Biochem. J.

(1966) 101, 103 103

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- wide) containing no midrib tissue were cut from the youngest
sistent with the scheme for photosynthesis proposed fully expanded leaf. All cuts were made under water to
by Calvin and co-workers (Calvin & Bassham, 1962) prevent the entry of air into the vascular tissue.
have been considered in recent reviews by Stiller
(1962) and Bassham (1964). The low activity of METHODS
ribulose 1,5-diphosphate carboxylase in some tissues Photosynthesi8 in 14CO2. A rectangular Perspex box
relative to the rate of photosynthesis, and the high (19 cm. x 16cm. x 9 cm.), sufficiently large to accommodate
concentrations of bicarbonate required by the six leaf segments, served as a photosynthesis chamber.
enzyme for maximum activity, raise doubts about The top of the chamber was detachable and split in half
its quantitative importance in photosynthetic fixa- lengthwise. Leaf segments were clamped vertically
tion of carbon dioxide (Racker, 1957; Peterkofsky & between the halves with about 1 cm. protruding from the
Racker, 1961; Stiller, 1962). Recently Kortschak, chamber and the bases of the segments dipping into water.
Hartt, & Burr (1965) reported that malate and Soft rubber on the adjoining edges of the top provided a
seal around the leaf segments but allowed them to be with-
aspartate were the major labelled products formed drawn from the chamber. Air, saturated with water vapour,
in sugar-cane leaves during short periods of photo- was pumped through the chamber and the air in the cham-
synthesis in 14CO2. The present studies confirm ber was circulated by a high-speed fan. Before the addition
this observation with another sugar-cane variety of 14(02 the segments were illuminated for at least 45min.
and provide information about the nature of the by a 400w Phillips HPL lamp. The light-intensity at the
primary carboxylation reaction and the pathway leaf surface, measured with a selenium cell, was 8200ft.-
of sugar formation. candles. From a calibration curve of the selenium cell
against a Kipp thermopile this intensity was equivalent to
MATERIALS 0.39cal./cm.2/min.
Immediately before the introduction of 14CO2 the air
3-Phosphoglyceric acid (tricyclohexylammonium salt) supply to the chamber was removed and exit holes were
and wheat-germ acid phosphatase were purchased from sealed with tape; 14C02 (0-86mc) was injected from a
Sigma Chemical Co. (St Louis, Mo., U.S.A.). Yeast invertase syringe bringing the CO2 concentration to 0.055%. At
was obtained from Difco Laboratories (Detroit, Mich., intervals individual leaf segments were transferred to 50ml.
U.S.A.). Ba14C03 (26.2mc/m-mole), L-[U-14C]malic acid of boiling 80% (v/v) ethanol and kept at this temperature
(15lImc/m-mole) and L-[U-14C]aspartic acid (6.1mc/m- for 3min. In certain experiments the segments were trans-
mole) were obtained from The Radiochemical Centre ferred to methanol-chloroform-2 0m-formic acid (12:5:3,
(Amersham, Bucks.). by vol.) at -80°, then held at -15° for 24hr. (Bieleski &
Leaf tissue. Field-grown sugar cane (hybrid variety, Young, 1963).
Pindar) was used. Sections (approx. 18cm. long, 15cm. A second procedure allowed both shorter treatments with
104 M. D. HATCH AND C. R. SLACK 1966
14CO2 and the killing of leaves while still exposed to 14CO2 solvents A and B a peak was obtained which co-chromato-
and light. After pre-illumination in the chamber leaf graphed with L-malate. With solvent B this was the only
segments were placed in a large test tube (vol. 60ml.). radioactive compound which moved a significant distance
14CO2 (45,tc) was injected and then boiling 80% (v/v) from the origin. The radioactivity eluted from chromato-
ethanol was poured into the tube either simultaneously or grams developed in solvent B co-chromatographed with
after 1-2sec. L-malate in solvents A, C and E. When samples of this
Extraction of tissue. Segments killed in the methanol- compound were treated with a fumarate hydratase prepara-
chloroform-formic acid mixture were extracted as described tion from wheat-germ 18-20% of the radioactivity was
by Bieleski & Young (1963). Segments killed in boiling located in fumaric acid at equilibrium. Similar values were
ethanol were homogenized and the insoluble material was obtained with L-[U-14C]malate.
re-extracted in sequence with 20ml. of boiling 80% (v/v) Aspartic acid. A compound which ran as a separate peak
ethanol, and twice with 15ml. of boiling water. Water and corresponding to L-aspartate was detected with solvent A.
ethanol extracts were combined. The remaining insoluble The eluted radioactivity co-chromatographed with L-
material was washed with a large volume of water followed by aspartate in solvents B and E, and the compound was
ethanol and then dried at 70° to a constant weight. Here- completely degraded by treatment with ninhydrin
after this material will be referred to as 'residue'. Radio- (Greenberg & Rothstein, 1957).
activity incorporated into segments is expressed as counts/ Phosphorylated compounds. With solvent A a separate
min./lOOmg. of the residue. area of radioactivity was obtained which corresponded in
Counting procedures. Samples of 0- ml. or less of the mobility to 3-phosphoglycerate and hexose monophos-
combined extracts containing at least 1000 counts/min. were phates. Occasionally a very small separate peak was
counted in a liquid-scintillating counter (model N664A; observed with a mobility corresponding to fructose 1,6-
Ekeo Electronics). A toluene-phosphor-ethanol solution diphosphate and ribulose 1,5-diphosphate.
