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Changes in Starch Formation and Activities of Sucrose Phosphate Synthase and Cytoplasmic Fructose-1,6-bisphosphatase in Response to Source-Sink Alterations

Authors:

Abstract

Short term experiments were conducted with vegetative soybean plants (Glycine max L. Merr. ;Ransom' or ;Arksoy') to determine whether sourcesink manipulations, which rapidly changed the ;demand' for sucrose and partitioning of photosynthetically fixed carbon into starch, were associated with alterations in activities of sucrose-P synthase and/or cytoplasmic fructose-1,6-bisphosphatase in leaf extracts. When demand for sucrose from a particular source leaf was increased by defoliation of other source leaves, starch accumulation was restricted and activities of both enzymes were markedly enhanced. When demand for sucrose from source leaves was limited by excision, starch accumulation in the detached leaves was increased while activity of sucrose-P synthase declined sharply. The consistent responsiveness of sucrose-P synthase activity to changes in demand for sucrose supports the contention that regulation of sucrose-P synthase is an integral component of the system which controls sucrose biosynthesis and partitioning of carbon between starch and sucrose biosynthesis in the light.
Plant
Physiol.
(1983)
72,
474-480
0032-0889/83/72/0474/07/$00.50/0
Changes
in
Starch
Formation
and
Activities
of
Sucrose
Phosphate
Synthase
and
Cytoplasmic
Fructose-1,6-bisphosphatase
in
Response
to
Source-Sink
Alterations'
Received
for
publication
November
29,
1982
and
in
revised
form
February
7,
1983
THOMAS
W.
RUFTY,
JR.
AND
STEVEN
C.
HUBER
United
States
Department
of
Agriculture,
Agriculture
Research
Service,
and
Departments
of
Crop
Science
and
Botany,
North
Carolina
State
University,
Raleigh,
North
Carolina
27650
ABSTRACT
Short
term
experiments
were
conducted
with
vegetative
soybean
plants
(Glycine
mar
L.
Mewf.
'Ransom'
or
'Arksoy')
to
determine
whether
source-
sink
manipulations,
which
rapidly
changed
the
'demand'
for
sucrose
and
partitioning
of
photosynthetically
fixed
carbon
into
starch,
were
associated
with
alterations
in
activities
of
sucrose-P
synthase
and/or
cytoplasmic
fructose-1,64Iisphatase
in
leaf
extracts.
When
demand
for
sucrose
from
a
particular
source
leaf
was
increased
by
defoliation
of
other
source
leaves,
starch
accumulation
was
restricted
and
activities
of
both
enzymes
were
markedly
enhanced.
When
demand
for
sucrose
from
source
leaves
was
limited
by
excision,
starch
accumulation
in
the
detached
leaves
was
increased
while
activity
of
sucrose-P
synthase
declined
sharply.
The
con-
sistent
responsiveness
of
sucrose-P
synthase
activity
to
changes
in
demand
for
sucrose
supports
the
contention
that
regulation
of
sucrose-P
synthase
is
an
integral
component
of
the
system
which
controls
sucrose
biosynthesis
and
partitoning
of
carbon
between
starch
and
sucrose
biosynthesis
in
the
light.
A
conceptual
framework
has
been
developed
to
account
for
regulation
of
partitioning
of
photosynthetically
fixed
carbon
be-
tween
biosynthesis
of
starch
and
sucrose
in
leaves
(7,
12,
24,
32).
In
this
view,
an
important
determinant
of
carbon
partitioning
into
starch
may
be
the
rate
of
sucrose
formation
and
the
related
generation
of
Pi
in
the
cytosol.
Cytoplasmic
Pi
exchanges
rapidly
across
the
chloroplast
envelope
with
triose
phosphates,
and
the
rate
of
this
countertransport
process
relative
to
the
rate
of
CO2
fixation
determines
to
a
large
extent
the
degree
to
which
carbon
is
diverted
from
starch
biosynthesis.
