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Photosynthesis by sugar-cane leaves. A new carboxylation reaction and the pathway of sugar formation

Authors:

Abstract

1. Radioactive products in detached leaf segments were examined after periods of steady-state photosynthesis in (14)CO(2). 2. After exposure to (14)CO(2) for approx. 1sec. more than 93% of the fixed radioactivity was located in malate, aspartate and oxaloacetate. After longer periods large proportions of the radioactivity appeared in 3-phosphoglycerate, hexose monophosphates and sucrose. Similar results were obtained with leaves still attached to the plant. 3. Radioactivity appeared first in C-4 of the dicarboxylic acids and C-1 of 3-phosphoglycerate. The labelling pattern in hexoses was consistent with their formation from 3-phosphoglycerate. 4. The reaction giving rise to C(4) dicarboxylic acid appears to be the only quantitatively significant carboxylation reaction. 5. Evidence is provided that the radioactivity incorporated into the C(4) dicarboxylic acid pool is transferred to sugars via 3-phosphoglycerate. A scheme is proposed to account for these observations.
Biochem.
J.
(1966)
101,
103
Photosynthesis
by
Sugar-cane
Leaves
A
NEW
CARBOXYLATION
REACTION
AND
THE
PATHWAY
OF
SUGAR
FORMATION
By
M.
D.
HATCH
AND
C.
R.
SLACK
David
North
Plant
Re8earch
Centre,
The
Colonial
Sugar
Refining
Co.
Ltd.,
Indooroopilly,
Queen8land,
Au8tralia
(Received
7
March
1966)
1.
Radioactive
products
in
detached
leaf
segments
were
examined
after
periods
of
steady-state
photosynthesis
in
14CO2.
2.
After
exposure
to
14CO2
for
approx.
1
sec.
more
than
93
%
of
the
fixed
radioactivity
was
located
in
malate,
aspartate
and
oxaloacetate.
After
longer
periods
large
proportions
of
the
radioactivity
appeared
in
3-phosphoglycerate,
hexose
monophosphates
and
sucrose.
Similar
results
were
obtained
with
leaves
still
attached
to
the
plant.
3.
Radioactivity
appeared
first
in
C-4
of
the
dicarboxylic
acids
and
C-i
of
3-phosphoglycerate.
The
labelling
patternin
hexoses
was
consistent
with
their
formation
from
3-phosphoglycerate.
4.
The
reaction
giving
rise
to
C4
dicarboxylic
acid
appears
to
be
the
only
quantitatively
significant
carboxylation
reaction.
5.
Evidence
is
provided
that
the
radioactivity
incorporated
into
the
C4
dicarboxylic
acid
pool
is
transferred
to
sugars
via
3-
phosphoglycerate.
A
scheme
is
proposed
to
account
for
these
observations.
Several
observations
which
appear
to
be
incon-
sistent
with
the
scheme
for
photosynthesis
proposed
by
Calvin
and
co-workers
(Calvin
&
Bassham,
1962)
have
been
considered
in
recent
reviews
by
Stiller
(1962)
and
Bassham
(1964).
The
low
activity
of
ribulose
1,5-diphosphate
carboxylase
in
some
tissues
relative
to
the
rate
of
photosynthesis,
and
the
high
concentrations
of
bicarbonate
required
by
the
enzyme
for
maximum
activity, raise
doubts
about
its
quantitative
importance
in
photosynthetic
fixa-
tion
of
carbon
dioxide
(Racker,
1957;
Peterkofsky
&
Racker,
1961;
Stiller,
1962).
Recently
Kortschak,
Hartt,
&
Burr
(1965)
reported
that
malate
and
aspartate
were
the
major
labelled
products
formed
in
sugar-cane
leaves
during
short
periods
of
photo-
synthesis
in
14CO2.
The
present
studies
confirm
this
observation
with
another
sugar-cane
variety
and
provide
information
about
the
nature
of
the
primary
carboxylation
reaction
and
the
pathway
of
sugar
formation.
MATERIALS
3-Phosphoglyceric
acid
(tricyclohexylammonium
salt)
and
wheat-germ
acid
phosphatase
were
purchased
from
Sigma
Chemical
Co.
(St
Louis,
Mo.,
U.S.A.).
