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Plant
Physiol.
(1984)
74,
389-394
0032-0889/84/74/0389/06/$01.00/0
Endosperm
Protein
Synthesis
and
L-[35SjMethionine
Incorporation
in
Maize
Kernels
Cultured
In
Vitro'
Received
for
publication
July
18,
1983
DAVID
E.
CULLY,
BURLE
G.
GENGENBACH*,
JANE
A.
SMITH,
IRWIN
RUBENSTEIN,
JAMES
A.
CONNELLY2,
AND
WILLIAM
D.
PARK3
Department
ofAgronomy
and
Plant
Genetics
(D.
E.
C.,
B.
G.
G.,
J.
A.
S.,
J.
A.
C.),
and
Department
of
Genetics
and
Cell
Biology
(I.
R.,
W.
D.
P.),
University
of
Minnesota,
St.
Paul,
Minnesota
55108
ABSTRACT
This
study
was
conducted
to
examine
protein
synthesis
and
L-(5Sj
methionine
incorporation
into
the
endosperm
of
Zea
mays
L.
kernels
developing
in
vitro.
Two-day-old
kernels
of
the
inbred
line
W64A
were
placed
in
culture
on
a
defined
medium
containing
10
microCuries
L-[5S]
methionine
per
milliliter
(13
milliCuries
per
millimole)
and
harvested
at
10,
15,
20, 25,
30,
35,
and
40
days
after
pollination.
Cultured
kernels
attained
a
final
endosperm
mass
of
120
milligrams
compared
to
175
milligrams
for
field-grown
controls.
Field
and
cultured
kernels
had
similar
concentrations
(microgram
per
miligram
endospern)
for
total
protein,
albumin
plus
globulin,
zein,
and
glutelin
fractions
at
most
kernel
ages.
Sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis
and
isoe-
lectric
focusing
patterns
for
endosperm
proteins
were
similar
for
field
and
cultured
kernels throughout
development.
By
15
days,
over
70%
of
the
LqiuSjmethionine
taken
up
was
present
in
endosperm
proteins.
Label
incorporation
visualized
by
fluorography
generally
foUlowed
the
protein
intensity
of
the
stained
gels.
The
high
methionine
content,
low
molecular
weight
zeins
(i.e.
15
and
9
kilodaltons)
were
highly
labeled.
Al
of
the
radioactivity
in
hydrolyzed
zein
samples
was
recovered
in
the
methionine
peak
indicating
minimal
conversion
to
L-jSSlcysteine.
The
procedure
described
here
is
suitable
for
long
term
culture
and
labeling
experiments
in
which
continued
kernel
development
is
required.
Protein
accumulation
is
an
important
process
in
seed
devel-
opment
because
the
resultant
reserves
ultimately
may
be
used
either
by
germinating
seedlings
or
by
humans
and
animals
as
a
source
of
nutrients.
In
corn
the
amount
of
protein
per
kernel
begins
to
increase
rapidly
approximately
10
to
15
d
after
pollination
(15).
Synthesis
of
two
classes
of
storage
proteins,
zeins
and
glutelins,
accounts
for
most
of
the
increase,
although
globulin
content
also
increases
slightly
during
kernel
development.
The
albumin
fraction
mostly
contains
enzymic
proteins
and
increases
rapidly
to
a
maximum
content
around 20
d
after
pollination
but
declines
during
the
later
stages
of
development.
Many
of
the
enzymes
involved
in
carbohydrate
metabolism,
starch
synthesis,
and
nitrogen
metab-
'Supported
by
the
United
States
Department
of
Agriculture
under
Grant
No.
5901-0410-8-0176
and
59-2271-0-1501
from
the
Competitive
Research
Grants
office
and
by
the
Minnesota
Agriculture
Experiment
Station.
Paper
No.
13519
Scientific
Journal
series.
2
Present
address:
Department
of
Biochemistry
and
Biophysics,
Uni-
versity
of
California,
Davis,
CA
95616.
3
Present
address:
Department
of
Biochemistry
and
Biophysics,
Texas
A
&
M
University,
College
Station,
TX
77840.
olism
increase
in
activity
prior
to
or
coincident
with
the
grain
filling
period
(13,
15,
16).
These
and
other
previous
studies
of
protein
deposition
in
developing
kernels
have
utilized
field
or
greenhouse-grown
corn
plants.
Methods
to
maintain
fertilized
corn
kernels
in
vitro
on
artifical
media
were
established
in
our
laboratory
as
a
model
system
to
examine
biochemical
and
physiological
aspects
of
kernel
growth
and
development
(1).
Previously,
we
have
shown
in
vitro
kernel
development
is
dependent
upon
temperature
and
genotype
(2,
4).
Recently,
Shimamoto
and
Nelson
(12),
by
supplying
radioac-
tive
leucine,
phenylalanine,
and
proline
in
the
kernel
culture
medium,
showed
that
most
of
the
radioactivity
was
retained
in
the
cob
tissues
while
about
4
to
9%
was
translocated
into
the
endosperm.