The
rate
of
sucrose
biosynthesis
and
Pi
generation
could
be
controlled
by
the
activities
of
SPS2
and/or
cytoplasmic
FBPase,
which
are
apparently
low
compared
to
activities
of
other
enzymes
in
the
sucrose
biosynthetic
pathway
(5,
11).
In
particular,
the
activity
of
SPS
in
leaf
extracts
has
been
found
to
be
consistently
correlated
negatively
with
leaf
starch
content
in
comparisons
among
different
species
(14),
and
when
specific
genotypes
were
'Cooperative
investigations
of
the
United
States
Department
of
Agri-
culture,
Agricultural
Research
Service,
and
North
Carolina
Agricultural
Research
Service,
Raleigh,
NC.
This
is
Paper
8613 of
the
Journal
Series
of
the
North
Carolina
Agricultural
Research
Service,
Raleigh,
NC
27650.
2Abbreviations:
SPS,
sucrose
phosphate
synthase;
FBPase,
fructose-1,6-
bisphosphatase;
F6P,
fructose
6-phosphate;
UDPG,
uridine
5'-diphospho-
glucose;
CER,
net
CO2
exchange
rate.
exposed
to
a
variety
of
environmental
and
nutritional
conditions
(16).
Although
there
is
some
evidence
to
suggest
that
partitioning
of
carbon
between
biosynthesis
of
starch
and
sucrose
is
programmed
genetically
(15),
partitioning
apparently
can
be
altered
by
'source-
sink'
manipulation
within
the
plant.
In
experiments
where
por-
tions
of
the
source
(carbon
exporting)
leaves
in
the
canopy
were
excised
or
shaded,
it
has
been
observed
that
sucrose
content
and
export
out
of
remaining
source
leaves
were
increased
during
time
periods
in
which
photosynthetic
rates
remained
relatively
stable
(3,
6,
30,
31).
Tissue
starch
has
sometimes
been
measured
and
observed
to
be
decreased
(31).
These
results
imply
that
partitioning
of
available
photoassimilate
into
starch
declined
when
'demand'
for
sucrose
was
increased
(7).
In
other
experiments
where
the
'sink
demand'
for
sucrose
was
decreased
by
girdling
or
fruit
removal,
photosynthetic
rates
have
been
observed
to
decrease
while
starch
accumulation
either
in-
creased
or
was
equal
to
that
in
control
plants
(4,
10,
18,
21,
27,
28).
This
suggests
that
partitioning
of
available
photoassimilate
into
starch
was
increased.
Source-sink
manipulations
which
result
in
rapid
adjustments
in
demand
for
sucrose
and
partitioning
of
carbon
into
starch
could
be
associated
with
alterations
in
the
activities
of
SPS
and/or
FBPase.
The
present
experiments
were
designed
to
investigate
this
hypothesis.
In
a
series
of
short
term
experiments,
demand
for
sucrose
was
either
increased
by
defoliation
of
source
leaves
or
decreased
using
detached
source
leaves.
Alterations
in
SPS
activity
were
consistently
observed
which
corresponded
with
changes
in
demand
for
sucrose
and
were
reciprocal
to
changes
in
starch
accumulation.
MATERIALS
AND
METHODS3
The
study
consisted
of
three
experiments
to
investigate
the
relationship
between
adjustments
in
demand
for
sucrose,
accu-
mulation
of
starch,
and
activities
of
SPS
and
FBPase
in
leaf
extracts
of
vegetative
soybean
plants
(Glycine
max
L.
Merrill,
'Ransom'
or
'Arksoy').
Plant
culture
conditions
were
similar
in
each
experiment.
The
plants
were
grown
in
a
standard
soil
mix
in
a
greenhouse
from
April
to
July,
1982.
