Differential Protein Accumulation in Banana Fruit during Ripening

Abstract

Banana (Musa acuminata, cv Dwarf Cavendish) proteins were extracted from pulp tissue at different stages of ripening and analyzed by two-dimensional electrophoresis. The results provide evidence of differential protein accumulation during ripening. Two sets of polypeptides have been detected that increase substantially in ripe fruit. These polypeptides were characterized as glycoproteins by western blotting and concanavalin A binding assays. Antibodies againts tomato polygalacturonase cross-react with one of these sets of proteins.

Full text

Plant
Physiol.
(1992)
98,
157-162
0032-0889/92/98/01
57/06/$01
.00/0
Received
for
publication
November
27,
1990
Accepted
August
5,
1991
Differential
Protein
Accumulation
in
Banana
Fruit
during
Ripening1
Eva
Dominguez-Puigjaner,
Miguel
Vendrell*,
and
M.
Dolors
Ludevid
Departamento
de
Biologia
Molecular
y
Agrobiologia
(E.D.
-P.,
M.
V.),
and
Departamento
de
Genetica
Molecular
(M.D.L.),
Centro
de
Investigaci6n
y
Desarrollo,
Consejo
Superior
de
Investigaciones
Cientificas,
Jordi
Girona
Salgado
18-26,
08034
Barcelona,
Spain
ABSTRACT
Banana
(Musa
acuminata,
cv
Dwarf
Cavendish)
proteins
were
extracted
from
pulp
tissue
at
different
stages
of
ripening
and
analyzed
by
two-dimensional
electrophoresis.
The
results
pro-
vide
evidence
of
differential
protein
accumulation
during
ripening.
Two
sets
of
polypeptides
have
been
detected
that
increase
substantially
in
ripe
fruit.
These
polypeptides
were
characterized
as
glycoproteins
by
westem
blotting
and
concanavalin
A
binding
assays.
Antibodies
againts
tomato
polygalacturonase
cross-react
with
one
of
these
sets
of
proteins.
enzyme
activity
observed
during
ripening
corresponds
to
de
novo
protein
synthesis
(12).
The
present
article
reports
the
isolation
of
total
proteins
from
banana
fruits.
Accumulation
of
specific
proteins
during
banana
ripening
has
been
observed.
One
of
this
proteins
immunoreacts
with
an
antiserum
against
tomato
polygalacturonase.
The
presence
of
this
enzyme
in
banana
ripe
fruit
is
discussed.
MATERIALS
AND
METHODS
Plant
Material
Ripening
involves
a
series
of
changes
during
the
early
stages
of
senescence.
After
a
phase
of
active
cell
division
and
further
cell
expansion,
fruit
growth
rate
declines
and
ripening
usually
begins.
The
physiological,
ultrastructural,
and
biochemical
changes
that
occur
at
this
time
are
highly
coordinated.
There
is
increasing
evidence
that
the
expression
of
specific
genes
is
required
for
normal
ripening
(1).
An
important
turnover
of
preexisting
proteins
and
the
de
novo
synthesis
of
RNAs
(6,
9,
10,
15)
and
proteins
(5,
11)
seems
to
be
essential
for
ripening
(2,
3).
One
of
these
proteins
is
polygalacturonase
(22).
It
has
been
widely
studied
as
one
of
the
specific
proteins
that
accu-
mulate
during
ripening.
The
de
novo
synthesis
of
polygalac-
turonase
occurs
after
ethylene
production
in
climacteric
fruits
(1
1).
There
are
three
isoenzymes
of
polygalacturonase
isolated
from
tomato
pericarp
(24,
26)
that
are
encoded
by
a
single
gene
(4).
The
banana
is
a
climacteric
fruit
like
the
apple,
pear,
peach,
tomato,
avocado,
and
others.
This
class
of
fruits
is
character-
ized
by
a
large
increase
in
ethylene
synthesis
at
the
onset
of
ripening.
It
is
believed
that
ethylene
regulates
the
expression
of
the
genes
involved
in
ripening.
Brady
and
O'Connell
(2)
demonstrated
an
increase
in
the
rates
of
turnover
of
proteins
in
banana
pulp
tissue.
