companion
enzymes.
Possibly,
this
reflects
a
regulatory
mechanism
for
glyox-
ysomal
synthesis
and/or
metabolism,
although
the
absence
of
lipase
activity
in
unimbibed
cotton
seeds
(10)
cannot
be
ignored
when
considering
regulation
of
oil
seed
metabolism.
Although
numerous
studies
deal
with
glyoxysomes
isolated
from
germinated
seedlings,
only
three
reports
exist
for
cotton
(8,
20,
25).
In
all
three
studies,
a
swing-out
rotor
was
used
for
the
isolations.
Prathapasenan
(20)
reported
equilibration
of
isocitrate
lyase
activity
in
a
main
protein
band
at
1.16
g/ml,
a
finding
similar
to
our
results.
In
the
other
reports,
adequate
separation
of
glyoxysomes
was
implied,
but
gradient
enzyme
profiles
were
not
published,
nor
were
data
given
on
enzyme
specific
activities,
or
contamination
from
other
organelles.
An
explanation
for
failures
with
swing-out
rotors
and
success
with
a
zonal
rotor
is
not
readily
apparent.
We
are
convinced
that
conditions
of
homogenization,
sample
application
and/or
gradient
construction
are
not
mainly
responsible
for
the
anomalies.
Numerous
modifications
of
these
conditions
have
been
attempted,
and
we
routinely
have
isolated
glyoxysomes
from
other
oil
seeds
by
using
swing-out
rotors.
Marker
enzyme
distributions
in
our
experiments
suggested
that
cotton
organelles
were
adhering
to
other
organelles
within
the
gradients.
A
major
advantage
of
zonal-rotor
centrifugation
is
the
elimination
of
tube
wall
effects.
Perhaps
weak
binding
factors,
unique
to
cotton
homogenates
or
the
glyoxysomal
membranes,
were
overcome
by
hydrodynamic
advantages
of
zonal-rotor
cen-
trifugations.
Distribution
of
marker
enzymes
on
the
zonal-rotor
gradients
generally
was
similar
to
that
reported
for
other
oil
seeds,
although
peak
glyoxysomal
activities
consistently
were
at
1.26
g/ml
(Fig.
2)
rather
than
at
1.25
g/ml
(Table
8.2,
Ref.
7).
When
recovery
of
isocitrate
lyase
activity
was
used
as
a
comparative
index
(8),
glyoxysome
recovery
from
cotton
(19%)
was
much
lower
than
from
castor
bean
(80%),
but
similar
to
that
from
watermelon,
cucumber,
or
sunflower
(13-25%).
Specific
activities
of
most
cotton
enzymes
were
2-
to
3-fold
lower
than
those
reported
for
other
sources
(7,
8,
12,
22).
A
notable
exception
in
all
our
experiments
was
a
10-fold
lower
specific
activity
for
malate
DH.
Comparisons
for
alanine
AT
are
not
possible
since
activity
was
reported
only
in
castor
bean
glyoxysomes
and
values
were
not
given
(27).
The
distribution
of
aspartate
AT
in
mitochondria
and
glyoxysomes
in
cotton
was
similar
to
that
in
castor
bean
and
sunflower
(9,
22,
27),
but
differed
from
that
in
dark-grown
cucumber
seedlings
where
activity
was
not
found
in
the
mitochondria
(15).
NADH
hydroxy-
pyruvate
reductase
activity
also
has
been
localized
in
glyoxysomes
of
sunflower,
castor
bean,
watermelon,
and
cucumber
(7).
Specific
activities
varied
substantially
among
the
species
(40-539
nmol
min-'
mg-'
protein).
The
value
for
sunflower
was
the
highest;
our
average
value
for
cotton
was
low,
similar
to
that
for
castor
bean
(9).
The
comparatively
low
specific
activities
of
cotton
enzymes
most
certainly
were
attributable
to
the
omnipresence
of
protein
body
fragments,
rather
than
to
poor
quality
of
the
glyoxysomes.
Isocitrate
lyase,
catalase,
and
the
two
aminotransferases
appear
to
be
matrix
enzymes
in
cotton
glyoxysomes
since
they
were
readily
solubilized
by
osmotic
shock
in
buffer
or
low
salt
(Table
III).
Subfractionation
of
alanine
AT
has
not
been
studied
previ-
ously,
and
aspartate
AT
localization
has
been
examined
only
in
castor
bean
glyoxysomes,
where
78%
was
released
in
buffer
(data
for
salt
treatment
were
not
given)
(9).
Per
cent
solubilization
of
malate
and
citrate
synthase
in
buffer
varied
widely
among
glyox-
ysomes
from
other
sources
(Table
7.14,
Ref.