Yeast
invertase
was
obtained
from
Difco
Laboratories
(Detroit,
Mich.,
U.S.A.).
Ba14C03
(26.2mc/m-mole),
L-[U-14C]malic
acid
(15lImc/m-mole)
and
L-[U-14C]aspartic
acid
(6.1mc/m-
mole)
were
obtained
from
The
Radiochemical
Centre
(Amersham,
Bucks.).
Leaf
tissue.
Field-grown
sugar
cane
(hybrid
variety,
Pindar)
was
used.
Sections
(approx.
18cm.
long,
15cm.
wide)
containing
no
midrib
tissue
were
cut
from
the
youngest
fully
expanded
leaf.
All cuts
were
made
under
water
to
prevent
the
entry
of
air
into
the
vascular
tissue.
METHODS
Photosynthesi8
in
14CO2.
A
rectangular
Perspex
box
(19
cm.
x
16cm.
x
9
cm.),
sufficiently
large
to
accommodate
six
leaf
segments,
served
as
a
photosynthesis
chamber.
The
top
of
the
chamber
was
detachable
and
split
in
half
lengthwise.
Leaf
segments
were
clamped
vertically
between
the
halves
with
about
1
cm.
protruding
from
the
chamber
and
the
bases
of
the
segments
dipping
into
water.
Soft
rubber
on
the
adjoining
edges
of
the
top
provided
a
seal
around
the
leaf
segments
but
allowed
them
to
be
with-
drawn
from
the
chamber.
Air,
saturated
with
water
vapour,
was
pumped
through
the
chamber
and
the
air
in
the
cham-
ber
was
circulated
by
a
high-speed
fan.
Before
the
addition
of
14(02
the
segments
were
illuminated
for
at
least
45min.
by
a
400w
Phillips
HPL
lamp.
The
light-intensity
at
the
leaf
surface,
measured
with
a
selenium
cell,
was
8200ft.-
candles.
From
a
calibration
curve
of
the
selenium
cell
against
a
Kipp
thermopile
this
intensity
was
equivalent
to
0.39cal./cm.2/min.
Immediately
before
the
introduction
of
14CO2
the
air
supply
to
the
chamber
was
removed
and
exit
holes
were
sealed
with
tape;
14C02
(0-86mc)
was
injected
from
a
syringe
bringing
the
CO2
concentration
to
0.055%.
At
intervals
individual
leaf
segments
were
transferred
to
50ml.
of
boiling
80%
(v/v)
ethanol
and
kept
at
this
temperature
for
3min.
In
certain
experiments
the
segments
were
trans-
ferred
to
methanol-chloroform-2
0m-formic
acid
(12:5:3,
by
vol.)
at
-80°,
then
held
at
-15°
for
24hr.
(Bieleski
&
Young,
1963).
A
second
procedure
allowed
both
shorter
treatments
with
103
M.
D.
HATCH
AND
C.
R.
SLACK
14CO2
and
the
killing
of
leaves
while
still
exposed
to
14CO2
and
light.
After
pre-illumination
in
the
chamber
leaf
segments
were
placed
in
a
large
test
tube
(vol.
60ml.).
14CO2
(45,tc)
was
injected
and
then
boiling
80%
(v/v)
ethanol
was
poured
into
the
tube
either
simultaneously
or
after
1-2sec.
Extraction
of
tissue.
Segments
killed
in
the
methanol-
chloroform-formic
acid
mixture
were
extracted
as
described
by
Bieleski
&
Young
(1963).
Segments
killed
in
boiling
ethanol
were
homogenized
and
the
insoluble
material
was
re-extracted
in
sequence
with
20ml.
of
boiling
80%
(v/v)
ethanol,
and
twice
with
15ml.
of
boiling
water.
Water
and
ethanol
extracts
were
combined.
The
remaining
insoluble
material
was
washed
with
a
large
volume
of
water
followed
by
ethanol
and
then
dried
at
70°
to
a
constant
weight.
Here-
after
this
material
will
be
referred
to
as
'residue'.
Radio-
activity
incorporated
into
segments
is
expressed
as
counts/
min./lOOmg.
of
the
residue.
Counting
procedures.