Plants
were
watered
as
necessary
in
the
morning
with
tap
water,
and
in
the
afternoon
of
alternate
days
with
approximately
600
ml
of
a
standard
nutrient
solution
which
contained
7.5
mm
N03
,
0.5
mm
H2PO4
,
3.0
mM
K+,
2.5
mm
Ca2+,
1.0
mM
Mg2+,
1.0
MM
s
42-,
and
trace
elements
3Mention
of
a
trademark
or
proprietary
product
does
not
constitute
a
guarantee
or
warranty
of
the
product
by
the
U.
S.
Department
of
Agri-
culture
and
does
not
imply
its
approval
to
the
exclusion
of
other
products
that
may
also
be
suitable.
474
SOURCE-SINK
ALTERATIONS
AND
SPS
0
0.5
1.0
TIME,
HOURS
1.5
2.0
0
0.5
1.0
1.5
2.0
TIME,
HOURS
25
L3
2C
T
s
IS
C,,
-)
D
10
D
),)
5
o
V
uV5
1.0
1.5
2.0
TIME,
HOURS
FIG.
1.
Changes
in
(A)
CER,
(B)
leaf
starch
accumulation,
and
(C)
leaf
sucrose
content
in
the
first
trifoliolate
of
Ransom
soybean
plants
following
defoliation
of
all
other
source
leaves.
The
first
trifoliolates
of
other,
intact
plants
were
used
as
controls.
glc,
glucose.
at
the
concentration
of
Hoagland
solution
(13).
Defoliation.
Twenty
Ransom
soybean
plants
were
grown
until
the
second
trifoliolate
had
expanded
fully.
At
this
growth
stage,
the
plants
were
growing
exponentially;
therefore,
requirements
for
sucrose
by
shoot
and
root
sinks
were
high.
At
10
AM
on
a
clear
day,
the
second
trifoliolate
and
primary
leaves
of
10
plants
were
excised,
leaving
the
first
trifoliolate
as
the
only
source
(carbon
exporting)
leaf.
At
this
time,
two
1.
1-cm
diameter
leaf
discs
were
removed
for
starch
analysis
from
the
first
trifoliolate
of
each
defoliated
plant
and
each of
the
10
intact,
control
plants.
The
discs
were
immediately
submerged
in
80%
ethanol
and
placed
in
a
freezer
at
-10°C.
The
experiment
lasted
2
h,
with
the
first
trifoliolate
of both
a
defoliated
and
a
control
plant
sampled
every
15
or
20
min
throughout.
At
each
sampling
time,
two
additional
leaf
discs
were
removed
as
before,
CER
was
measured
(see
below),
and
then
the
first
trifoliolate
was
excised,
weighed,
and
immedi-
ately
frozen
at
-80°C
for
later
enzyme
analysis.
Detached
Leaves.
In
the
initial
experiment,
Ransom
soybean
plants
were
grown
until
the
leaf
canopy
was
closed.
At
10
AM
on
a
clear
day,
six
fully
expanded
trifoliolates
were
randomly
selected
from
canopies
of
three
plants.
The
trifoliolates
were
excised
at
the
base
of
the
petiole
and
immediately
placed
into
500-ml
beakers
filled
with
distilled
H20,
where
the
petioles
were
re-cut
under
water.
A
leaf
was
harvested
just
after
excision
and
every
0.5
h
over
a
2.5-h
experimental
period;
the
petiole
was
removed,
fresh
weight
was
measured,
and
the
leaf
was
immediately
frozen
at
-80°C
for
later
analysis
of
enzymes.
Leaf
discs
were
taken
at
excision
and
prior
to
harvest
for
determination
of
starch
accu-
mulation,
and
CER
was
measured
at
the
time
of
harvest.
A
second
experiment
was
conducted
with
a
uniform
population
of 20
Arksoy
soybean
plants
which
had
nine
fully
expanded
trifoliolates
and
were
close
to
canopy
closure.
