Plant
Physiol.
(1972)
49,
789-793
Sugar
Transport
in
Immature
Internodal
Tissue
of
Sugarcane
II.
MECHANISM
OF
SUCROSE
TRANSPORT'
Received
for
publication
December
6,
1971
JOHN
E.
BOWEN
Department
of
Plant
Physiology,
Hawaii
Agricultural
Experiment
Station,
University
of
Hawaii,
Hilo,
Ha-
waii
96720
JAMES
E.
HUNTER
Department
of
Plant
Pathology,
Hawaii
Agricultural
Experiment
Station,
University
of
Hawaii,
Hilo,
Hawaii
96720
ABSTRACT
The
mechanism
by
which
sucrose
is
transported
into
the
inner
spaces
of
immature
internodal
parenchyma
tissue
of
sugarcane
(Saccharum
officinarum
L.
var.
H
49-5)
was
studied
in
short
term
experiments
(15
to
300
seconds).
Transport
of
sucrose,
glucose,
and
fructose
was
each
characterized
by
a
Vmax
of
1.3
Amoles/gram
fresh
weight-2
hours,
and
each
of
these
three
sugars
mutually
and
competitively
inhibited
trans-
port
of
the
other
two.
When
'4C-glucose
was
supplied
exoge-
nouslv,
"C-glucose
6-phosphate
and
"C-glucose
were
the
first
labeled
compounds
to
appear
in
the
tissue;
no
"C-sucrose
was
detected
until
after
60-second
incubation.
After
15-second
in-
cubation
in
"C-sucrose,
all
intracellular
radioactivity
was
in
glucose,
fructose,
glucose
6-phosphate,
and
fructose
6-phos-
phate;
trace
amounts
of
"C-sucrose
were
found
after
30
seconds
and
after
5
minutes,
71
%
of
the
intracellular
radioactivity
was
in
sucrose.
Although
it
was
possible
that
sucrose
was
trans-
ported
intact
into
the
inner
space
and
then
immediately
hydro-
lyzed,
it
was
shown
that
the
rate
of
hydrolysis
under
these
conditions
was
too
low
to
account
for
the
rate
of
hexose
accu-
mulation.
Pretreatment
of
the
tissue
with
rabbit
anti-invertase
antiserum
eliminated
sucrose
transport,
but
had
no
effect
on
glucose
transport.
Since
the
antibodies
did
not
penetrate
the
plasmalemma,
it
was
concluded
that
sucrose
was
hydrolyzed
by
an
invertase
in
the
free
space
prior
to
transport.
The
glucose
and
fructose
moieties,
or
their
phosphorylated
derivatives,
were
then
transported
into
the
inner
space
and
sucrose
was
resynthe-
sized.
No
evidence
for
the
involvement
of
sucrose
phosphate
in
transport
was
found
in
these
experiments.
Hydrolysis
external
to
the
plasmalemma
appears
to
be
a
prerequisite
for
metabolic
utilization
of
exogenously
supplied
sucrose
in
some
higher
plant
cells
(12,
17,
18)
and
fungi
(6,
15).
Conversely,
other
higher
plant
tissues
store
sucrose
but
have
low
or
undetectable
invertase
activity
(16),
implying
that
hydrolysis
of
sucrose
prior
to
uptake
is
not
necessary
in
these
tissues.
I
Journal
Series
No.
1400
of
the
Hawaii
Agricultural
Experiment
Station.
Sugarcane
presents
an
enigma
insofar
as
sucrose
transport
is
concerned.
In
early
studies
of
sucrose
absorption
and
ac-
cumulation
in
immature
storage
parenchyma
tissue
of
sugar-
cane,
Bieleski
(3)
reported
that
sucrose
was
absorbed
without
prior
hydrolysis
and
that
its
uptake
was
characterized
by
a
Km
value
distinct
from
that
of
glucose
and
fructose.
However,
Sacher
et
al.
(19)
reported
that
sucrose
was
inverted
in
passing
from
the
external
solution
to
the
storage
compartment
where
it
reappeared
as
sucrose.
Support
for
this
contention
was
gained
from
the
observation
of
Hatch
and
Glasziou
(11)
that
when
("C-fructosyl)-labeled
sucrose
moved
from
the
vascular
tissue
to
the
parenchyma,
it
initially
was
broken
down
and
subsequently
resynthesized
with
random
labeling
of
the
glu-
cose
and
fructose
moieties.
To
explain
these
observations,
Sacher
et
al.
(19)
proposed
that
sucrose
was
accumulated
via
a
sucrose
derivative
that
could
be
formed
from
sucrose
only
via
the
hexose
moieties.