Samples
of
0-
ml.
or
less
of
the
combined
extracts
containing
at
least
1000
counts/min.
were
counted
in
a
liquid-scintillating
counter
(model
N664A;
Ekeo
Electronics).
A
toluene-phosphor-ethanol
solution
(Ziegler,
Chleck
&
Brinkerhoff,
1957)
was
used
for
these
aqueous
samples.
The
counting
efficiency
was
55%
and
quenching
was
not
significant.
Weighed
samples
(approx.
10mg.)
of
the
dried
residue
were
counted
in
steel
planchets
with
a
Geiger-Muller
tube.
In
the
range
used
counts
were
proportional
to
the
weight
of
the
sample.
The
counting
efficiency
of
this
method
was
determined
by
liquid-scintillation
counting
of
similar
residue
samples
suspended
in
toluene
containing
phosphor
and
2%
(w/v)
thixin.
Identification
and
estimation
of
radioactivity
in
individual
compounds
Chromatography
8olvents
and
counting
procedure.
For
identifying
individual
components
and
determining
the
amount
of
the
radioactivity
in
each
compound
samples
of
extracts
were
chromatographed
on
paper
with
one
or
more
of
the
following
solvent
systems:
A,
butan-l-ol-propionic
acid-water
(10:5:7,
by
vol.)
(Benson
et
al.
1950);
B,
pentan-l-ol
saturated
with
5M-formic
acid
(Aronoff,
1956,
p.
122);
C,
propan-l-ol-aq.
NH3
(sp.gr.
0.90)-water
(6:3:1,
by
vol.)
(Bieleski
&
Young,
1963);
D,
ethyl
acetate-
pyridine-water
(8:2:1,
by
vol.)
(White
&
Secor,
1953);
E,
phenol
saturated
with
water
(Benson
et
al.
1950).
For
subsequent
references
to
these
solvents
the
letter
prefixing
each
will
be
used.
Whatman
no.
1
paper
was
used
and
for
solvents
A,
C
and
E
the
paper
was
previously
washed
with
oxalic
acid.
The
location
and
the
amount
of
radioactivity
in
individual
compounds
or
groups
of
compounds
was
determined
with
a
chromatogram
strip-counter
and
the
area
of
individual
peaks
measured
with
a
planimeter.
When
quantities
of
individual
compounds
were
required
samples
of
extracts
were
chromatographed
as
a
band
and
eluted
from
developed
chromatograms.
Marker
compounds
on
chromatograms
were
detected
as
follows:
organic
acids
and
amino
acids
with
ninhydrin
(Aronoff,
1956,
p.
120),
phosphorylated
compounds
with
ammonium
molybdate
(Bandurski
&
Axelrod,
1951)
and
sugars
with
p-anisidine.
Malic
acid.
When
extracts
were
chromatographed
in
solvents
A
and
B
a
peak
was
obtained
which
co-chromato-
graphed
with
L-malate.
With
solvent
B
this
was
the
only
radioactive
compound
which
moved
a
significant
distance
from
the
origin.
The
radioactivity
eluted
from
chromato-
grams
developed
in
solvent
B
co-chromatographed
with
L-malate
in
solvents
A,
C
and
E.
When
samples
of
this
compound
were
treated
with
a
fumarate
hydratase
prepara-
tion
from
wheat-germ
18-20%
of
the
radioactivity
was
located
in
fumaric
acid
at
equilibrium.
Similar
values
were
obtained
with
L-[U-14C]malate.
Aspartic
acid.
A
compound
which
ran
as
a
separate
peak
corresponding
to
L-aspartate
was
detected
with
solvent
A.
The
eluted
radioactivity
co-chromatographed
with
L-
aspartate
in
solvents
B
and
E,
and
the
compound
was
completely
degraded
by
treatment
with
ninhydrin
(Greenberg
&
Rothstein,
1957).
Phosphorylated
compounds.
With
solvent
A
a
separate
area
of
radioactivity
was
obtained
which
corresponded
in
mobility
to
3-phosphoglycerate
and
hexose
monophos-
phates.
Occasionally
a
very
small separate
peak
was
observed
with
a
mobility
corresponding
to
fructose
1,6-
diphosphate
and
ribulose
1,5-diphosphate.