However,
in
banana
fruits,
little
is
known
about
the
synthesis
of
particular
proteins
not
previ-
ously
present
in
preclimacteric
stages.
Recent
studies
on
eth-
ylene
and
O2
effects
in
bananas
suggest
that
the
increase
in
'This
work
was
supported
by
grant
ALI-88-0138
from
Comisi6n
Interministerial
de
Ciencia
y
Technologia,
and
grant
AR/88
from
the
Comissi6
Interdepartamental
de
Recerca
e
Innovaci6
Tocnologica
Generalitat
de
Catalunya.
E.D.-P.
is
a
recipient
of
a
fellowship
from
the
Ministerio
Educati6n
y
Ciencia.
157
Immature
fruits
of
banana
(Musa
acuminata,
cv
Dwarf
Cavendish)
were
obtained
from
Tenerife
(Canary
Islands).
Fruits
were
dipped
in
fungicide
solution
(1%o
benlate,
3%o
dithane,
w/v)
and
placed
in
ventilated
jars
with
a
known
flow
of
humidified
air
at
22°C.
The
respiration
rate
of
the
fruit
tissue,
determined
as
CO2
production,
was
measured
by
con-
necting
the
effluent
air
from
each
respiration
jar
to
an
infrared
gas
analyzer.
Ethylene
measurements
were
made
with
a
gas
chromatograph
fitted
with
a
flame
ionization
detector.
Fruit
samples
at
determinate
ripening
stages
were
immediately
frozen
in
liquid
nitrogen
and
stored
at
-80°C.
Preparation
of
Protein
Extracts
To
extract
total
protein,
frozen
pulp
tissues
at
specific
ripening
stages
were
ground
in
a
mortar
under
liquid
N2.
The
powder
was
delipidized
in
an
acetone:hexane
mixture
(51:49,
v/v)
and
centrifuged
at
6000g
for
20
min
at
4°C.
The
final
pellet
was
vacuum
dried
until
a
fine
flour
was
obtained,
then
stored
at
-40°C.
Because
the
main
difficulty
in
obtaining
protein
extracts
from
banana
fruit
is
to
get
rid
of
other
interfering
compounds,
we
applied
the
following
extraction
procedure:
equally
weighted
flour
samples
were
first
extracted
with
buffer:
0.25
M
Tris-HCl,
pH
8.4,
0.2
M
glycine,
0.4%
(w/v)
SDS,
and
10%
(v/v)
2-mercaptoethanol.
The
supernatants
obtained
after
centrifugation
(12,000g
for
20
min
at
4°C)
were
immediately
treated
with
an
equal
volume
of
phenol
(previously
equili-
brated
with
1
M
Tris,
pH
8).
Both
the
phenolic
phase
and
the
interphase
were
recovered
and
washed
four
times
with
10
mM
Tris,
1
mm
EDTA
(pH
7.5).
The
proteins
in
the
phenolic
phase
were
precipitated
with
four
volumes
of
methanol/0.
1
M
ammonium
acetate
at
-20°C
overnight.
The
pellet
was
then
dried
and
dissolved
in
a
sample
buffer:
60
mm
Tris-HCl
(pH
6.8),
10%
(v/v)
glycerol,
2%
(w/v)
SDS,
and
5%
(v/v)

DOMINGUEZ-PUIGJANER
ET
AL.
2-mercaptoethanol
for
one-dimensional
SDS-PAGE,
or
in
a
lysis
buffer
(9.5
M
urea,
2%
[v/v]
Nonidet
P-40,
and
2%
[v/
v]
of
a
mixture
of
ampholines
[Pharmacia],
pH
range
3-10
and
5-8)
for
two-dimensional
electrophoresis.
Protein
con-
centrations
were
measured
by
the
Lowry
method
as
modified
by
Peterson
(21)
using
BSA
as
a
standard.
Electrophoresis
and
Immunoblot
Total
proteins
in
the
pulp
extracts
were
separated
by
SDS-
PAGE
on
a
15%
(w/v)
acrylamide,
0.4%
(w/v)
bisacrylamide
slab
gel
(1.5
x
160
x
200
mm)
according
to
the
Laemmli
method
(14).