This
derivative,
thought
to
be
sucrose
phosphate
(phosphorylated
at
the
C-6
position
of
fructose),
then
moved
across
the
limiting
membrane,
presumably
the
tonoplast,
and
ultimately
into
the
vacuole
(9,
19).
In
previous
studies
of
transmembrane
transport
of
sucrose
into
immature
parenchyma
tissue
of
sugarcane,
uptake
meas-
urements
and
identification
of
the
sugar
components
of
the
tissue
were
made
after
4
hr
(7-9,
19),
and
in
one
case
after
24
to
36
hr
(3).
Extensive
metabolism
of
sugars
transported
and
accumulated
during
these
time
periods
precludes
any
conclu-
sions
about
the
initial
reactions
in
the
transport
process.
Another
facet
of
sucrose
transport
was
studied
recently
by
Maretzki
and
Thom
(14)
with
cell
suspension
cultures
of
sugar-
cane.
Specifically,
they
did
not
observe
any
transmembrane
movement
of
sucrose
into
these
cells
(14).
However,
these
cells
typically
have
very
low
levels
of
invertase
activity
external
to
the
plasmalemma
(Maretzki,
personal
communication),
a
fact
that
may
be
interpreted
as
evidence
that
sucrose
is
transported
only
after
being
hydrolyzed.
Direct
evidence
for
prerequisite
extracellular
hydrolysis
of
sucrose
before
transport
into
immature
sugarcane
storage
tis-
sue
is
reported
herein.
Furthermore,
the
glucose
and
fructose
moieties,
or
their
phosphorylated
derivatives,
apparently
are
transported
into
the
cells,
rather
than
sucrose
or
sucrose
phos-
phate
as
proposed
earlier
(3,
9,
19).
MATERIALS
AND
METHODS
Tissue
discs
(6
mm
diameter
X
75
,u
thick)
were
cut
from
immature
internodal
parenchyma
tissue
of
12-month-old
sugar-
cane
(Saccharum
officinarum
L.
var.
H49-5).
The
discs
were
789
BOWEN
AND
HUNTER
cut
to
these
dimensions
to
facilitate
rapid
equilibration
of
the
intercellular
spaces
with
"C-sugar
solutions.
After
incubation
of
0.5
g
fresh
weight
of
discs
for
15
to
300
sec,
the
tissue
was
rinsed
for
15
sec
which
removed
98%
of
the
"free
space"
sugars.
Methods
of
cutting
and
preparing
tissue
discs,
incubat-
ing
in
"C-sugar-0.5
mm
CaSO,
solutions,
and
measuring
"C-
activity
were
described
previously
(4).
To
determine
the
dis-
tribution
of
"C
among
the
intracellular
sugars,
the
tissue
was
frozen
in
liquid
nitrogen
immediately
after
rinsing
and
was
extracted
subsequently
by
grinding
with
50%
ethanol.
After
ethanol
was
removed
under
N2,
the
aqueous
extract
was
ad-
justed
to
pH
8
and
applied
to
a
1
X
30
cm
anion
exchange
column
(AG
1
X
4
(Cl),
200-400
mesh).
Free
sugars
and
sugar
phosphates
were
eluted
from
the
column
with
an
NH,Cl/
K2B,0r
8H20
gradient
(2).
Flow
rate
was
50
ml/hr,
and
10-ml
fractions
were
collected.
Radioactivity
was
determined
on
an
aliquot
of
each
fraction
(4).
Fractions
containing
free
sugars
were
pooled,
concentrated,
and
separated
by
paper
chromatog-
raphy
(4).
A
protein
preparation
of
high
invertase
activity
was
ob-
tained
from
immature
storage
parenchyma
by
the
methods
of
Hatch
et
al.
(10)
and
Alexander
(1).
This
preparation
was
par-
tially
purified
by
(NH4)2SO,
precipitation.
The
40
to
50%
(NH,)2SO,
fraction
manifested
the
highest
invertase
activity
and
thus
was
used
throughout
this
study.
Invertase
activity
was
determined
by
measuring
colorimetrically
the
glucose
formed
from
sucrose
by
the
"Glucostat"
method
(Worthington
Bio-
chemical
Corporation).
The
reaction
mixture
contained
20
,tmoles
of
sucrose,
10
,umoles
of
phosphate-citrate
buffer,
pH
5.5,
and
0.5
mg
of
enzyme
protein
in
a
total
volume
of
1
ml.
Reactions
were
initiated
by
adding
sucrose
and
were
run
for
2
hr
at
28
C.
Protein
was
measured
by
the
method
of
Lowry
et
al.
(13).