Their
analyses
also
indicated
that
20
to
30%
of
the
radioactivity
in
water
or
TCA
extracts
of
the
dried
endosperm
was
recovered
as
the
original
amino
acid.
They
did
not
determine
the
distribution
of
the
radioactive
amino
acids
into
endosperm
protein
fractions.
To
most
validly
and
effectively
utilize
in
vitro
kernel
develop-
ment
as
a
model
system
for
biochemical
studies,
endosperm
protein
synthesis
and
accumulation
by
kernels
maintained
in
vitro
needs
to
be
compared
with
that
of
field-grown
kernels.
Incorporation
of
radioactive
methionine
into
endosperm
pro-
teins
of
cultured
kernels
also
was
measured
to
facilitate
these
comparisons.
We
report
here
that
endosperm
development
and
protein
synthesis
in
cultured
corn
kernels
can
be
equivalent
to
that
of
field-grown
kernels.
MATERIALS
AND
METHODS
Plant
Material.
Plants
of
the
maize
(Zea
mays
L.)
inbred
line,
W64A,
were
grown
in
the
field
at
St.
Paul
in
1980.
Ear
shoots
were
bagged
before
silking
and
the
date
of
silk
emergence
was
recorded.
Self-
or
sib-pollinations
were
made
4
d
after
silking
and
ears
were
removed
for
culture
2
d
after
pollination.
Culture
Procedure.
Ears
were
dissected
aseptically
as
described
previously
(1)
except
that
the
silks
were
removed.
The
basal
two-
thirds
of
the
ear
was
sectioned
into
blocks
of
six
kernels
(two
rows
of
three)
and
then
five
kernels
were
cut
away
from
the
subtending
cob.
Five
or
six
cob
pieces
were
seated
into
culture
media
in
25
x
100
mm
Petri
dishes
such
that
the
media
contacted
only
the
cob
tissue.
Kernels
were
incubated
in
the
dark
at
29±
1°C.
The
medium
contained
the
following
in
,umol/l:
KH2PO4,
3750;
MgCl2.6H20,
1500;
CaCl2.2H20,
1200;
H3BO3,
100;
MnSO4.H20,
100;
ZnSO4.7H20,
10;
KI,
5;
Na2MoO4.2H20,
1;
CuSO4.5H20,
0.1;
CoCl266H20,
0.1;
and
Fe-EDTA,
50.
The
medium
also
contained
0.4
mg/l
thiamine-HCl,
0.044
mg/l
folic
acid,
1.2
mg/l
niacin,
150
g/l
sucrose,
and
the
following
L-amino
389
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
Plant
Physiol.
Vol.
74,
1984
acids
in
mg/l:
Asp,
500;
Thr,
198;
Ser,
347;
Glu,
1761;
Pro,
621;
Gly,
168;
Ala,
476;
Cys,
160;
Val,
330;
Met,
117;
Ile,
237;
Leu,
1009;
Tyr,
262;
Phe,
387;
Lys,
88;
His,
164;
Arg,
150;
Asn,
300;
Gln,
292;
and
Trp,
204.
The
pH
was
adjusted
to
5.8,
5.5
g/l
agar
was
added,
and
the
media
was
autoclaved
(15
p.s.i.,
120C)
for
20
min.
After
cooling,
50-ml
aliquots
were
dispensed
into
Petri
dishes.
L-[35S]Methionine
(New
England
Nuclear)
was
filter
ster-
ilized
and
added
to
autoclaved
media
to
give
10
MCi/ml
and
a
specific
activity
of
13
mCi/mmol
at
the
start
of
the
culture
period.
Endosperm
Samples.
Beginning
at
10
d
after
pollination
and
at
subsequent
5-d
intervals
until
40
d
after
pollination,
kernels
were
removed
from
the
cob
and
the
embryos
excised.
The
endosperms
plus
pericarps
(referred
to
simply
as
endosperm)
were
frozen
on
dry
ice
and
stored
at
-80°C
until
processed.
Intact
frozen
endosperms
were
lyophilized,
weighed
individually,
and
ground
on
a
Torit
reciprocating
shaker
for
1
min.
Dry
meal
was
stored
at
-80°C
until
extracted.
Extraction
of
SDS-Soluble
Proteins.
Ten
mg
of
dry
meal
were
weighed
into
a
plastic,
1.4-ml
centrifuge
tube
and
defatted
twice
with
1.0
ml
acetone
for
30
min
at
4°C
on
a
tube
rotator.
After
centrifuging
(4
min,
15,000g),
the
supernatant
was
discarded
and
the
meal
was
then
extracted
with
0.1
ml
Laemmli
sample
buffer
(5)
for
30
min
at
room
temperature.
After
centrifuging
for
4
min,
the
supernatant
was
transferred
to
a
clean
tube,
the
meal
was
subjected
to
a
second
extraction,
and
the
supernatants
were
combined.