An
equal
amount
of
protein
(80
tig)
was
loaded
in
each
lane.
Gels
were
fixed
in
50%
(w/v)
TCA
and
stained
with
Coomassie
blue.
In
two-dimensional
electrophoresis
(NEPHGE2
x
SDS-PAGE),
the
first
dimension,
NEPHGE
was
performed
according
to
the
method
of
O'Farrell
et
al.
(20)
as
modified
by
Meyer
and
Chartier
(18).
Equal
protein
quantities
(160
kig)
were
loaded
in
cylindrical
gels
(length
7
cm,
internal
diameter
1.5
mm)
containing
3.7%
acrylamide,
0.21%
bisacrylamide,
9.5
M
urea,
2%
(v/v)
Nonidet
P-40,
and
0.8%
(v/v)
ampholines
(Pharmacia,
3-10
and
5-8
pH
range).
Samples
were
placed
on
the
acid
end
of
the
gel
and
covered
with
an
overlay
solution
(8
M
urea,
5%
[v/v]
Nonidet
P-40,
5%
[v/v]
2-mercaptoethanol,
and
1%
[v/v]
ampholines
[3-
10
and
5-8
pH
range])
and
run
for
30
min
at
100
V,
45
min
at
200
V,
75
min
at
300
V,
and
60
min
at
500
V.
Gels
were
equilibrated
in
sample
buffer
(60
mm
Tris
HC1
[pH
6.8],
10%
[v/v]
glycerol,
2%
[w/v]
SDS,
and
5%
[v/v]
2-mercaptoetha-
nol)
for
20
min.
In
the
second
dimension,
SDS-PAGE-equil-
ibrated
gels
(1.5
x
7.3
x
10
cm)
were
run
on
15%
polyacryl-
amide
slab
gels
for
3
h
at
120
V.
Standard
molecular
mass
markers
(from
Sigma)
were
as
follows:
BSA
(68
kD),
ovalbu-
min
(45
kD),
carbonic
anhydrase
(30
kD),
soybean
trypsin
inhibitor
(21
kD),
and
lysozyme
(14
kD).
Immunoblotting
of
two-dimensional
electrophoresed
pro-
teins
was
performed
as
described
(16).
Antiserum
to
tomato
polygalacturonase
was
used
at
a
1:1000
dilution.
Immune
complexes
were
detected
using
peroxidase-conjugated
antirab-
bit
immunoglobulin
G
and
4-chloro-
1-naphtol
as
substrate.
Preimmune
rabbit
antiserum
was
used
as
a
control.
Detection
of
Glycoproteins
The
proteins
resolved
in
two-dimensional
electrophoresis
(NEPHGE
x
SDS-PAGE)
were
transferred
to
nitrocellulose
sheets
(0.45
,um,
Schleicher
and
Schuell)
as
described
by
Towbin
et
al.
(23).
Transferred
glycoproteins
were
localized
on
the
blot
after
the
procedure
described
by
Clegg
(7)
to
locate
glycoproteins
that
bind
concanavalin
A.
Essentially,
the
sheets
were
first
incubated
with
concanavalin
A
(Pharmacia)
and
then
with
the
enzyme
glycoprotein,
horseradish
peroxidase
(Sigma),
and
developed
with
O-dianisidine
as
a
substrate.
Duplicate
sheets
were
incubated
only
with
peroxidase,
leav-
ing
out
the
concanavalin
A
incubation
to
detect
possible
concanavalin
A
proteins
in
the
extracts.
To
discard
endoge-
nous
peroxidases,
a
second
control
was
made,
exposing
nitro-
cellulose
membranes
to
the
peroxidase
substrate.
2Abbreviation:
NEPHGE,
nonequilibrium
pH
gradient
electro-
phoresis.
A
*-
4
Co,
w
U
CNH,
w
2
2
ol
B
kD
M
-
-
*
.1
_
-A
.
.1.
2
-1
0
A
B
C
D
.v
4
5
*
:f
,7.,2
30
<
30q3
<
2.
21
*
4W_
_
23
14_
Figure
1.
A,
Respiration
rates
and
ethylene
production
of
whole
banana
fruit.