For
production
of
anti-invertase
antiserum,
a
preparation
of
sugarcane
protein
in
phosphate-buffered
0.86%
NaCl
solution
was
injected
into
the
marginal
ear
vein
of
a
rabbit.
Six,
12.
18,
24,
24,
and
24
mg
of
protein
were
injected
on
day
1,
4,
6,
8,
11,
and
13,
respectively.
On
day
15,
an
intraperitoneal
booster
injection
of
100
mg
of
protein
was
given.
The
injections
on
days
13
and
15
were
given
with
0.1
ml
of
antihistamine
(For-
tamine
solution,
Fort
Dodge
Laboratories,
Fort
Dodge,
Iowa)
to
prevent
anaphylaxis.
The
rabbit
was
bled
by
cardiac
punc-
ture
10
days
after
the
last
injection.
The
precipitin
titer
of
the
antiserum
was
determined
as
the
highest
2-fold
dilution
that
produced
discernible
lines
of
pre-
cipitation
in
a
precipitin
ring
test.
The
tubes
were
incubated
at
37
C
for
4
hr
and
final
readings
were
made
after
refrigeration
overnight.
Immunodiffusion
assays
were
conducted
at
room
temperature
in
60
X
15
mm
Petri
dishes
containing
a
4-mm
layer
of
0.7%
agar
supplemented
with
15
mm
sodium
azide
and
15
mM
NaCl.
For
enzymic
reactions
with
the
crude
protein
preparation
the
antiserum
was
added
to
the
reaction
system
3
min
before
su-
crose
was
added
and
was
present
during
the
incubation
period.
In
sugar
transport
experiments,
tissue
sections
were
incubated
in
the
antiserum
for
10
min,
after
which
the
tissue
was
rinsed
in
0.5
mM
CaSO,
solution
three
times
for
1
min
each,
and
transferred
to
"C-sugar
solutions.
Normal
serum
taken
from
the
rabbit
before
it
was
im-
munized
served
as
a
control
in
these
experiments.
In
no
in-
stance
was
there
a
significant
effect
attributable
to
the
presence
of
the
control
serum
when
tested
at
the
same
dilutions
as
the
antiserum,
nor
was
there
any
evidence
of
protease
activity
in
the
incubation
medium
under
these
experimental
conditions.
D-Glucose-`C
(U),
D-fructose-"C
(U),
sucrose-"C
(U)
and
UDP-glucose-`C
(U)
were
products
of
Amersham/
Searle
Corp.
The
(lC-glucosyl)-labeled
sucrose
was
synthesized
en-
zymically
using
a
UDP-glucose
fructose
transglucosylase
prepa-
ration
from
sugarcane
(19).
The
sucrose
was
purified
by
paper
chromatography
(4).
A
sample
of
the
sucrose
was
treated
with
yeast
invertase
and
the
products
rechromatographed.
Only
the
glucose
moiety
was
labeled.
The
sucrose
preparation
contained
no
other
labeled
compounds.
All
experiments
were
replicated
at
least
three
times,
and
the
data
were
generally
reproducible
within
+-5%
unless
stated
otherwise.
RESULTS
AND
DISCUSSION
Kinetics
of
Sugar
Transport.
The
concentrations
of
glucose
and
fructose
which
gave
one-half
the
maximum
"C
uptake
were
6.7
and
8.4
mm,
respectively,
and
the
V,,.
value
for
both
hexoses
was
1.3
,umoles/g
fresh
weight-2
hr
(4).
Sucrose
up-
take
also
is
characterized
by
an
apparent
Vn,.x
of
1.3
icmoles/g
fresh
weight-2
hr,
as
determined
from
a
double
reciprocal
plot
in
the
present
study.
However,
the
Kmt
for
sucrose
transport
varied
widely
among
different
tissue
preparations,
making
im-
possible
a
meaningful
estimation
of
this
parameter,
although
the
Kmii
values
for
glucose
and
fructose
were
readily
reproduci-
ble.
Thus,
it
was
hypothesized
that
one
or
more
rate-limiting
steps
may
precede
transmembrane
transport
of
sucrose
into
this
tissue,
and
that
the
rate
of
this
limiting
reaction
may
vary
with
different
tissue
preparations.
Since
Hatch
and
co-workers
(9,
19)
found
acid
invertase
activity
in
the
free
space
to
vary
with
different
tissue
preparations
from
18
to
69%
of
the
total
invertase
activity
in
the
tissue,
and
moreover,
since
invertase
has
been
implicated
in
sucrose
transport
into
sugarcane
tissue
(9,
19),
it
was
considered
that
activity
of
this
enzyme
may
be
the
rate-limiting
factor
in
sucrose
transport.