7).
In
castor
bean,
the
enzymes
were
membrane-bound
(approximately
10%o
released),
but
could
be
dissociated
by
KCI
or
detergent.
In
maize
scutellum,
96%
malate
synthase
was
released
from
glyoxysomes
in
buffer,
86
Plant
Physiol.
Vol.
68,
1981
www.plant.org on February 17, 2015 - Published by www.plantphysiol.orgDownloaded from
Copyright © 1981 American Society of Plant Biologists. All rights reserved.

GLYOXYSOMAL
ENZYMES
IN
COTTON
SEEDS
and
89%
citrate
synthase
was
solubilized
from
pine
megagameto-
phyte
glyoxysomes
(7).
The
two
cotton
enzymes
behaved
similarly,
mimicking
pumpkin
malate
synthase
and
cucumber
citrate
syn-
thase
(22)
(approximately
65%
released
in
buffer
alone).
Malate
DH
was
not
released
quantitatively
from
any
osmotically
shocked
glyoxysomes
(7),
but
was
readily
removed
from
membranes
with
increased
ionic
strength.
Cotton
malate
DH
behaved
somewhat
differently.
A
higher
proportion
was
released
by
simple
shock
(81%
versus
33-75%
for
castor
bean,
cucumber,
maize,
and
pump-
kin),
but
little
more
activity
was
released
with
0.2
M
KCI.
Hydroxy-
pyruvate
reductase
appeared
tightly
bound
to
cotton
glyoxysome
membranes
as
opposed
to
the
matrix
localization
in
castor
bean
(93%
released
in
the
absence
of
KCI)
(9).
Data
for
this
enzyme
in
other
oil
seeds
is
lacking.
Comparative
sub-organelle
localization
of
hydroxyacyl-CoA
DH
and
other
,8
oxidation
enzymes
in
cotton
is
given
in
another
paper
(19).
Acknowledgments-We
thank
A.
Reynolds
and
H.
Le
for
technical
assistance.
Special
thanks
are
due
Dr.
John
S.
Choinski,
Jr.
for
his
many
hours
of
examining
pellets
and
gradient
fractions
with
the
electron
microscope.
The
cooperation
and
support
of
the
Western
Cotton
Research
Laboratory,
Science
and
Education
Admin-
istration,
United
States
Department
of
Agriculture,
Phoenix
is
also
greatly
appreci-
ated.
LITERATURE
CITED
1.
BEEVERS
H
1979
Microbodies
in
higher
plants.
Annu
Rev
Plant
Physiol
30:
159-
193
2.
CHOINSIU
JS
JR,
RN
TRELEASE
1978
Control
of
enzyme
activities
in
cotton
cotyledons
during
maturation
and
germination.
II.
Glyoxysomal
enzyme
de-
veloi5ment
in
embryos.
Plant
Physiol
62:
141-145
3.
COOPER
TG,
H
BEEVERS
1969
Mitochondria
and
glyoxysomes
from
castor
bean
endosperm.
J
Biol
Chem
244:
3507-3513
4.
CuRRY
RA,
IP
TING
1976
Purification,
properties,
and
kinetic
observations
on
the
isoenzymes
of
NADP
isocitrate
dehydrogenase
of
maize.
Arch
Biochem
Biophys
176:
501-509
5.
DILWORTH
MF,
LS
DuRE
III
1978
Developmental
biochemistry
of
cotton
seed
embryogenesis
and
germination.
X.
Nitrogen
flow
from
arginine
to
asparagine
in
germination.
Plant
Physiol
61:
698-702
6.
FREVERT
J,
W
KoLLER,
H
KINDL
1980
Occurrence
and
biosynthesis
of
glyoxy-
somal
enzymes
in
ripening
cucumber
seeds.
Hoppe-Seyler's
Z
Physiol
Chem
361:
1557-1565
7.
GERHARDT
B
1978
Microbodies/Peroxisomen
pflanzlicher
Zellen.
Springer-Ver-
lag,
Wien,
NY
8.
HUANG
AHC
1975
Comparative
studies
of
glyoxysomes
from
various
fatty
seedlings.
Plant
Physiol
55:
870-874
9.
HUANG
AHC,
H
BEEVERS
1973
Localization
of
enzymes
within
microbodies.
J
Cell
Biol
58:
379-389
10.
HUANG
AHC,
RA
MOREAU
1978
Lipases
in
the
storage
tissues
of
peanut
and
other
oil
seeds
during
germination.
Planta
141:
111-116
11.
IHLE
JN,
LS
DuRE
III
1972
The
developmental
biochemistry
of
cottonseed
embryogenesis
and
germination
III.
Regulation
of
the
biosynthesis
of
enzymes
utilized
in
germination.