Data
represent
the
average
of
four
fruits.
Days
were
referred
from
the
start
of
the
respiratory
climacteric.
Arrows
indicate
the
four
stages
at
which
samples
were
collected.
A,
B,
C,
and
D
correspond
to
the
preclimacteric,
early
climacteric,
climacteric,
and
postclimateric,
respectively.
B,
SDS-PAGE
of
total
proteins
extracted
from
banana
pulp.
Lanes
A,
B,
C,
and
D
correspond
to
samples
at
different
stages
of
ripening
as
shown
in
A.
Eighty
micrograms
protein
were
applied
to
each
lane.
The
molecular
mass
markers
are
in
lane
M.
Black
arrows
show
polypeptides
in
kD
that
decrease
during
ripening,
and
white
arrows
the
polypeptides
in
kD
that
increase
during
this
process.
Gels
were
stained
with
Coomassie
blue.
Plant
Physiol.
Vol.
98,
1992
158
iA
3

BANANA
DIFFERENTIAL
PROTEIN
ACCUMULATION
DURING
RIPENING
RESULTS
Protein
Analysis
in
Banana
Pulp
As
shown
in
Figure
1A,
the
ripening
process
in
banana
begins
with
an
increase
in
respiration
and
ethylene
produc-
tion.
The
CO2
production
peak
appears
about
1
d
after
the
ethylene
peak.
It
subsequently
declines
as
the
fruit
enter
the
postclimacteric
period.
To
determine
the
changes
in
protein
content
and/or
accu-
mulation
during
ripening,
total
proteins
were
extracted
at
different
stages
of
the
process.
The
arrows
in
Figure
1A
correspond
to
preclimacteric
(A),
early
climacteric
(B),
cli-
macteric
(C),
and
postclimacteric
(D)
stages.
Samples
of
iden-
tical
dry
weight
from
fruits
at
these
four
stages
were
collected
and
processed
for
protein
extraction.
Many
classic
total
pro-
tein
extraction
methods
were
used.
However,
due
to
the
special
characteristics
of
the
banana
fruit
tissue
(high
content
of
polyphenols
and
polysaccharides),
the
use
of
the
Tris-SDS/
phenol
extraction
method
improved
the
recovery
of
total
proteins.
The
absolute
protein
values
obtained
from
each
sample
and
expressed
in
mg/g
fresh
weight
were
3.7,
3.25,
kD
68-
45-
2.15,
and
1.40;
these
values
correspond
respectively
to
the
stages
indicated
above
(A,
B,
C,
and
D).
To
examine
the
differential
accumulation
of
banana
pro-
teins
during
ripening,
samples
of
proteins
extracted
were
subjected
to
SDS-PAGE
analysis
(Fig.
lB).
As
the
fruit
rip-
ened,
many
changes
were
observed.
Some
polypeptides,
for
example,
one
of
23
kD
that
was
prominent
in
immature
fruit,
decreased
with
ripening.
Others,
like
relatively
abundant
poly-
peptides
(28,
30,
and
42
kD)
increased
from
the
preclimacteric
to
the
postclimacteric
stage.
The
30
kD
polypeptide
appeared
to
have
reached
its
lowest
concentration
during
the
climacteric
stage,
but
a
slight
increase
was
observed
at
the
ripened
stage.
Two-dimensional
electrophoresis
(NEPHGE/SDS-PAGE)
protein
analysis
improved
the
resolution
of
major
polypep-
tides.
Figure
2
shows
that
the
main
proteins
remain
present
in
the
four
stages.
However,
there
are
significant
changes
in
the
relative
concentration
of
some
polypeptides.
Thus,
the
protein
present
at
the
preclimacteric
stage
(spot
12)
increased
in
the
course
of
ripening,
but
another
(spot
10)
decreased.
The
levels
of
polypeptides
9
and
11
fluctuate.
Protein
accu-
mulation
was
observed
at
the
preclimacteric
stage,
decreasing
A
30-
21--
14-
68-
45-
LU
Ao
0
Co
en
+
C
30-
21-
14-
pH--
5
.~~~~~~~~~~~~~~~~~~~~~~~~~
,--
...~",---
a
7
6
75
4
Figure
2.