Extraction
of
Protein
Fractions.
Fifty
to
70
mg
meal
was
defatted
twice
with
2.5
ml
acetone
(with
agitation
30
min,
4°C)
and
then
air
dried.
Protein
fractions
were
then
extracted
from
the
defatted
meal
according
to
the
basic
procedure
of
Osborne
and
Mendel
(10)
modified
as
follows.
Each
extraction
step
was
performed
twice
with
2.5
ml
of
solvent
by
mixing
vigorously
and
placing
tubes
on
a
rotator
for
30
min.
The
albumin
plus
globulin
fraction
was
extracted
with
0.5
M
NaCl
at
4°C.
After
NaCl
extraction,
the
pelleted
meal
was
washed
with
water
at
4°C
and
the
supernatant
discarded.
Zeins,
including
the
zein
2
fraction
(14),
were
then
extracted
with
70%
ethanol
and
1%
2-mercap-
toethanol
at
room
temperature.
The
glutelin
fraction
remaining
in
the
meal
was
then
extracted
with
0.05
N
NaOH
at
room
temperature.
All
solutions
contained
1
mM
PMSF4
(Sigma)
to
inhibit
protease
activity,
centrifugations
were
at
1200g
for
5
min,
and
samples
were
tightly
capped
and
stored
at
-20
or
-80°C.
Protein
Quantification.
Fractions
containing
25
to
100
yg
protein
were
assayed
according
to
the
method
of
Lowry
et
al.
(8)
using
BSA
as
the
standard.
Glutelin
samples
were
assayed
di-
rectly.
Albumin
plus
globulin
samples
were
concentrated
by
TCA
precipitation,
resuspended
in
one-tenth
volume
1
N
NaOH,
and
subsequently
diluted
to
0.1
N
NaOH
before
assay.
Zein
samples
were
lyophilized
and
taken
up
in
70%
ethanol
before
assay.
Based
on
the
distribution
of
radioactivity,
at
least
98%
of
the
extracted
zein
was
recovered
when
samples
were
processed
in
the
above
manner.
SDS-PAGE.
Samples
from
SDS
extractions
were
heated
to
100°C
for
2
min,
centrifuged,
and
applied
to
sample
wells
of
1.5-
mm
thick
slab
gels
consisting
of
a
1-cm,
3%
acrylamide
stacking
gel
and
a
10-cm,
12.5%
acrylamide
separating
gel
according
to
Laemmli
(5).
Electrophoresis
was
at
35
mamps
constant
current
for
4
h.
Zein
samples
were
lyophilized,
resuspended
in
Laemmli
sam-
ple
buffer
to
a
concentration
of
1
Mg/,ul,
and
electrophoresed
on
12.5%
acrylamide
gels.
Albumin
plus
globulin
and
glutelin
sam-
ples
were
treated
similarly
following
TCA
precipitation,
resus-
pension
in
one-tenth
volume
1
N
NaOH,
and
subsequent
dilution
'Abbreviations:
PMSF,
phenylmethylsulfonyl
chloride;
TEMED,
N,
N,N',N'-tetramethylethylenediamine;
IEF,
isoelectric
focusing.
to
0.1
N
NaOH
with
Laemmli
sample
buffer.
Concentrations
were
adjusted
so
that
about
25
Mg
of
protein
from
each
sample
was
applied
to
the
gel.
Gels
were
stained
with
Coomassie
Blue
for
at
least
2
h
and
destained
in
25%
ethanol
and
7%
acetic
acid
before
photography
or
fluorography.
Isoelectric
Focusing.
Zein
samples
from
the
original
extraction
containing
about
100
Mg
protein
were
lyophilized
and
resus-
pended
in
25
,l
sample
buffer
consisting
of
6
M
urea,
10
mM
Tris/glycine
(pH
8.5),
and
2%
2-mercaptoethanol.
The
resulting
solution
was
applied
to
0.2
x
10
x
42
cm
IEF
gels
consisting
of
4%
acrylamide,
0.1%
bis-acrylamide,
6
M
urea,
and
2%
pH
6-8
ampholytes
(Bio-Rad)
and
focused
as
described
by
Righetti
et
aL
(11)
for
2
h
at
12
w
(constant)
and
then
for
30
min
at
15
w
(constant).
Fluorography.
Stained
gels
containing
35S-labeled
samples
were
treated
with
EnHance
(New
England
Nuclear),
dried
onto
What-
man
3MM
paper,
and
fluorographed
(6)
for
7
to
14
d
using
preflashed
Kodak
XAR-5
film.
Scintillation
Counting.
Samples
(10-50
I)
were
counted
in
10
ml
Aquasol
scintillation
fluid
(New
England
Nuclear)
in
a
Beck-
man
LS
8000
counter.
Counts
were
corrected
for
35S
half-life
and
counting
efficiency.
Identification
of
"S-Labeled
Amino
Acids.