When
the
major
peak
was
eluted
and
co-chromatographed
with
marker
compounds
in
solvent
C
the
radioactivity
corresponded
with
3-phosphoglycerate,
glucose
6-phosphate
and
fructose
6-phosphate.
After
treatment
with
acid
phosphatase
all
the
radioactivity
was
recovered
in
com-
pounds
which
co-chromatographed
with
glyceric
acid,
glucose
and
fructose
in
solvents
B,
C
and
D.
In
some
experiments
traces
of
glyceric
acid
were
detected
when
the
original
extracts
were
chromatographed
in
solvents
A
and
B.
Sucrose.
After
longer
periods
of
exposure
to
14C02
a
labelled
compound
appeared
which
chromatographed
with
sucrose
in
solvent
A.
This
compound
co-chromatographed
with
sucrose
in
solvent
D
and
after
treatment
with
invertase
the
radioactivity
was
located
equally
between
glucose
and
fructose.
Proportions
of
radioactivity
in
individual
compounds.
The
proportion
of
the
total
radioactivity
in
malate
was
calcu-
lated
from
chromatograms
developed
in
solvent
B
and
checked
with
solvent
A
and
the
proportions
of
aspartate
and
sucrose
were
obtained
with
solvent
A.
After
determining
the
proportion
for
the
total
phosphorylated
compounds
with
solvent
A
the
amount
of
radioactivity
in
3-phospho-
glycerate
and
glucose
6-phosphate
plus
fructose
6-phosphate
was
calculated
from
chromatograms
run
in
solvents
B
or
D
after
treatment
of
this
fraction
with
phosphatase.
Oxaloacetic
acid.
With
the
standard
procedure
employed
radioactive
oxaloacetate
was
not
detected.
Hence
the
following
procedure
was
used.
Leaves
were
exposed
to
14CO2
in
a
test
tube
as
previously
described
and
after
approx.
1
sec.
photosynthesis
was
stopped
by
pouring
one
of
the
following
solutions
into
the
tube:
(1)
80%
(v/v)
ethanol
containing
10mg.
of
2,4-dinitrophenylhydrazine
and
0-2N-HCI
at
-
80°,
(2)
the
same
solution
at
820,
(3)
the
same
solution
at
820
but
without
2,4-dinitrophenylhydrazine.
The
subsequent
procedure
was
the
same
as
already
described
except
that
the
concentrated
extracts
were
extracted
with
chloroform
to
obtain
phenylhydrazones
(Aronoff,
1956,
p.
133).
The
radioactivity
in
the
chloroform
extract
was
determined
and
samples
were
chromatographed
with
authentic
oxaloacetate
2,4-dinitrophenylhydrazone
in
the
following
solvents:
butan-l-ol-water-ethanol
(5:4:1,
by
vol.),
butan-l-ol
saturated
with
M-NaHCO3,
0-1
M-potassium
104 1966
PHOTOSYNTHESIS
BY
SUGAR-CANE
LEAVES
glycinate,
pH8-4,
and
butan-1-ol-ethanol-0-5N-NH3
(7:1:2,
by
vol.)
described
by
Block,
Durram
&
Zweig
(1958).
With
the
chloroform
extract
from
the
extract
of
leaf
killed
at
-
80°
at
least
90%
of
the
radioactivity
co-chromato-
graphed
with
the
marker
in
all
solvents.
When
a
portion
of
the
chloroform
extract
was
mixed
with
authentic
2,4-
dinitrophenylhydrazone
of
oxaloacetate
and
recrystallized
twice
the
specific
activity
of
the
derivative
remained
constant.
Insoluble
compound8.
The
insoluble
residue
was
treated
with
72%
(v/v)
H2SO4
to
hydrolyse
cellulose
(Jermyn,
1955).
Essentially
all
the
radioactivity
was
recovered
in
a
com-
pound
which
co-chromatographed
with
glucose
in
solvents
C
and
D.
When
the
material
was
hydrolysed
with
6N-HCI
(Pirie,
1955)
no
radioactivity
was
detected
in
amino
acids.
Degradation
of
labelled
product8
Malic
acid.
Radioactivity
in
the
C-1
plus
C-4
and
the
C-2
plus
C-3
of
malate
was
determined
by
MnO2
oxidation
(Friedemann
&
Kendall,
1928).