Two-dimensional
electrophoretic
patterns
of
total
proteins
during
banana
ripening.
Figure
2A
through
D
corresponds
to
samples
at
different
stages
of
ripening
as
shown
in
Figure
1A
and
are
the
same
used
in
Figure
1
B.
NEPHGE
was
carried
out
in
the
first
dimension
(3-10
pH
range
ampholites)
and
SDS-PAGE
in
the
second
dimension
(15%
acrylamide).
The
molecular
mass
markers
are
indicated
on
the
right
in
kD.
Lateral
arrows
(black
and
white)
correspond
to
polypeptides
that
increase
and
decrease,
respectively.
White
arrows
indicate
polypeptides
lacking
in
comparison
with
other
gels
in
the
figure.
Apparent
isoelectric
points
are
indicated
on
the
bottom.
Numbered
spots
are
referred
to
in
the
text.
159

DOMINGUEZ-PUIGJANER
ET
AL.
kD
68-1
e
a
45-.
w
nI
30-~~~~~~~~~~~~~~~~~~~~0
21-
+
14
-
pH-
5
Figure
3.
Detection
of
specific
glycoproteins
in
postclimacteric
ba-
nana
fruit.
Two-dimensional
gel
electrophoresis
(see
Fig.
2D)
trans-
ferred
to
nitrocellulose
sheet
and
visualized
with
the
concanavalin
A-
peroxidase
method.
at
high
ethylene
levels
in
the
fruit
but
increasing
in
the
postclimacteric
stage.
Polypeptides
5,
6,
7,
8,
and
13
remained
fairly
constant
during
ripening.
Note
that
the
two
sets
of
proteins
(spots
3
and
4
of
28
kD
and
spots
17-19
of
42
kD)
whose
levels
were
very
low
in
nonripening
fruit
increased
with
the
onset
of
ripening.
These
sets
of
polypeptides
are
resolved
in
more
than
one
spot
by
two-dimensional
gel
electrophoresis.
In
the
case
of
the
28
kD
polypeptides
(spots
3
and
4)
with
apparent
isoelectric
point
4.9
and
5.2,
respectively,
spot
4
was
more
abundant
than
spot
3.
The
42
kD
polypeptides
(spots
17-19)
did
not
resolve
as
defined
spots
but
as
broad
bands.
The
most
abundant
poly-
peptide
from
these three
proteins
was
the
most
basic
(spot
19).
In
fact,
it
was
the
first
to
be
observed
in
the
previous
stages
(B
and
C).
The
high
molecular
mass
region
contains
a
complex
of
minor
polypeptides.
Qualitative
differences
were
observed
among
the
four
stages
of
ripening.
The
most
evident
of
these
was
the
disappearance
of
the
triplet
of
polypeptides
14, 15,
and
16
in
the
climacteric
peak
and
postclimacteric
stages.
Characterization
of
High
Mannose
Glycoproteins
As
shown
in
Figures
1B
and
2,
some
specific
polypeptides
increase
and
accumulate
during
ripening
(see
spots
3,
4,
12,
13,
17-19).
To
examine
the
presence
of
carbohydrate
moiety
in
these
proteins,
a
lectin-binding
assay
was
used.
Total
pro-
teins
of
postclimacteric
fruit
were
transferred
to
nitrocellulose
sheets
and
incubated
with
concanavalin
A
(Fig.
3).
Because
concanavalin
A
is
a
lectin
with
affinity
for
mannose,
glucose,
N-acetylglucosamine,
and
sorbose
residues
(7),
the
stained
polypeptides
could
contain
one
of
these
glycoconjugates.
Thus,
the
three
42
kD
polypeptides
resolved
as
broad
spots
are
glycoproteins.
From
one-dimensional
SDS-PAGE-con-
canavalin
A
binding
protein
assays,
it
had
previously
been
observed
that
the
42
kD
band
was
a
doublet
(data
not
shown).
A
comparison
of
Figures
2
and
3
shows
that
these
three
proteins
are
located
alternatively
between
other
spots
stained
only
by
Coomassie
blue.