Protein
fractions
from
kernels
cultured
in
the
presence
of
L-[35S]methionine
were
oxidized
with
performic
acid
and
hydrolyzed
in
sealed
evacuated
ampoules
with
6
N
HCI
at
10
OC
for
24
h
(9).
They
were
then
lyophilized,
taken
up
in
0.2
N
Na-citrate
(pH
2.2),
centrifuged,
and
20
to
80
Al
of
the
supernatant
applied
to
a
Dionex
amino
acid
analyzer.
Fractions
(0.5
ml)
were
collected,
neutralized
with
50
Mi
10
N
HCI,
and
mixed
with
10
ml
Aquasol
scintillation
fluid
for
counting;
dpm
were
assigned
to
specific
amino
acid
peaks
based
on
the
retention
times.
RESULTS
Kernel
Development.
Kernels
grown
in
culture
had
a
faster
rate
of
endosperm
dry
matter
accumulation
than
field-grown
kernels
for
the
first
20
d
(Fig.
1).
Dry
matter
accumulation,
however,
persisted
over
a
longer
period
in
field-grown
kernels.
Endosperm
dry
matter
of
the
35
and
40
d
cultured
kernels
was
more
variable
than
at
other
sampling
dates
(Fig.
1;
Table
I).
We
attempted
to
maximize
kernel
dry
weight
and
minimize
the
160
-
C,)
0
01)
E
1201-
80
-
40
-
10 15
20
25 30
days
after
pollination
35
40
FIG.
1.
Endosperm
growth
for
field
(O-O)
and
cultured
(@--*)
W64A
corn
kernels.
Each
point
is
the
mean
of
42
isolated
endosperms
for
field
kernels
and
17
to
36
endosperms
for
cultured
kernels.
a
a
I
a
I
a
390
CULLEY
ET
AL.
I
.
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
IN
VITRO
CULTURE
LABELING
OF
CORN
ENDOSPERM
PROTEINS
Table
I.
Endosperm
Protein
Concentration
and
Distribution
in
/"SJMethionine-Labeled
Cultured
Kernels
and
in
Field-Grown
Kernels
of
W64A
Corn
Endosperm
Mass
Albumin
+
Zein
Glutelin
Total
Protein
Age
Globulin
Cult
Field
Cult
Field
Cult
Field
Cult
Field
Cult
Field
d
mg
ug/mg
meal
10
18
±
I"
10
±
1
25
44
6
2
23
24
54
70
15
47±1
24±2.
20±1
30±1
34±3 25±3
33±1
46±4
88±6
102±6
20
83±2
58±3
11
±
1
18±
1
56±3
49±2
40±3
42±
1
107±5
109±4
25
118±2
92±3
8±
1
14±3
69±4
55±4
48±4
48±2
125±4
117±4
30
152±8
116±5
10±2
13±
1
62±4
60±2
39±3
37±2
112±3
112±
1
35
139±23
143±3
13±2
11
±
1
56±4
55±2
40±4
35±
1
108±
10
102±3
40
129±
19
176±2
6±
1
5±
1
51
±8
71
±4
36±3
32±4
93±8
108±7
a
Mean
±
SE
for
three
single
kernel
samples
from
each
treatment,
except
10
d
where
six
kernels
were
bulked
for
protein
extraction.
Table
II.
L-[35S]Methionine
Uptake
and
Distribution
of
Radioactivity
in
Endosperm
Protein
Fractions
of
Cultured
W64A
Kernels
Albumin
Fraction
SDS
Age
TCA
Soluble
+
Zein
Glutelin
Total'
Extractionb
Globulin
d
1O-'
X
dpm/mg
mealc
10
243
58
23
77
401
572
15
115±27
66±4
91
±2
112±
1
384±28
327±78
20
72±4
42±9
122±8
152±
17
388±
15
451
±33
25
65±9
48±4
187±
14
250±8
550±
11
506±20
30
61
±
11
78±22
201
±20
190±33
530±31
436±31
35
56±3 52±7
155±8
179±21
440±
18
339±8
40
35±5
31±12
155±15
172±10
393±26
337±42
a
Solubilized
residue
meal
following
this
extraction
sequence
contained
less
than
8%
of
the
radioactivity.
b
Meal
extracted
with
Laemmli
buffer,
residue
after
SDS
extraction
contained
less
than
15%
of
the
radioactivity.
c
Mean
±
SE
for
three
single
kernel
samples, except
10
d
where
six
kernels
were
bulked.
FIELD
CULTURE
METHIONINE
I
kD
10
15
20
25
30
35
40
10
15
20
25
30
35
40
94-
68-
40-
f
*-
*-
30-^
s
1
21-
14-
10
15
20
25
30
3
5
40
A*
A_
4_
............
E=
_|
.-
a.
.......
_
Jo_
_g,,
:''ffl'J4.
.:/
_E _ i,, a. . e
e1* 16 6 *
*w
w
iR.V
WF
t
A......
....
a!:
OW"Z
_m
FIG.
2.