Samples
of
the
C02
and
acetaldehyde,
trapped
in
sodium
hydroxide
and
sodium
bisulphite
respectively,
were
counted
in
the
liquid-
scintillation
counter
by
using
the
procedure
described
for
aqueous
samples.
The
C-i
of
malate
was
determined
by
a
modification
of
the
Von
Peckmann
reaction
described
by
Racusen
&
Aronoff
(1953).
Trials
with
L-[U-14C]malate
established
the
exact
conditions
for
the
complete
release
of
the
C-1
as
CO.
Samples
of
malate
containing
at
least
35,000
disintegrations/min.
were
dried
in
a
combustion
flask
with
L-malate
carrier
and
heated
at
800
for
45min.
with
5ml.
of
100%
H2SO4
in
the
combustion
apparatus
for
a
Nuclear-Chicago
Dynacon
electrometer.
The
CO
released
was
then
flushed
into
a
250ml.
ion
chamber
and
counted.
Similar
samples
were
subjected
to
combustion
with
Van
Slyke
reagent
to
determine
the
total
radioactivity.
Aspartic
acid.
Radioactivity
in
the
C-1
plus
C-4
and
the
C-2
plus
C-3
of
aspartate
was
determined
by
treatment
with
ninhydrin
(Greenberg
&
Rothstein,
1957).
Glyceric
acid.
Glyceric
acid
was
isolated
from
3-phospho-
glycerate
by
chromatography
in
solvent
B
after
treatment
with
acid
phosphatase.
The
glycerate
was
decarboxylated
by
oxidation
with
ceric
sulphate
(Zelitch,
1965).
Radio-
activity
in
the
C02,
trapped
in
N-NaOH,
and
in
the
residual
acetic
acid
was
determined
with
a
liquid-scintillation
counter.
Hexo8ee.
After
treatment
of
sucrose
with
invertase
and
hexose
monophosphates
with
acid
phosphatase
the
resulting
glucose
and
fructose
were
isolated
from
chromatograms
developed
in
solvent
D
and
converted
into
their
common
osazone.
This
derivative
was
degraded
to
give
the
C-1,
C-2
and
C-3
as
the
mesoxaldehyde
osazone,
the
C-4
and
C-5
as
formic
acid
and
the
C-6
as
formaldehyde
(Aronoff,
1956,
p.
110).
RESULTS
IncorporaWon
of
radioactivity
from
14CO2
a8
a
function
of
time.
Experimental
conditions
were
chosen
to
produce
as
nearly
as
possible
a
steady
state
of
photosynthesis
under
physiological
condi-
tions
of
concentration
of
carbon
dioxide
and
light-
intensity.
Studies
by
K.
T.
Glasziou
&
J.
C.
Waldron
in
our
Laboratory
have
shown
that
the
rate
of
photosynthesis
of
leaf
segments,
prepared
as
described
in
the
Methods
section,
is
low
when
first
exposed
to
light.
The
rate
increases
to
a
steady
value
after
about
30min.
and
is
maintained
for
several
hours.
In
our
experiments
leaves
were
exposed
to
light
in
a
humidified
stream
of
air
for
at
least
45min.
before
the
addition
of
14CO2.
The
intensity
of
visible
light
at
the
leaf
surface
was
approx.
40%
of
that
in
direct
sunlight.
The
total
fixation
of
14C0O2
(Fig.
1)
and
the
radio-
activity
in
individual
compounds
(Figs.
2
and
3)
were
determined
for
leaves
exposed
to
14CO2
for
periods
of
up
to
150sec.
Over
this
period
the
rate
of
14CO2
fixation
remained
linear.
The
changing
proportions
of
radioactivity
in
individual
com-
pounds
is
shown
in
Fig.
2.
Three
trends
are
apparent,
the
continued
fall
of
the
first-labelled
products,
malate
and
aspartate,
the
rise
then
fall
of
3-phosphoglycerate
and
hexose
monophosphates,
and
the
steady
increase
of
the
percentage
of
the
total
radioactivity
in
the
end
products
sucrose
and
a
glucan.
The
plot
of
total
radioactivity
in
individual
compounds
(Fig.