J
Biol
Chem
247:
5048-5055
12.
KOLLER
W,
J
FREVERT,
H
KINDL
1979
Albumins,
glyoxysomal
enzymes,
and
globulins
in
dry
seeds
of
Cucumis
sativus.
qualitative
and
quantitative
analysis.
Hoppe-Seyler's
Z
Physiol
Chem
360:
167-176
13.
KOLLER
W,
J
FREVERT,
H
KINDL
1979
Incomplete
glyoxysomes
appearing
at
a
late
stage
of
maturation
of
cucumber
seeds.
Z
Naturforsch
34:
1232-1236
14.
LINSKENs
HF,
BD
SANWAL,
MV
TRAcEY
1964
Triose
phosphate
isomerase.
Mod
Methods
Plant
Anal
7:
533-534
15.
Liu
KDF,
AHC
HUANG
1977
Subcellular
localization
and
developmental
changes
of
aspartate-a-ketoglutarate
transaminase
isozymes
in
the
cotyledons
of
cucumber
seedlings.
Plant
Physiol
59:
777-782
16.
LowRY
OH,
NJ
ROSEBROUGH,
AL
FARR,
RJ
RANDALL
1951
Protein
measure-
ment
with
the
Folin
phenol
reagent.
J
Biol
Chem
193:
265-275
17.
MIERNYK
JA,
RN
TRELEASE,
JS
CHOINSKI
JR
1979
Malate
synthase
activity
in
cotton
and
other
ungerminated
oilseeds.
Plant
Physiol
63:
1068-1071
18.
MIERNYK
JA,
RN
TRELEASE
1981
Role
of malate
synthase
in
citric
acid
synthesis
by
maturing
cotton
embryos:
A
proposal.
Plant
Physiol
67:
875-881
19.
MIERNYK
JA,
RN
TRELEASE
1981
Control
of
enzyme
activities
in
cotton
cotyle-
dons
during
maturation
and
germination.
IV.
ft-oxidation.
Plant
Physiol
67:
341-346
20.
PRATHAPASENAN
G
1972
Localization
of
isocitrate
lyase
in
germinating
seeds
of
cotton
(Gossypium
herbaceum
L.).
Biochem
J
128:
54
p
21.
RADIN
JW,
RN
TRELEASE
1976
Control
of
enzyme
activities
in
cotton
cotyledons
during
maturation
and
germination.
I.
Nitrate
reductase
and
isocitrate
lyase.
Plant
Physiol
57:
902-905
22.
SCHNARRENBERGER
C,
A
OESER,
NE
TOLBERT
1971
Development
of
microbodies
in
sunflower
cotyledons
and
castor
bean
endosperm
during
germination.
Plant
Physiol
48:
566-574
23.
SHIROYA
T
1963
Metabolism
of
raffmose
in
cotton
seeds.
Phytochemistry
2:
33-
46
24.
SINGER
TP,
G
OESTREICHER,
P
HOGUE,
J
CONTREIRAS,
I
BRANDAO
1973
Regu-
lation
of
succinate
dehydrogenase
in
higher
plants.
Plant
Physiol
52:
616-621
25.
SMITH
EW,
RC
FITES
1973
The
influence
of
chilling
temperature
alteration
of
glyoxysomal
succinate
levels
on
isocitratase
activity
from
germinating
seedlings.
Biochem
Biophys
Res
Commun
55:
647-654
26.
TRELEASE
RN,
JA
MIERNYK,
JS
CHOINSIu
JR,
SJ
BORTMAN
1980
Enzyme
synthesis
in
developing
cotton
embryos.
In
JR
Mauney,
ed,
Proceedings
of
the
Beltwide
Cotton
Production
Research
Conference,
Cotton
physiology-A
Treatise,
Sect
II,
Boll
Development.
National
Cotton
Council
Publication,
pp
355-366
27.
WIGHTMAN
F,
JC
FOREST
1978
Reviews:
Properties
of
plant
aminotransferases.
Phytochemistry
17:
1455-1471
28.
YATSU
LY,
TF
JACKS
1968
Association
of
lysosomal
activity
with
aleurone
grains
in
plant
seeds.
Arch
Biochem
Biophys
124:
466-471
29.
YOULE
RJ,
AHC
HUANG
1976
Development
and
properties
of
fructose
1,6-
bisphosphatase
in
the
endosperm
of
castor-bean
seedlings.
Biochem
J
154:
647-652
Plant
Physiol.
Vol.
68,
1981
87
www.plant.org on February 17, 2015 - Published by www.plantphysiol.orgDownloaded from
Copyright © 1981 American Society of Plant Biologists. All rights reserved.