Total
endosperm
proteins
from
field
(A)
and
cultured
(B)
kernels
sampled
at
5-d
intervals
between
10
and
40
d.
Proteins
were
obtained
by
SDS
buffer
extraction
of
dried
endosperm
meal.
Samples
were
electrophoresed
on
SDS
12.5%
acrylamide
gels;
each
lane
represents
the
protein
in
0.6
mg
meal.
The
patterns
of
methionine
incorporation
(C)
were
obtained
by
fluorography
of
gel
B
for
8
d.
391
I
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Plant
Physiol.
Vol.
74,
1984
(:)TTA
L
e
ALBUMIN
,.
I
N
!
f
Gi
H
..
I?
':
.Pigo
AA
.
r
i4B:
..................
r
;jt|||4J2B§etBoi
a:
_i
a
.: ,.' : " - Is ^
... >Si
Baza...
...
'=
and.
:
|
-
t.w
1
.!.......
....
,.,
| @ X. Z t'S,,j. _
@&,';-*
X
L:
*_
_
-
-or I.. ..... .par
FIG.
3.
Total
endosperm
protein,
albumin
plus
globulin,
zein,
and
glutelin
fractions
from
30-d
field
and
cultured
kernels.
Samples
were
electrophoresed
on
SDS
12.5%
acrylamide
gels
(A-L)
or
separated
by
IEF
(M-O).
For
each
set
of
three
lanes,
the
field
sample
is
on
the
left,
the
cultured
sample
in
the
middle,
and
the
fluorograph
on
the
right.
Total
SDS-extractable
proteins
(A-C),
albumin
plus
globulin
(D-F),
zein
(G-I
and
M-O),
and
glutelin
(J-L).
variation
by
culturing
one
kernel
on
a
cob
segment
that
normally
would
have
six
kernels.
The
data
in
Figure
1
indicate
that
final
endosperm
average
dry
weight
in
culture
is
about
70%
of
that
in
the
field.
The
heaviest
cultured
kernels
were
selected
for
further
chemical
analyses
and
comparisons
with
field-grown
kernels.
Protein
Quantity.
Endosperms
from
L-[35S]methionine
labeled
kernels
were
selected
for
protein
extraction
and
analysis
for
each
sampling
date.
Total
endosperm
protein
concentrations
(mg
protein/mg
meal)
and
distribution
into
solubility
classes
were
similar
for
cultured
and
field
kernels
(Table
I).
Total
protein
concentration
peaked
around
day
25
and
declined
slightly
during
maturation
for
kernels in
both
treatments.
The
concentration
of
albumin
plus
globulin
declined
throughout
development
al-
though
field
kernels
were
initially
at
a
higher
level.
Cultured
kernels
accumulated
zein
more
rapidly
during
early
development
than
field
kernels,
but
at
40
d
field
kernels
had
higher
zein
concentrations.
Little
difference
in
glutelin
concentration
was
found
throughout
development.
The
distributions
of
protein
into
the
various
fractions
(calculated
as
per
cent
of
total
protein,
data
not
shown)
were
similar
for
cultured
and
field
kernels
for
most
of
the
sampling
dates.
During
the
later
part
of
development,
zein
and
glutelin
accounted
for
52
to
55%
and
33
to
39%
of
the
protein,
respectively,
in
both
treatments.
L-I3SIMethionine
Uptake.
Extraction
with
Laemmli
sample
buffer
(SDS
extract)
solubilized
over
85%
of
the
radioactivity
in
endosperm
meal.
Sequential
extraction
of
the
three
protein
frac-
tions
was
on
the
average
slightly
more
efficient
than
SDS
extrac-
tion
leaving
less
than
8%
of
the
radioactivity
in
the
residue.
The
initial
acetone
wash
accounted
for
about
1%
of
the
radioactivity
in
the
meal.
An
added
third
extraction
with
each
solvent
in
the
extraction
procedure
yielded
only
3%,
2%,
and
4%
more
dpm
for
the
albumin
plus
globulin,
zein,
and
glutelin
fractions,
re-
spectively.
Approximately
60%
of
the
radioactivity
at
day
10
was
found
in
the
TCA-soluble
portion
of
the
0.5
M
NaCl
extract
(Table
II).
By
day
20,
the
relative
and
total
amounts
of
radioac-
tivity
were
increased
in
both
the
zein
and
glutelin
fractions.
Calculations
of
specific
activity
(dpm/gg
protein)
from
data
in
Tables
I
and
II
indicated
that
the
zein
fraction
incorporated
about
the
same
amount
of
radioactivity
per
Ag
protein
through-
out
development.
The
glutelin
fraction
showed
an
increase
in
specific
activity
by
day
25
suggesting
that
proteins
with
different
methionine
content
were
synthesized
during
endosperm
devel-
opment.
Conversion
of
L-I35SIMethionine
to
L-I35SCysteine.
The
zein
8.-
,)8
-
4(e.)-
.4
392
CULLEY
ET
AL.