3)
shows
the
asymptotic
trend
of
the
early
products
and
intermediates
and
the
exponential
rise
in
the
radioactivity
associated
with
end
products.
When
a
similar
experiment
was
carried
out
in
the
dark
the
rate
of
14CO2
fixation
was
only
0-4%
of
that
in
the
light.
Even
after
5min.
in
14C02
radioactivity
was
located
almost
entirely
in
aspartate
and
malate
whereas
no
radio-
activity
was
detected
in
3-phosphoglycerate
or
hexose
monophosphates.
Our
conditions
of
leaf
pretreatment
and
light-intensity
differed
from
those
used
by
Kortschak
et
al.
(1965).
Hlowever,
our
45r
5)
0
0
.0
:
0
50
100
Time
(sec.)
150
Fig.
1.
Total
14C
incorporated
into
leaf
segments
as
a
function
of
time.
The
radioactivity
in
the
residue
is
included
and
the
counts
are
for
a
55%
counting
efficiency.
Other
details
are
described
in
the
Methods
section.
Bioch.
1966,
101
Vol.
101
105
M.
D.
HATCH
AND
C.
R.
SLACK
results
with
the
variety
Pindar
were
qualitatively
similar
to
those
obtained
by
these
workers
with
the
variety
H37-1933.
The
effect
of
different
killing
procedures
on
the
distribution
of
radioactivity
in
individual
com-
pounds
was
examined
(Table
1).
Results
were
very
similar
when
leaves
were
killed
either
in
boiling
80%
(v/v)
ethanol
or
in
the
methanol-chloroform-formic
acid
mixture
at
-80°.
In
these
experiments
leaf
segments
were
transferred
from
the
chamber
to
the
killing
mixture.
To
determine
if
any
modification
of
the
labelling
pattern
occurred
during
transfer,
leaf
segments
were
exposed
to
14CO2
in
test
tubes
and
0
i
0
.4
0
~io
0
S
~
44.
Q.
I-
10
4-
o
1
--
to
--
-4)o
P-4
Time
(sec.)
Fig.
2.
Proportion
of
the
total
radioactivity
in
individual
compounds
for
periods
in
14CO2
up
to
15Osec.
Data
are
from
the
same
experiment
as
described
in
Fig.
1.
Details
of
procedures
for
indentifying
and
counting
individual
compounds
are
described
in
the
Methods
section.
0,
Malate+aspartate;
*,
3-phosphoglycerate;
A,
hexose
monophosphates;
A,
sucrose;
*,
glucan.
0
20 40 60
80
Time
(sec.)
Fig.
3.
Time-course
for
the
total
radioactivity
incorporated
into
individual
compounds.
Data
are
from
the
same
experi-
ment
as
described
in
Figs.
1
and
2.
0,
Malate+
aspartate;
Cl,
malate;
*,
3-phosphoglycerate;
A,
hexose
mono-
phosphates;
A,
sucrose;
U,
glucan.
Table
1.
Effect
of
different
killing
procedure8
on
the
diteribution
of
radioactivity
in
individual
compound8
In
Expt.
1
leaf
segments
were
supplied
with
14CO2
in
the
chamber,
and
at
the
times
shown
segments
were
removed
and
placed
in
boiling
80%
(v/v)
ethanol
or
methanol-chloroform-formic
acid
(MCF)
at
-80.
In
Expt.
2
leaves
were
supplied
with
14CO2
in
large
test-tubes
and
after
4
sec.
one
was
killed
by
adding
boiling
80
%
(v/v)
ethanol
and
the
other
was
transferred
to
another
tube
containing
the
same
mixture.
Other
details
are
described
in
the
Methods
section.
Expt.
Time
in
no.
14CO2
(sec.)
1
30
30
60
60
10-6
x
Total
14C
incorporated
(counts/min./
Killing
100mg.
of
residue)
method
8-9
Ethanol
10.1
MCF
19-9
Ethanol
19-3
MCF
Percentage
of
total
14C
in
individual
compounds
3-Phospho-
Hexose
mono-
Malate
Aspartate
glycerate
phosphates
Sucrose
Glucan
30
15
36
14-5
2-5
1-0
32
16
34
15
2-0
1-0
26
8-5
27
25
8-5
4-0
27
10
30
23
6-0
4.5
2-6
Ethanol
added
to
tube
2-3
Leaf
removed
t
ethanool
53
25
56
25
15
14
7
00
5
0
0
106
1966
2
4
4
PHOTOSYNTHESIS
BY
SUGAR-CANE
LEAVES
then
killed
either
by
the
direct
addition
of
boiling
80%
(v/v)
ethanol
or
by
transferring
them
to
the
same
mixture.