Unfortunately,
we
were
unable
to
distinguish
in
two-dimensional
electrophoresis
the
doublet
previously
observed
in
one-dimensional
electrophoresis.
These
three
glycoproteins
could
correspond
to
glycosylated
forms
of
only
one
protein
presenting
charge
heterogeneity.
However,
a
single
polypeptide
could
always
be
resolved
in
more
than
one
spot
due
to
different
glycosylation
contents.
The
acidic
28
kD
proteins
were
also
highly
glycosylated,
as
was
observed
by
concanavalin
A
binding
experiments.
En-
dogenous
peroxidases
or
concanavalin
A
proteins
that
could
interfere
in
the
assay
were
not
detected.
Accumulation
of
a
Polygalacturonase-Related
Protein
in
Ripe
Banana
Fruits
One
of
the
ripening
events
that
occurs
in
climacteric
fruits
after
the
synthesis
of
ethylene
is
polygalacturonase
production
(1
1).
To
determine
whether
one
of
the
glycoproteins
charac-
terized
in
ripe
fruits
was
banana
polygalacturonase,
an
im-
munoblotting
assay
was
carried
out
on
total
proteins.
Results
are
shown
in
Figure
4.
Total
banana
proteins
from
the
po-
stclimacteric
stage
resolved
by
two-dimensional
electropho-
resis
were
incubated
with
tomato
polygalacturonase
anti-
serum.
The
results
indicate
a
specific
immunoreaction
of
the
serum
with
five
polypeptides
of
42
kD.
At
the
top
of
the
figure
I
*
iS
18
19
X
toi
a
A.*.
4.
kD
68
1i
18
19
,....i
45-
30-
c.S
Ce,1
21
-
14
-
pH-
5
6
Figure
4.
Immunodetection
of
polygalacturonase
in
ripe
fruit.
Total
protein
extracts
of
mature
banana
pulp
were
resolved
by
two-dimen-
sional
electrophoresis
and
transferred
to
nitrocellulose
sheets.
Blot
was
incubated
with
anti-polygalacturonase
serum
(dilution
1:1000)
and
immunoreactive
bands
labeled
with
peroxidase
conjugate.
Mo-
lecular
mass
markers
are
indicated
on
the
left.
Inset
at
the
top
of
the
figure
shows
the
Coomassie
blue-stained
gel.
Arrows
show
the
spots
that
correspond
to
17,
18,
and
19
spots,
positively
reacted
with
the
anti-polygalacturonase
serum.
160
Plant
Physiol.
Vol.
98,
1992

BANANA
DIFFERENTIAL
PROTEIN
ACCUMULATION
DURING
RIPENING
(see
inset
corresponding
to
Coomassie
blue
staining
of
the
same
gel
transferred),
it
can
be
seen
that
the
immunoreactive
polypeptides
were
the
glycoproteins
17,
18,
and
19,
plus
two
more
acidic
polypeptides
of
identical
mol
wt.
However,
we
could
not
distinguish
at
this
resolution
level
whether
each
spot
was
a
doublet.
The
results
suggest
that
the
broad
spots
observed
in
Figures
2
and
3
could,
in
fact,
be
more
than
one
protein.
No
cross-reaction
against
other
polypeptides
was
observed
with
this
immune
serum.
No
reaction
with
preim-
mune
serum
was
detected.
DISCUSSION
Ripening
of
fruits
is
associated
with
novel
translational
and/or
transcriptional
events.
In
bananas,
there
have
been
few
reports
about
protein
changes
associated
with
fruit
rip-
ening.
Brady
and
O'Connell
(2)
reported
that
most
of
the
increment
in
protein
synthesis
early
in
the
climacteric
rise
resulted
in
an
increase
in
the
turnover
and
the
replacement
of
preexisting
species
of
protein.
On
the
basis
of
accumulation
and/or
activity
changes
of
different
cell
wall
enzymes,
De
Leo
and
Sacher
(8)
correlated
increasing
activities
of
acid
phos-
phatase
with
synthesis
de
novo.
Our
present
results
indicate
that
there
is
a
differential
protein
accumulation
during
banana
ripening
and
some
specific
proteins
of
ripe
fruit
have
been
detected.