Interactions
in
Sugar
Transport.
Glucose
and
fructose
are
transported
into
the
inner
spaces
of
immature
sugarcane
paren-
chyma
tissue
slices
via
the
same
pathway
(3,
4,
7).
However,
agreement
has
not
been
reached
upon
whether
sucrose
also
is
transported
by
this
system
(3,
9,
19).
Transport
differs
in
sugarcane
cellular
suspension
cultures
in
that
glucose
and
fructose
do
not
compete
for
uptake
(14).
The
possible
occurrence
of
interactions
and
mutual
competi-
tions
in
the
transport
of
glucose,
fructose,
and
sucrose
was
in-
vestigated
in
a
series
of
factorially
designed
experiments.
Each
solution
contained
0.5
mM
CaSO,;
1
mm
"C-glucose,
"C-fruc-
tose,
or
"C
(U)-sucrose;
and
except
in
the
controls,
one
or
more
of
the
above
three
'2C-sugars
at
1
mM.
When
the
uptake
of
glucose,
fructose
and
sucrose
after
5
min
was
examined
in
the
presence
of
each
of
the
other
sugars,
each
sugar
mutually
interfered
with
uptake
of
the
others
(Table
I).
Additional
experiments
demonstrated
that
each
of
these
three
sugars
competitively
inhibited
the
transport
of
the
others
as
determined
from
double
reciprocal
plots.
Therefore,
it
was
concluded
that
the
transport
of
glucose,
fructose,
and
sucrose
apparently
is
effected
through
the
same
mechanism,
i.e.,
the
same
carrier
sites,
thus
supporting
the
findings
of
Bieleski
(3).
No
answer
can
be
advanced
from
these
data
to
the
question
of
the
form
in
which
sucrose
is
transported,
i.e.,
whether
hydro-
lytic
cleavage
occurs
prior
to
or
during
transport.
Distribution
of
"C
in
Intracellular
Sugars
as
a
Function
of
Time
and
Exogenous
Sugar
Supplied.
Tissue
discs
(0.5
g)
were
cut,
washed,
and
immersed
in
1
mm
"C-glucose
for
up
to
1
min,
or
in
1
mm
"C
(U)-sucrose
or
1
mm
("C-glucosyl)-labeled
sucrose
for
up
to
5
min
to
accumulate
labeled
sugars
in
the
inner
space.
Discs
were
removed
from
the
"C-sugar
solutions
at
intervals,
rinsed
for
15
sec,
and
frozen
for
later
extractions.
Thus
there
was
a
15-sec
lapse
between
sampling
and
cessation
of
metabolic
activity.
The
data
in
Table
II
have been
corrected
for
lapsed
time.
i.e..
tissue
samples
were
actually
removed
790
Plant
Physiol.
Vol.
49,
1972
SUCROSE
TRANSPORT
IN
TISSUE
SECTIONS.
II
from
the
bathing
solution
15
sec
prior
to
the
times
stated.
In-
tracellular
sugars
were
extracted
from
the
frozen
tissue,
sepa-
rated
into
free
and
phosphorylated
sugar
fractions,
and
indi-
vidual
components
of
the
two
fractions
were
separated
and
quantitatively
estimated
by
radioassay.
After
15-sec
incubation
in
"C
(U)-glucose,
the
tissue
con-
tained,
on
a
gram
fresh
weight
basis,
0.16
nmole
"C-glucose-
6-P
and
0.14
nmole
"C-glucose
(Table
II).
Earlier
it
was
re-
ported
(4)
that
as
much
as
82%
of
the
intracellular
radioac-
tivity
was
in
the
phosphorylated
sugar
fraction
under
similar
conditions.
In
the
present
detailed
studies,
however,
it
was
observed
that
the
size
of
the
phosphorylated
"C-sugar
fraction
varied
from
53
to
70%
of
the
total
intracellular
radioactivity
with
different
tissue
preparations,
although
in
replicate
experi-
ments
with
tissue
discs
from
the
same
preparation,
the
magni-
tude
and
composition
of
the
free
sugar
and
sugar
phosphate
fractions
were
very
reproducible
(within
+5%).
The
glucose-6-
P/glucose
ratio
in
the
experiment
reported
in
Table
II
is
the
lowest
found
in
any
experiment.
After
30
and
60
sec
the
intra-
cellular
concentrations
of
"C-glucose
and
"C-glucose-6-P
had
increased
and
radioactivity
also
began
to
appear
in
fructose,
sucrose,
glucose-1-P,
fructose-6-P,
and
fructose-1,6-diP
(Ta-
ble
II).