The
two
procedures
gave
almost
identical
results
(Table
1).
To
confirm
that
the
results
obtained
were
not
due
to
an
alteration
of
the
normal
photosynthetic
process
caused
by
detaching
and
cutting
the
leaves
experiments
were
conducted
with
leaves
on
the
plant.
The
apical
25
cm.
of
the
youngest
fully-
expanded
leaf
of
plants
was
exposed
to
14CO2
in
a
sealed
chamber
or,
for
short-term
experiments,
an
oO
0
0
0
x
IS
P-
0
10
20
30
40
50
60
'
200
Time
(sec.)
Fig.
4.
Changes
in
the
distribution
of
radioactivity
after
transfer
of
leaf
segments
from
14CO2
to
carbon
dioxide.
After
15sec.
in
14CO2
leaf
segments
were
removed
simul-
taneously
from
the
chamber
to
a
stream
of
air
without
changing
the
distance
from
the
light
source.
At
intervals
individual
segments
were
killed
and
analysed
as
described
in
the
Methods
section.
0,
Malate+
aspartate;
o],
malate;
*,
3-phosphoglycerate;
A,
hexose
monophosphates;
A,
sucrose;
*,
glucan;
+,
total
radioactivity.
open
test
tube
(see
the
Methods
section).
A
time-
course
study
over
a
period
from
4sec.
to
90sec.
in
14CO2
gave
results
which
were
almost
identical
with
those
obtained
with
leaf
sections.
When
an
attached
leaf
was
exposed
to
14CO2
for
2sec.
and
killed
by
the
direct
addition
of
boiling
80%
(v/v)
ethanol
the
proportions
of
the
incorporated
radio-
activity
in
malate,
aspartate,
3-phosphoglycerate
and
hexose
monophosphates
were
54%,
37%,
7%
and
2%
respectively.
We
established
that
oxaloacetate
would
be
completely
destroyed
during
our
isolation
and
chromatography
procedures.
Hence
attempts
were
made
to
identify
radioactive
oxaloacetate
in
leaf
extracts
as
its
2,4-dinitrophenylhydrazone.
Only
killing
at
-
80°
in
the
presence
of
the
reagent
gave
reproducible
results
(Table
2).
A
much
smaller
proportion
of
the
total
radioactivity
was
recovered
in
the
derivative
when
leaves
were
killed
in
boiling
80%
(v/v)
ethanol
containing
2,4-dinitrophenyl-
hydrazine.
Table
2.
Radioactivity
in
oxaloacetate
after
different
killing
procedures
Leaves
were
exposed
to
14CO2
for
approx.
1
sec.
and
then
killed
either
at
820
or
-80Q
in
the
presence
of
2,4-dinitro-
phenylhydrazine
or
without
the
reagent
(see
the
Methods
section).
Oxaloacetate
was
isolated
and
counted
as
its
2,4-dinitrophenylhydrazone.
Percentage
of
total
14C
Compound
Malate
Aspartate
Oxaloacetate
3-Phosphoglycerate
Other
compounds
With
reagent
at
-80°
54
36
3.4
5-2
3*0
With
reagent
at
820
57
32
0-6
8-0
2-4
Without'
reagent
at
820
47
42
0
6-0
5-0
Table
3.
Changes
in
the
distribution
of
radioactivity
in
leaf
segments
after
transfer
from
14C02
in
the
light
to
carbon
dioxide
in
the
dark
Illuminated
segments
were
exposed
to
14CO2
for
9sec.
At
9sec.
the
chamber
was
darkened
with
a
shutter
and
humidified
air
was
flushed
through
at
a
rate
of
l1./sec.
After
3sec.
the
first
leaf
segment
was
killed.
Radioactive
compounds
were
extracted
and
analysed
as
described
in
the
Methoods
section.
Percentage
of
total
14C