The
analysis
of
these
differences
was
especially
difficult
in
banana
because
of
the
presence
of
large
amounts
of
starch
(about
90%
of
the
dry
matter),
polyphenolics
in
green
fruit
tissue,
and
soluble
sugars
in
the
ripe
fruit
(17).
The
main
proteins
of
the
four
stages
studied
in
the
ripening
process
were
present
in
all
the
stages.
The
qualitative
differences
observed
may
be
related
to
the
synthesis
and
hydrolysis
of
proteins
involved
in
the
ripening
process
(3).
Most
of
them
could
be
constitutive,
structural,
or
storage
proteins.
However,
our
data
provide
evidence
of
stage-specific
accumulations
of
particular
polypeptides.
The
most
significant
changes
were
detected
in
the
last
two
stages
(C
and
D).
Two
polypeptides
of
28
and
42
kD
were
present
in
the
early
climacteric
stage
but
increased
dramatically
after
the
peak
of
ethylene
produc-
tion.
The
main
reports
of
protein
induced
by
ripening
are
focused
in
cell
wall
hydrolytic
enzymes
(12,
25).
Recently,
it
has
been
shown
that
cellulase
(5)
and
polygalacturonase
(13)
increase
when
ripening
of
avocado
begins.
We
have
observed
that
one
of
the
stage-specific
polypeptides
(42
kD)
cross-reacts
with
tomato
polygalacturonase
antibodies
(kindly
given
by
Dr.
G.A.
Tucker).
The
42
kD
proteins
are
resolved
in
seven
spots
by
two-dimensional
gel
electrophore-
sis.
However,
the
antibody
against
tomato
polygalacturonase
recognizes
only
five
of
them.
In
lectin-binding
experiments,
we
have
observed
that
three
of
the
five
cross-reacting
polypep-
tides
bind
concanavalin
A,
suggesting
that
these
proteins
contain
a
high
mannose
glycan
moiety.
These
kinds
of
glycans
are
characteristic
of
N-glycosylation
modification
of
proteins
in
the
endoplasmic
reticulum.
The
heterogeneity
of
charge
observed
both
in
immunoblots
and
in
lectin-binding
experi-
ments
could
indicate
the
presence
of
different
glycoproteins
immunorelated
or
the
presence
of
only
one
polypeptide
with
different
levels
of
glycosylation.
The
data
presented
here
suggest
that
the
42
kD
polypeptides
could
be
polygalacturonase-related
proteins.
The
immunolog-
ical
properties
of
these
polypeptides
indicate
common
anti-
genic
determinants
with
tomato
polygalacturonase.
It
should
be
noted
that
no
other
cross-reaction
was
observed
in
the
total
ripe
banana
extracts.
In
addition,
the
presence
of
neutral
amino
sugars
was
previously
described
in
tomato
polygalac-
turonase.
The
carbohydrate
portion
of
the
tomato
enzyme
contains
mannose,
fucose,
xylose,
and
N-acetyl-glucosamine
(19).
However,
the
analysis
of
an
enzymatic
activity
related
to
these
polypeptides
would
be
the
approach
to
use
to
dem-
onstrate
the
presence
of
polygalacturonase
in
ripe
banana
fruits.
It
was
interesting
to
note
that
another
ripened
stage-specific
protein
(28
kD)
accumulated
in
large
amounts
in
postclimac-
teric
fruit.
Recent
experiments
in
vivo
carried
out
in
our
laboratory
indicate
that
this
protein
was
not
present
in
early
climacteric
stages,
but
that
it
was
highly
synthesized
de
novo
in
ripe
fruit.
However,
the
function
of
this
polypeptide
is
unknown
and
no
equivalent
product
was
detected
in
other
fruits.
Antibodies
raised
against
this
protein
will
provide
us
with
a
tool
to
characterize
it.
ACKNOWLEDGMENTS
The
authors
are
grateful
to
Dr.
G.A.
Tucker
for
generously
provid-
ing
the
tomato
polygalacturonase
antibody
and
to
Dr.
J.
Hernandez
for
the
supply
of
bananas.
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Vol.
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1992
162