When
"C
(U)-sucrose
was
supplied
exogenously,
most
of
the
intracellular
radioactivity
was
concentrated
in
glucose,
glucose-
6-P,
and
fructose
after
15
sec
(Table
II).
No
radioactivity
was
detected
in
sucrose
until
after
30
sec,
when
6%
of
the
total
"C
appeared
in
this
sugar.
The
"C-sucrose
concentration
in-
creased
sharply
through
5
min
when
71%
of
the
intracellular
"C-activity
was
in
this
sugar
(Table
II).
The
absence
of
radio-
activity
in
sucrose
until
after
a
30-sec
incubation
may
indicate
that
sucrose
is
not
transported
as
the
disaccharide,
or
at
least
that
the
transport
rate
for
sucrose
is
considerably
less
than
that
of
glucose
and
fructose.
When
("C-glucosyl)-labeled
sucrose
was
supplied
to
the
tis-
sue
discs,
after
15
sec,
43
and
57%
of
the
intracellular
radio-
activity
was
contained
in
glucose
and
glucose-6-P,
respectively
(Table
II).
After
30
sec
"C-fructose
and
"C-fructose-6-P
were
detected,
indicating
that
glucose
is
converted
readily
to
fruc-
tose
by
this
tissue.
The
experiment
with
"C-glucose
further
supports
this
conclusion.
In
other
respects,
the
distribution
of
"C
from
exogenously
supplied
("C-glucosyl)-labeled
sucrose
was
quite
similar
to
that
from
"C
(U)-sucrose
and
"C-glucose
(Table
II).
The
possibility
that
sucrose
may
be
hydrolyzed
after
trans-
port
into
the
storage
compartment,
i.e.,
the
vacuole,
was
con-
sidered
in
the
following
experiment.
Tissue
discs
were
incu-
bated
in
1
mm
"C
(U)-sucrose
for
5
min
and
rinsed.
A
tissue
sample
was
removed
for
analysis,
and
the
remainder
was
placed
in
0.5
mm
CaSO,-l
mm
"C-sucrose
solution
to
accumu-
late
sugar
for
an
additional
2
hr.
Tissue
samples
were
taken
at
intervals
during
this
period.
After
a
5-min
incubation
in
"C
(U)-sucrose,
71
%
of
the
intracellular
radioactivity
was
in
su-
crose,
3%
in
glucose,
6%
in
fructose,
and
20%
in
phosphate
derivatives
of
glucose
and
fructose
(Table
II).
After
a
2-hr
incubation
in
'C-sucrose,
64%
of
the
total
intracellular
radio-
activity
still
remained
in
sucrose.
The
radioactivity
of
glucose
and
fructose
had
increased
slightly
after
2
hr,
comprising
7%
and
9%
of
the
total
radioactivity,
respectively
(Fig.
1).
Over
the
2-hr
period
the
total
intracellular
radioactivity
decreased
by
6%.
Glasziou
(8)
calculated
the
half-time
(t0
2)
for
inversion
of
"C-sucrose
to
glucose
and
fructose
in
the
inner
space
to
be
about
6
hr,
and
the
turnover
time
about
8.6
hr.
Similar
esti-
mates
calculated
from
the
present
data
using
Glasziou's
formu-
lae
(8)
were
a
t1,2
of
5.4
hr
and
a
turnover
time
of
7.8
hr.
These
values
can
be
utilized
to
demonstrate
that
intracellular
hydroly-
Table
I.
Mutual
Effects
of
Glucose,
Fructose,
and
Sucrose
on
their
Absorption
by
Immature
Interniodal
Tissue
of
Sugarcanie
The
concentration
of
each
sugar
was
1
mM;
CaSO4,
0.5
mM;
pH
6.5;
and
the
temperature
was
28
C.
The
absorption
period
was
5
min,
and
the
tissue
was
0.5
g
fresh
weight.
"
4C
Absorption
in
Terms
of
'4C-Sugar
Supplied
Sugars
Present_______________
Glucose
Fructose
Sucrose
enmoles/g
fresh
wIS
min
Glucose
6.3
-
-
Fructose
-
6.5
-
Sucrose
3.1
Glucose
+
fructose
3.2
4.4
Glucose
+
sucrose
5.7
1.0
Fructose
+
sucrose
-
5.4
2.5
Glucose
+
fructose
+
sucrose
2.7
4.2
0.7
Table
II.
4C
Distribution
Among
Sugars
int
Immature
Inzternodal
Parenichyma
Tissue
of
Sugarcane
as
a
Functionz
of
Time
and
"4C-Sugar
Supplied
Exogenously
The
exogenous
sugar
concentration
was
1
mM;
0.5
mm
CaSO4;
pH
6.5;
and
the
temperature
was
28
C.