LO
B
LiN
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IN
VITRO
CULTURE
LABELING
OF
CORN
ENDOSPERM
PROTEINS
..I
a
i..
I
CULTURE
D
E
F
,--f
m
..:X....X.
A:::*.
*qe
B:';t.
:fiX.
i,
FIG.
4.
SDS
electrophoretic
comparison
of
extraction
methods
for
endosperm
glutelins.
Protein
remaining
in
the
meal
after
zein
extraction
was
solubilized
by
SDS
buffer
(A
and
D)
or
by
0.05
N
NaOH
(B
and
E).
Samples
were
adjusted
to
contain
about
25
mg
protein
per
lane.
Lanes
C
and
F
show
the
SDS
solubilized
protein
remaining
in
meal
after
the
0.05
N
NaOH
extractions
of
B
and
E,
respectively.
C
and
F
were
loaded
with
4-fold
more
extract
than
A,
B,
D,
and
E.
fractions
of
three
35
d
kernels
were
hydrolyzed
under
conditions
which
converted
cysteine
to
cysteic
acid
and
methionine
to
methionine
sulfone.
All
of
the
recovered
radioactivity
(82%
recovery)
was
in
the
methionine
sulfone
peak
fractions
(data
not
shown).
No
other
amino
acid
peak
fraction
contained
radioactiv-
ity
exceeding
the
background.
These
results
indicated
that
the
amino
acid
pool
utilized
for
zein
synthesis
did
not
contain
significant
amounts
of
L-[35S]cysteine.
Electrophoretic
Comparisons
of
Endosperm
Proteins.
To
de-
termine
the
variability
for
polypeptide
profiles
among
single
kernels
from
the
same
treatment,
we
extracted
total
SDS-soluble
protein
from
three
field-grown
and
three
cultured
35
d
kernels.
Two
samples
of
each
extract
were
run
on
SDS-polyacrylamide
gels
(not
shown).
There
was
no
discernible
variation
between
the
two
samples
from
each
kernel.
Polypeptide
profiles
among
the
field
kernels
also
appeared
identical,
but
minor
variation
in
the
concentration
of
several
polypeptides
was
observed
among
dif-
ferent
cultured
kernels.
These
differences
in
polypeptide
concen-
trations
on
stained
gels
coincided
with
the
variation
in
intensity
of
bands
on
fluorographs
(not
shown).
Based
on
the
high
recovery
of
radioactivity
from
labeled
meal
extracted
with
SDS
buffer,
the
polypeptides
obtained
should
represent
the
majority
of
the
en-
dosperm
proteins.
Patterns
for
SDS-extracted
proteins
from
10
to
40
d
field
and
cultured
kernels
are
shown
in
Figure
2,
A
and
B.
The
two
major
mol
wt
species
of
zein
(located
between
the
21
and
30
kD
markers)
were
present
by
15
d
in
cultured
kernels
and
by
20
d
they
were
the
predominant
polypeptides
in
kernels
from
both
treatments.
Some
of
the
high
mol
wt
polypeptides
were
not
evident
in
the
10-d
kernels
but
were
in
15-d
kernels.
Other
than
earlier
initiation
of
zein
synthesis
in
cultured
kernels,
the
profiles
of
SDS-extracted
polypeptides
were
similar
for
cultured
and
field
kernels
at
the
same
age.
The
fluorograph
(Fig.
2C)
of
the
gel
shown
in
Figure
2B
shows
the
distribution
of
L-[35S]methionine
in
endosperm
proteins
dur-
ing
kernel
development.
Proteins
isolated
from
10
d
endosperms
contained
little
radioactivity
in
comparison
with
the
older
ker-
nels.
By
day
15,
L-[35S]methionine
was
incorporated
into
poly-
peptides
ranging
in
size
from
90
kD
to
less
than
10
kD.
Two
methionine-rich
polypeptides
of
about
16
to
19
kD
and
one
of
about
9
kD
can
be
identified
by
comparing
the
intensity
of
bands
on
the
fluorograph
(Fig.
2C)
with
the
corresponding
bands
on
the
gel
stained
for
protein
(Fig.
2B).
Electrophoretic
profiles
of
albumin
plus
globulin,
zein,
and
glutelin
protein
fractions
from
30
d
field
and
cultured
kernels
are
shown
in
Figure
3.
The
albumin
plus
globulin
fraction
comprised
about
10%
of
the
total
protein
and
two
prominent
bands
corresponding
to
mol
wt
of
27
and
11
kD
were
observed
(Fig.
3,
D
and
E).
The
27
kD
band
increased
in
intensity
between
15
and
20
d
and
then
decreased
slightly
through
40
d
(gels
not
shown).
The
11
kD
band
was
present
in
significant
amounts
at
10
and
15
d,
but
otherwise
showed
a
developmental
pattern
similar
to
the
27
kD
band.
Label
incorporation
(Fig.