The
sixth
trifoliolate
475
50
40C
T
cm
~0
2C
C-)
Il
0
-1I
I
I
I
__
-
CONTROL
-
DEFOLIATED
l
I-
I
1
II
I
I
1
CONTROL
,
aw
O
DEFOLIATED
II
I
I
I
I
20
U
E
X
16
*2
i
e
C-
CP
12
X
0
8
<
en
LLI
4
Cf)
U,
0
II
-
C
NI
I
I
I
CON
ROL
a-
0
Er
_
I
0
o
0
'
_-DEFELIATED
0
I
I
I
_
rW
^
=.
I
I
I
I
I
il
4
Plant
Physiol.
Vol.
72,
1983
30
=
25
n
0.
y
20
L-
15
F-
V(I
0.
Cl)
0
Q5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0
24T
0
20
,-
4
_t
12
>:
8
CD
4
TIME,
HOURS
TIME,
HOURS
FIG.
2.
Activities
of
(A)
SPS
and
(B)
cytoplasmic
FBPase
in
extracts
of
the
first
trifoliolate
of
Ransom
soybean
plants
which
had
been
defoliated
of
all
other
source
leaves
and
of
the
first
trifoliolate
of
intact
(control)
plants.
was
excised
from
10
of
the
plants
and
re-cut
in
distilled
H20
as
before.
The
sixth
trifoliolate
of
the
remaining
10
plants
was
left
attached
and
used
as
a
control.
All
leaves
were
fully
exposed
to
light
throughout.
A
detached
and
a
control
leaf
were
harvested
as
described
previously
at
intervals
(specified
in
results)
over
a
4-h
experimental
period.
Photosynthesis
Measurements.
Photosynthetic
rates
were
meas-
ured
using
a
Beckman
model
865
differential
IR
CO2
analyzer
equipped
with
a
clamp-on
Plexiglas
cuvette
enclosing
the
upper
and
lower
surfaces
of
a
10-cm2
area
of
the
appropriate
leaf.
Air
at
the
same
temperature
and
CO2
concentration
of
the
ambient
air
was
passed
through
the
cuvette
for
30
to
45
s
at
a
flow
rate
of
1.5
I/min.
Differences
between
CO2
concentration
in
incoming
and
exhaust
air
streams
were
monitored
and
used
to
calculate
CER.
Leaves
always
were
fully
exposed
to
light.
Starch
Analysis.
Leaf
discs
were
extracted
with
hot
80%o
ethanol
until
the
tissue
was
pigment
free.
Particulates
including
starch
were
pelleted
by
centrifugation
and
then
suspended
in
1.0
ml
of
0.2
N
KOH
and
placed
in
boiling
water
for
30
min.
After
cooling,
the
pH
of
the
mixture
was
adjusted
to
about
pH
5.5
with
200
pl
of
1.0
N
acetic
acid.
To
each
sample,
1.0
ml
of
dialyzed
amyloglu-
cosidase
(from
Aspergillus
oryzae,
Sigma)
solution
(35
units/ml
in
50.0
mm
Na
acetate
buffer,
pH
4.5)
was
added,
and
the
tubes
were
incubated
at
55°C
for
30
min.
After
digestion,
the
tubes
were
placed
in
boiling
water
for
1
min
and
centrifuged,
and
the
glucose
in
the
supernatant
was
analyzed
enzymically
using
hexokinase
and
glucose-6-P
dehydrogenase
(17).
Enzyme
Extraction
and
Assays.
The
frozen
leaf
tissue
was
ground
with
a
Polytron
high
speed
homogenizer
in
a
grind
me-
dium
(8.0
ml
of
medium/g
fresh
weight)
containing
50.0
mm
Hepes-NaOH
(pH
7.5),
5.0
mM
MgCI2,
1.0
mm
EDTA,
2.5
mM
DTT,
2%
polyethylene
glycol
20
(w/v),
and
1%
BSA
(w/v).
The
brei
was
then
filtered
through
eight
layers
of
cheesecloth,
and
cells
were
disrupted
by
passage
through
a
French
pressure
cell
(330
kg!
cm2).
Debris
was
pelleted
by
centrifugation
at
38,000g
for
10
mi
and
enzyme
assays
were
conducted
on
the
supernatant
fluid.