Concn
of
Intracellular
Sugars
and
Sugar
Phosphates
Seconds
Glucose
Fruc-
Su-
G-6-P
G-1-P
F-6-P
F-i,
6-
tose
crose
diP
nmoles/g
fresh
weigh
Incubated
in
14C
(U)-glucose
15
0.14
0 0
0.16
0
0
0
30
0.25
0.03
0
0.27
0
0.06 0.02
60
0.23
0.05
0.36
0.38
0.06
0.11
0.06
Incubated
in
14C
(U)-sucrose
15
0.05
0.05
0
0.05
0
0.02
0
30
0.05
0.07 0.02 0.09
0
0.09
0
60
0.09
0.06
0.15
0.13
0.02 0.22
0.02
300
0.10
0.20
2.30
0.23
0.07
0.16
0.20
Incubated
in
(14C-glucosyl)-labeled
sucrose
15
0.06
0
0
0.08
0
0
0
30
0.08
0.04
0
0.11
0
0.02
0
60
0.10
0.07
0.16
0.14
0.03
0.08
0.03
300
0.15
0.24
2.08
0.12
0.06
0.24
j
0.15
sis
of
sucrose
occurred
too
slowly
to
account
for
the
rate
of
"C-glucose
and
"C-fructose
accumulation
in
this
tissue.
The
rate
of
apparent
sucrose
transport
into
this
tissue
was
3.1
nmoles/g
fresh
weight
5
min
(Table
I).
Since
ti,2
for
inversion
of
"C-sucrose
was
5.4
hr,
the
5-min
incubation
period
was
equivalent
to
approximately
0.02
t,,2.
During
a
time
period
equal
to
0.02
tq,2,
during
which
sucrose
was
transported
appar-
ently
at
a
rate
of
3.1
nmoles/g
fresh
weight,
the
loss
of
"C
from
sucrose
solely
from
turnover
of
the
intracellular
sucrose
pool
would
be
equivalent
to
(0.02)
(0.5)
(3.1
nmoles),
or
0.03
nmole
of
"C-sucrose.
Therefore,
the
maximum
amounts
of
"C-glucose
and
"C-fructose
that
could
arise
from
intracellular
hydrolysis
of
"C-sucrose
after
transport
would
be
0.03
nmole
of
each,
assuming
that
no
interconversions
of
glucose
and
fruc-
tose
occurred.
Such
interconversions
do
occur,
however,
so
as-
suming
for
example
that
all
"C-fructose
derived
from
"C-su-
crose
was
converted
to
"C-glucose,
the
maximum
"C-glucose
concentration
derived
from
"C-sucrose
would
be
0.06
nmole/g
fresh
weight-
5
min.
When
tissue
sections
were
incubated
in
"C
(U)-sucrose
for
791
Plant
Physiol.
Vol.
49,
1972
BOWEN
AND
HUNTER
5
min,
3
%
of
the
total
intracellular
"4C
appeared
in
free
glucose
and
6%
in
fructose
at
the
end
of
the
incubation
period.
These
values
are
equivalent
to
0.10
nmole
of
"4C-glucose
and
0.20
nmole
of
14C-fructose.
As
seen
above,
the
maximum
"4C-hexose
concentration
derivable
from
14C-sucrose
was
0.06
nmole,
so
intracellular
hydrolysis
of
14C-sucrose
could
account
for
only
20%
of
the
total
intracellular
free
"4C-hexoses.
If
the
phos-
phorylated
14C-hexoses
in
the
inner
space
are
considered,
less
than
7%
of
the
"4C-hexoses
can
be
attributed
to
"4C-sucrose
hydrolysis.
Thus,
it
may
be
concluded
that
14C-glucose,
14C-
fructose,
and
their
phosphorylated
derivatives
in
the
inner
space
cannot
be
derived
totally
from
hydrolysis
of
'4C-sucrose
by
invertase
subsequent
to
transport.
These
observations
render
unlikely
the
contention
that
sucrose
is
transported
intact
into
the
inner
space
(3).
Rather,
a
more
plausible
explanation
would
be
that
sucrose
is
hydrolyzed
extracellularly,
the
glucose
and
fructose
moieties
transported
across
the
membrane,
and
su-
crose
is
then
resynthesized
in
the
inner
space.
This
hypothesis
explains
the
present
data
as
well
as
the
earlier
findings
of
other
investigators
(9,
11,
19).
Effect
of
Rabbit
Anti-invertase
Antiserum
on
Sucrose
and
Glucose
Transport.