3F)
closely
paralleled
the
apparent
protein
concentration;
there
were
no
polypeptides
with
an
unusually
high
or
low
methionine
content
in
the
albumin
plus
globulin
fraction.
Zein
profiles
on
SDS
gels
(Fig.
3,
G
and
H)
appeared
identical
for
field
and
cultured
kernels.
Previous
amino
acid
analyses
(7)
have
shown
that
the
24
kD
zein
class
had
a
higher
methionine
concentration
than
the
21
kD
zein
class.
The
labeling
intensity
(Fig.
31)
is
consistent
with
this
observation.
The
three
smallest
zein
polypeptides
also
have
a
much
higher
methionine
concen-
tration
than
the
two
larger
classes
as
evidenced
by
the
label
incorporation.
The
zein
IEF
patterns
(Fig.
3,
M
and
N)
were
similar,
although
several
minor
bands
were
more
distinct
in
the
culture
sample
(Fig.
3N)
than
in
the
field
sample
(Fig.
3M).
This
observation
was
not
consistent
across
other
age
kernels
(gels
not
shown).
Most
zein
species
separated
by
IEF
had
about
the
same
methio-
nine
incorporation,
but
one
species
nearest
the
acidic
end
had
a
very
high
concentration
and
several
species
nearer
the
alkaline
end
appeared
to
have
relatively
low
methionine
concentration
(Fig.
30).
The
fluorographic
patterns
were
consistent
for
cultured
kernels
throughout
development
(gels
not
shown).
Alkaline
extraction
of
the
glutelin
fraction
was
used
in
this
study
so
that
protein
concentration
could
be
readily
measured.
SDS
electrophoresis
after
2
to
4
months
storage
of
this
fraction
resulted
in
a
pattern
with
few
distinct
polypeptides.
The
field
and
culture
patterns,
however,
were
similar
(Fig.
3,
J
and
K).
Label
incorporation
(Fig.
3L)
was
generally
consistent
with
the
poly-
peptide
pattern.
Glutelins
extracted
with
0.5
N
NaOH
may
be
subject
to
proteolysis
and
amino
acid
degradation
or
deamination,
espe-
cially
after
prolonged
storage.
After
the
initial
study
of
field
and
cultured
kernels
was
completed,
we
tested
the
SDS
buffer
extrac-
tion
(see
"Materials
and
Methods")
on
endosperm
meal
after
FIELD
A
B
C
94
-
68-
40-
30-~
21-
14-I
393
w
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Plant
Physiol.
Vol.
74,
1984
both
the
standard
zein
and
glutelin
extraction
steps.
When
protein
remaining
in
the
meal
after
the
standard
zein
extraction
was
solubilized
with
SDS
buffer,
a
gel
pattern
was
obtained
with
many
distinct
species
ranging
in
mol
wt
from
less
than
10
kD
to
greater
than
90
kD
(Fig.
4,
A
and
D).
Similar
patterns
were
obtained
from
glutelins
extracted
by
the
standard
method
(Fig.
4,
B
and
E).
The
gel
in
Figure
4
was
run
1
d
after
the
extracts
were
made
and
provided
a
much
more
distinct
pattern
than
for
glutelins
electrophoresed
after
several
months
of
-80°C
storage
in
0.05
N
NaOH
(Fig.
3,
J
and
K).
Little
SDS-soluble
protein
(Fig.
4,
C
and
F)
remained
in
the
meal
following
the
standard
glutelin
extraction.
Results
for
field
and
cultured
kernels
were
nearly
identical.
Thus,
SDS
treatment
of
meal
following
zein
extraction
yields
a
protein
fraction
which
is
similar
to
that
of
freshly
extracted
glutelin
and
which
should
not
contain
a
signifi-
cant
amount
of
residual
proteins
normally
not
included
in
the
glutelin
fraction.
Similar
gel
patterns
were
obtained
when
SDS
glutelin
extracts
were
run
after
2
months
storage
at
-80C
(not
shown).
SDS
extraction
is
easier
to
use
for
electrophoresis
pur-
poses
and
provides
better
storage
conditions
than
NaOH
extrac-
tion.
DISCUSSION
In
vitro
culture
of
corn
kernels
is
an
appropriate
procedure
for
studying
maize
endosperm
proteins
throughout
kernel
develop-
ment.
Kernels
of
the
inbred
line
W64A
grew
well
in
the
described
culture
conditions
and
the
final
endosperm
mass
obtained
70%
of
that
of
comparable
field-grown
kernels.
Similar
developmental
patterns
were
noted
previously
by
Jones
et
al.
(4)
in
an
assessment
of
temperature
effects
on
kernel
development
and
carbohydrate
synthesis.
Endosperm
protein
concentration
(,ug/mg
meal)
of
cultured
W64A
kernels
was
comparable
to
that
of
field
kernels
throughout
the
developmental
period.
The
quantitative
distri-
bution
of
endosperm
proteins
among
solubility
classes
(Table
I)
and
the
composition
within
classes
(Figs.