Sucrose-P
synthase
was
assayed
by
measurement
of
fructose-6-
P-dependent
formation
of
sucrose
(+sucrose-P)
from
UDP-glu-
cose
(2).
The
assay
mixture
(70
pl)
contained
7.5
mm
UDP-glucose,
7.5
mm
fructose-6-P,
5
mM
MgCl2,
50.0
mm
Hepes-NaOH
(pH
7.5),
and
an
aliquot
of
leaf
extract.
The
assay
mixture
for
sucrose
synthase
was
the
same,
except
that
fructose
replaced
fructose-6-P.
Mixtures
were
incubated
at
25°C
for
10
min,
and
reactions
were
terminated
by
addition
of
70
pL1
1.0
N
NaOH.
Unreacted
fructose-
6-P
(or
fructose)
was
destroyed
by
placing
the
tubes
in
boiling
water
for
10
min.
After
cooling,
0.25
ml
of
0.1%
(v/v)
resorcinol
in
95%
ethanol
and
0.75
ml
of
30%Yo
HCI
were
added,
and
the
tubes
were
incubated
at
80°C
for
8
min
(25).
The
tubes
were
allowed
to
cool
and
were
centrifuged
at
l,500g
for 5
min,
and
the
A520
was
measured.
Cytoplasmic
FBPase
was
assayed
by
a
continuous
spectropho-
tometric
assay.
The
1.0-ml
reaction
mixture
contained
50
mM
Hepes-NaOH
(pH
7.0),
5
mM
MgCl2,
100
,UM
FBP,
0.2
mM
NADP,
2
units
each
of
phosphoglucoisomerase
and
glucose-6-P
dehydro-
genase,
1
unit
of
phosphogluconate
dehydrogenase,
and
an
aliquot
of
leaf
extract
(under
these
conditions,
only
the
cytoplasmic
FBPase
is
active;
Ref.
11).
Estimates
of
Mg2+-independent
FBPase
activity
were
obtained
by
conducting
the
assay
in
the
presence
of
10
mm
EDTA
(D.
M.
Pharr
and
S.
C.
Huber,
submitted).
The
tissue
extract
also
was
used
for
hexose
and
sucrose
deter-
minations.
Following
centrifugation,
an
aliquot
of
the
extract
was
removed,
diluted
with
redistilled
H20,
boiled
for
1
min,
and
analyzed
enzymically
(17).
RESULTS
Defoliation
of
Source
Leaves.
Defoliation
of
all
source
leaves
except
the
first
trifoliolate
had
little
discernible
effect
on
the
photosynthetic
rate
of
this
remaining
source
leaf
(Fig.
IA),
but
markedly
affected
accumulation
of
leaf
starch
(Fig.
1B).
While
the
first
leaf
of
control
plants
accumulated
starch
continually,
accu-
mulation
in
the
same,
source
leaf
of
defoliated
plants
was
severely
restricted.
Defoliation
and
the
related
decrease
in
accumulation
of
starch
apparently
were
associated
with
increased
formation
and
export
of
sucrose.
As
noted
previously,
in
similar
defoliation
studies
which
employed
14C
labeling,
increased
formation
and
export
of
sucrose
have
been
observed
consistently
(3,
6,
30,
31).
In
the
present
experiment,
after
an
initial
decline,
the
tissue
sucrose
content
was
increased
in
the
second
h
after
defoliation
and
stabi-
lized
at
a
level
which
exceeded
the
sucrose
content
in
leaves
of
-
1I
I
I
I-
0~~~~~~~
DEfOLIATED
_s
°
-e
3
.10~~
I
Io
I
;;:;7CONRO
0
I
I
I
I
I
DEFOUATED
I
_ m
I
I
-
O.