The
rabbit
antiserum
yielded
a
single
band
in
the
immunodiffusion
assay,
a
surprising
result
since
the
sugarcane
protein
preparation,
i.e.,
the
antigen,
was
not
pure
invertase.
It
can
only
be
assumed
that
the
contaminant
proteins
were
either
nonantigenic
or
were
present
in
concentrations
too
low
to
elicit
an
antibody
response.
One-half
milliliter
of
the
rabbit
antiserum
to
sugarcane
protein
diluted
1
:32
almost
completely
precipitated
100
,ug
of
the
protein
preparation
and
also
inactivated
its
invertase.
Normal
serum
had
no
effect
on
the
invertase
reaction.
A
titer
of
1024,
expressed
as
the
recipro-
cal
of
the
antiserum
dilution,
was
obtained
with
the
antiserum.
Purified
y-globulin
isolated
from
this
antiserum
(5)
reacted
in
100
TOTAL14
C
ACTII
90
~~~~~E
XTRACT
90
so-
D
'.
LJ
"
J 70
~~~~~~~SUCROSE
z
10
FRUCTOSE
0
0
0
I
2
HOURS
FIG.
1.
Hydrolysis
of
14C
sucrose
in
the
inner
space
of
immature
storage
parenchyma
of
sugarcane.
Tissue
discs
were
incubated
in
'4C
(U)-sucrose
for
5
min
and
rinsed
to
remove
"4C-sugars
from
the
free
space.
Tissue
was
then
transferred
to
0.5
mM
CaSO4-1
mM
12C-
sucrose
solution
for
2
hr.
Temperature
was
28
C,
pH
6.5.
Table
III.
Effect
of
Rabbit
Anitisera
to
Suigarcanze
Proteinis
onl
Sucrose
anid
Glucose
Absorptioni
by
Immature
Storage
Parenchyllma
Tissute
of
Siugarcane
External
sugar
concentration
was
1
mM;
CaSO4,
0.5
mu;
pH
6.5;
and
the
temperature
was
28
C.
The
absorption
period
was
5
min.
Sugar
Supplied
Other
Additives
14C
Activity
in
Terms
of
Sugar
Supplied
nm'oles/g
fresh
.J.5!
m
m
'4C
(U)-Glucose
'4C
(U)-Sucrose
(Glucosyl-'4C)
-labeled
sucrose
None
1
mm
Sucrose
Antibody
1
mNu
Sucrose
+
antibody
None
1
mNi
Glucose
Antibody
1
mui
Glucose
+
antibody
None
1
mNi
Glucose
Antibody
1
mm
Glucose
+
antibody
6.2
5.7
6.0
6.2
3.2_
1.0
0.1
0
3.1
1.1
0.1
0.1
the
same
manner
as
the
crude
antiserum,
except
that
the
former
was
more
active.
To
test
the
crude
antiserum
for
nonspecific
binding
of
glu-
cose
and
fructose,
the
antiserum,
diluted
1:
32,
was
placed
in
dialysis
tubing
and
suspended
in
either
10
/SM
glucose
or
fruc-
tose.
After
12
hr
at
4
C,
the
sugar
concentrations
inside
the
dialysis
tube
and
in
the
external
solution
were
measured.
For
both
sugars
the
concentrations
were
similar
on
both
sides
of
the
membrane,
indicating
that
neither
glucose
nor
fructose
was
bound
to
the
components
of
the
antiserum.
Antiserum
to
the
sugarcane
protein
preparation
was
added
to
intact
immature
sugarcane
parenchyma
tissue
discs
which
contained
acid
invertase
activity
in
the
free
space.
The
antibody
was
removed
readily
from
the
bathing
solution,
as
demon-
strated
by
the
diminished
ability
of
the
solution
to
precipitate
protein.
No
reaction
of
normal
rabbit
serum
was
observed
in
vitro
or
with
intact
tissue.
The
effects
of
rabbit
anti-sugarcane-protein
antiserum
upon
transport
of
'4C-glucose.
'4C
(U)-sucrose,
and
(14C-glucosyl)-
labeled
sucrose
are
summarized
in
Table
III.
The
experiment
was
thrice
replicated
with
no
significant
variation
in
results.
As
would
be
expected
if
hydrolysis
of
sucrose
by
invertase
was
a
prerequisite
to
transport,
pretreatment
of
the
tissue
discs
with
the
antiserum
reduced
accumulation
of
1"C
from
14C
(U)-su-
crose
and
("4C-glucosyl)-labeled
sucrose
by
95%
or
more
in
the
5-min
absorption
period.
The
antiserum
had
no
significant
effect
upon
'4C-glucose
transport.