2,
3,
and
4)
also
were
similar
for
field
and
cultured
kernels.
During
the
10-
to
30-d
period,
cultured
endosperms
synthesized
substantially
more
zein
and
glutelin
per
endosperm
than
field
kernels
(from
data
in
Table
I,
calculations
not
shown).
Storage
protein
synthesis
apparently
kept
pace
with
the
accumulation
of
storage
carbohydrates
in
cultured
kernels.
Jones
et
al.
(4)
found
that
starch
concentration
in
cultured
kernels
reached
the
maximum
about
10
d
earlier
(30
d)
than
field
kernels
and
attributed
the
difference
to
cooler
field
temperatures.
Most
of
the
L-[35S]methionine
taken
up
by
10
d
kernels
(after
8
d
on
labeled
culture
medium)
was
present
in
the
TCA-soluble
fraction
(Table
II).
By
15
d,
a
substantial
amount
of
radioactivity
had
been
incorporated
into
zeins
and
glutelins.
Methionine
uptake
and
incorporation
per
mg
meal
was
about
25%
higher
at
25
and
30
d
than
at
10
to
20
d.
This
indicates
faster
endosperm
dry
matter
accumulation
at
later
stages
of
development.
We
found
no
indication
that
L-[35S]methionine
had
been
metabo-
lized
to
L-[35S]cysteine
in
mature
cultured
kernels.
Assuming
an
initial
specific
activity
of
13
mCi/mmol
and
no
conversion,
then
the
amount
of
radioactivity
in
the
zein
fraction
was
equal
to
80
to
1
5
nmol
methionine/mg
zein.
These
values
are
in
the
range
reported
for
the
methionine
content
of
zein
extracted
from
protein
bodies
(120
nmol/mg
zein)
(7),
for
total
zein
(46
nmol/
mg
zein)
(3),
and
for
the
zein
1
fraction
(24
and
95
nmol/mg)
solubilized
by
alcohol
(14).
The
methionine
concentrations
re-
ported
(
14)
for
the
zein
2
fraction
(i.e.
zeins
extractable
by
alcohol
plus
2-mercaptoethanol)
were
much
higher
(307
and
440
nmol
methionine/mg
zein
2).
Zein
2
is
about
33%
of
the
total
zein
(14)
and
is
enriched
for
lower
mol
wt
polypeptides.
Radioactively
labeled
endosperm
tissue
was
also
beneficial
in
devising
and
monitoring
the
procedures
we
used
for
protein
extraction
from
a
small
amount
of
endosperm
(50-100
mg)
obtained
from
individual
kernels.
The
number
of
extractions
required
to
efficiently
extract
each
solubility
class
was
more
easily
monitored
by
recovery
of
radioactivity
than
by
protein
analyses
according
to
Lowry
et
al.
(8).
Recovery
of
individual
protein
fractions
after
precipitation
or
freeze
drying
also
was
determined
by
dpm
recovery.
Shimamoto
and
Nelson
(12)
labeled
kernels
for
7
d
(between
12
and
19
d
post-pollination)
with
L-[U-'4C]leucine,
L-[U-'4C]
phenylalanine,
and
L-[U-`4C]proline.
The
radioactivity
in
their
medium
(,uCi/ml)
was
only
1
to
4%
of
that
used
in
this
study,
and
the
uptake
into
endosperm
tissue
(cpm/mg)
also
was
sub-
stantially
lower.
Their
results
indicated
that
cob
tissue
had
about
10-
to
20-fold
more
radioactivity
(cpm/mg)
than
endosperm
tissue
and
that
radioactivity
in
the
cob
and
endosperm
was
distributed
among
amino
acids
in
addition
to
the
original
one.
Although
we
did
not
analyze
the
cob
tissue,
we
found
that
the
radioactivity
in
endosperm
proteins
was
recovered
as
L-[35]S
methionine.
In
this
study
and
that
of
Shimamoto
and
Nelson
(12),
kernels
were
labeled
continuously
for
a
relatively
long
time;
the
earliest
sampling
was
after
7
or
8
d
of
culture.
In
preliminary
experi-
ments,
we
found
little
radioactivity
in
endosperm
of
kernels
labeled
for
less
than
2
d
(unpublished
results).
We
also
have
found
that
long
term
normal
kernel
development
is
dependent
on
the
presence
of
the
cob
tissue;
2
to
5
d
kernels
removed
from
the
cob
generally
did
not
undergo
endosperm
development
when
cultured.
Pulse
or
pulse-chase
labeling
experiments
likely
could
be
conducted
with
kernels
removed
from
the
cob
tissue
at
various
times
during
development
and
then
analyzed
after
a
short
time.
Our
data
indicate
that
the
procedure
described
is
suitable
for
experiments
in
which
long
term
normal
kernel
development
is
necessary
or
beneficial.
Acknowledgment-We
thank
C.
M.
Donovan
for
assistance
with
the
amino
acid
analyses.
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