°po
CONTROL
476
RUFTY
AND
HUBER
SOURCE-SINK
ALTERATIONS
AND
SPS
I1
I
I
AECR
I
I
I
0
1
2
HOURS
AFTER
EXCISION
1
E
T
30
0
._
cr
0
20
-0'a
E
_
10
cr
w
cn
3
p
_
3c
e._
0I
oF
=
100
x
I
0
0
80
-5
E
,:
60
-J
40
0
1
2
3
HOURS
AFTER
EXCISION
FIG.
3.
Changes
in
(A)
CER
and
leaf
starch
content,
and
(B)
leaf
sugar
content
in
mature
trifoliolates
from
Ransom
soybean
plants
following
excision.
glc,
glucose.
C-)
C,)
a-
(I)
.-30
o
20
a.
0
E
30
-
20
co'
"I..-
0,
10
Cs
Xa.
b
EE
o
.
0
1
2
3
0
1
2
3
HOURS
AFTER
EXCISION
HOURS
AFTER
EXCISION
FIG.
4.
Activities
of
(A)
SPS
and
(B)
cytoplasmic
FBPase
in
leaf
extracts
from
mature
trifoliolates
from
Ransom
soybean
plants
following
excision.
control
plants
by
12
to
20%o
(Fig.
lC).
The
leaf
sucrose
content
generally
has
been
found
to
be
closely
related
with
the
steady
state
rate
of
sucrose
formation
and
export
out
of
source
leaves
(9);
therefore,
the
response
pattern
observed
is
consistent
with
in-
creased
sucrose
biosynthesis
and
equilibration
of
the
transient
sucrose
pools
in
the
leaf
with
the
enhanced
rate
of
sucrose
export.
The
measured
decrease
in
net
synthesis
of
starch
and
presumed
increase
in
sucrose
formation
and
export
in
defoliated
plants
was
associated
with
markedly
increased
activities
of both
SPS
(Fig.
2A)
and
cytoplasmic
FBPase
(Fig.
2B)
in
leaf
extracts.
Increased
enzyme
activities
relative
to
control
plants
were
apparent
within
the
first
h
after
defoliation
and
were
maximized
at
the
end
of
2
h.
The
stimulation
of
SPS
activity
ranged
from
50
to
77%
relative
to
activity
in
leaves
of
control
plants
during
the
second
h
of
the
experiment,
whereas
total
activity
of
FBPase
was
enhanced
33
to
45%.
Crude
soybean
leaf
extracts
contain
both
Mg2e-dependent
and
independent
FBPase
activities
(Pharr
and
Huber,
submitted).
The
Mg2+-dependent
enzyme
is
thought
to
be
involved
in
sucrose
biosynthesis
in
the
cytoplasm.
The
FBPase
activities
in
Figure
2B
represent
the
summation
of
Mg2e-dependent
and
independent
activities.
Both
were
increased
similarly
by
defoliation
(data
not
shown).
Decreased
partitioning
of
carbon
into
starch
and
increased
activities
of
SPS
and
FBPase
have
been
observed
in
a
number
of
other,
similar
defoliation
experiments
with
vegetative
soybean
plants
(data
not
shown).
In
four
experiments,
the
maximum
enhancement
of
SPS
activities
during
the
2-h
time
interval
after
defoliation
was
74
8%
relative
to
activities
of
controls.
Maximum
enhancement
of
FBPase
activities
has
been
more
variable,
ranging
from
5
to
45%
relative
to
controls,
with
a
mean
enhancement
of
24
±
20%o.
In
each
experiment,
increases
in
SPS
activities
were
substantially
larger
than
increases
in
FBPase
activities.
Detached
Leaves.
Two
additional
experiments
were
conducted
in
which
source
leaves
were
detached
from
the
plant
in
order
to
severely
limit
the
demand
for
sucrose,
and
thus
increase
partition-
ing
of
photosynthate
into
starch.
It
has
been
reported
previously
that
detached
leaves
accumulate
considerable
amounts
of
starch
(20).
In
an
initial
experiment,
the
photosynthetic
rate
of
detached
source
leaves
remained
stable
for
about
1
h
after
excision
and
then
declined