CONCLUSIONS
According
to
the
scheme
developed
by
Glasziou
(8)
and
Sacher
et
al.
(19),
exogenously
supplied
sucrose
is
hydrolyzed
by
an
invertase
in
the
free
space
of
immature
storage
paren-
chyma
of
sugarcane
prior
to
accumulation
in
the
storage
com-
partment
of
the
cell.
It
was
proposed
that
the
glucose
and
fructose
moieties
from
sucrose
were
subsequently
phos-
phorylated
and
converted
to
sucrose
phosphate
(8,
19),
and
that
it
was
sucrose
phosphate
which
was
transported
ultimately
into
the
vacuole.
While
the
present
data
support
the
finding
that
sucrose
is
hydrolyzed
prior
to
transport,
no
evidence
for
sucrose
phosphate
transport
was
found
after
5-min
incubation.
792
Plant
Physiol.
Vol.
49,
1972
SUCROSE
TRANSPORT
I}
Rather,
data
are
presented
to
indicate
that
after
inversion
of
sucrose,
glucose,
and
fructose
or
their
phosphorylated
deriva-
tives
are
transported
into
the
cellular
storage
compartment
be-
fore
sucrose
is
resynthesized.
It
is
probable
that
this
disparity
in
results
is
attributable
to
the
great
difference
in
time
that
the
tissue
was
incubated
in
exogenous
'4C-sucrose,
i.e.,
15
sec
to
5
min
in
the
present
study
vs.
4
hr
in
the
earlier
ones
(8,
19).
Since
glucose,
fructose,
and
sucrose
are
metabolized
readily
by
the
sugarcane
tissue,
the
premise
was
accepted
that
the
shorter
incubation
period
would
provide
more
meaningful
data
relative
to
the
initial
reactions
at
the
transport
sites.
LITERATURE
CITED
1.
ALEXANDER,
A.
G.
1965.
Hydrolytic
proteins
of
sugarcane:
the
acid
invertases.
J.
Agr.
Univ.
Puerto
Rico
49:
287-307.
2.
BEDETTrI,
G.,
G.
AGNOLO,
AND
F.
POCCHIARI.
1970.
Anion
exchange
chromatog-
raphy
of
glycolysis
intermediates.
J.
Chromat.
49:
53-56.
3.
BIELESKI,
R.
L.
1962.
The
physiology
of
sugar-cane.
V.
Kinetics
of
sugar
accumulation.
Aust.
J.
Biol.
Sci.
15:
429-444.
4.
BOWEN,
J.
E.
1972.
Sugar
transport
in
immature
internodal
tissue
of
sugarcane.
I.
Mechanism
and
kinetics
of
accumulation.
Plant
Physiol.
49:
82-86.
5.
BOYD,
W.
C.
1956.
Fundamentals
of
Immunology.
Interscience
Publishers
Inc.,
New
York.
pp.
69-70.
6.
FUENTE-SANCHEZ,
G.
AND
A.
SOLS.
1962.
Transport
of
sugars
in
yeasts.
II.
Mechanisms
of
utilization
of
disaccharides
and
related
glycosides.
Biochim.
Biophys.
Acta
56:
49-62.
q
TISSUE
SECTIONS.
II
793
7.
GLASZIOIJ,
K.
T.
1960.
Accumulation
and
transformation
of
sugars
in
sugar
cane
stalks.
Plant
Physiol.
35:
895-901.
8.
GLAsziou,
K.
T.
1961.
Accumulation
and
transformation
of
sugars
in
stalks
of
sugar
cane.
Origin
of
glucose
and
fructose
in
the
inner
space.
Plant
Physiol.
36:
175-179.
9.
HATCH,
M.
D.
1964.
Sugar
accumulation
by
sugar-cane
storage
tissue:
the
role
of
sucrose
phosphate.
Biochem.
J.
93:
521-526.
10.
HATCH,
M.
D.,
J.
A.
SACHER,
ANXD
K.
T.
GLAsziou.
1963.
Sugar
accumulation
cycle
in
sugar
cane.
I.
Studies
on
enzymes
of
the
cycle.
Plant
Physiol.
38:
338-343.
11.
HATCH,
M.
D.
AND
K.
T.
GLASZIOU.
1964.
Direct
evidence
for
translocation
of
sucrose
in
sugarcane
and
stems.
Plant
Physiol.
39:
180-184.
12.
HELLEBUST,
J.
A.
AND
D.
F.
FORWARD.
1962.
The
invertase
of
corn
radicle
and
its
activity
in
successive
stages
of
growth.
Can.
J.
Bot.
40:
113-126.
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