(Ziegler, Chleck & Brinkerhoff, 1957) was used for these When the major peak was eluted and co-chromatographed
aqueous samples. The counting efficiency was 55% and with marker compounds in solvent C the radioactivity
quenching was not significant. corresponded with 3-phosphoglycerate, glucose 6-phosphate
Weighed samples (approx. 10mg.) of the dried residue and fructose 6-phosphate. After treatment with acid
were counted in steel planchets with a Geiger-Muller tube. phosphatase all the radioactivity was recovered in com-
In the range used counts were proportional to the weight of pounds which co-chromatographed with glyceric acid,
the sample. The counting efficiency of this method was glucose and fructose in solvents B, C and D. In some
determined by liquid-scintillation counting of similar experiments traces of glyceric acid were detected when the
residue samples suspended in toluene containing phosphor original extracts were chromatographed in solvents A and B.
and 2% (w/v) thixin. Sucrose. After longer periods of exposure to 14C02 a
labelled compound appeared which chromatographed with
Identification and estimation of radioactivity in sucrose in solvent A. This compound co-chromatographed
individual compounds with sucrose in solvent D and after treatment with invertase
the radioactivity was located equally between glucose and
Chromatography 8olvents and counting procedure. For fructose.
identifying individual components and determining the Proportions of radioactivity in individual compounds. The
amount of the radioactivity in each compound samples of proportion of the total radioactivity in malate was calcu-
extracts were chromatographed on paper with one or more lated from chromatograms developed in solvent B and
of the following solvent systems: A, butan-l-ol-propionic checked with solvent A and the proportions of aspartate and
acid-water (10:5:7, by vol.) (Benson et al. 1950); B, sucrose were obtained with solvent A. After determining the
pentan-l-ol saturated with 5M-formic acid (Aronoff, 1956, proportion for the total phosphorylated compounds with
p. 122); C, propan-l-ol-aq. NH3 (sp.gr. 0.90)-water (6:3:1, solvent A the amount of radioactivity in 3-phospho-
by vol.) (Bieleski & Young, 1963); D, ethyl acetate- glycerate and glucose 6-phosphate plus fructose 6-phosphate
pyridine-water (8:2:1, by vol.) (White & Secor, 1953); was calculated from chromatograms run in solvents B or D
E, phenol saturated with water (Benson et al. 1950). For after treatment of this fraction with phosphatase.
subsequent references to these solvents the letter prefixing Oxaloacetic acid. With the standard procedure employed
each will be used. Whatman no. 1 paper was used and for radioactive oxaloacetate was not detected. Hence the
solvents A, C and E the paper was previously washed with following procedure was used. Leaves were exposed to
oxalic acid. 14CO2 in a test tube as previously described and after
The location and the amount of radioactivity in individual approx. 1 sec. photosynthesis was stopped by pouring one of
compounds or groups of compounds was determined with a the following solutions into the tube: (1) 80% (v/v) ethanol
chromatogram strip-counter and the area of individual containing 10mg. of 2,4-dinitrophenylhydrazine and
peaks measured with a planimeter. When quantities of 0-2N-HCI at - 80°, (2) the same solution at 820, (3) the same
individual compounds were required samples of extracts solution at 820 but without 2,4-dinitrophenylhydrazine.
were chromatographed as a band and eluted from developed The subsequent procedure was the same as already described
chromatograms. except that the concentrated extracts were extracted with
Marker compounds on chromatograms were detected as chloroform to obtain phenylhydrazones (Aronoff, 1956,
follows: organic acids and amino acids with ninhydrin p. 133). The radioactivity in the chloroform extract was
(Aronoff, 1956, p. 120), phosphorylated compounds with determined and samples were chromatographed with
ammonium molybdate (Bandurski & Axelrod, 1951) and authentic oxaloacetate 2,4-dinitrophenylhydrazone in the
sugars with p-anisidine. following solvents: butan-l-ol-water-ethanol (5:4:1, by
Malic acid. When extracts were chromatographed in vol.), butan-l-ol saturated with M-NaHCO3, 0-1 M-potassium
Vol. 101 PHOTOSYNTHESIS BY SUGAR-CANE LEAVES 105
glycinate, pH8-4, and butan-1-ol-ethanol-0-5N-NH3 Waldron in our Laboratory have shown that the
(7:1:2, by vol.) described by Block, Durram & Zweig (1958). rate of photosynthesis of leaf segments, prepared as
With the chloroform extract from the extract of leaf killed described in the Methods section, is low when first
at - 80° at least 90% of the radioactivity co-chromato- exposed to light. The rate increases to a steady
graphed with the marker in all solvents. When a portion of
the chloroform extract was mixed with authentic 2,4- value after about 30min. and is maintained for
dinitrophenylhydrazone of oxaloacetate and recrystallized several hours. In our experiments leaves were
twice the specific activity of the derivative remained exposed to light in a humidified stream of air for at
constant. least 45min. before the addition of 14CO2. The
Insoluble compound8. The insoluble residue was treated intensity of visible light at the leaf surface was
with 72% (v/v) H2SO4 to hydrolyse cellulose (Jermyn, 1955). approx. 40% of that in direct sunlight.
Essentially all the radioactivity was recovered in a com- The total fixation of 14C0O2 (Fig. 1) and the radio-
pound which co-chromatographed with glucose in solvents activity in individual compounds (Figs. 2 and 3)
C and D. When the material was hydrolysed with 6N-HCI were determined for leaves exposed to 14CO2 for
(Pirie, 1955) no radioactivity was detected in amino acids. periods of up to 150sec. Over this period the rate of
14CO2 fixation remained linear. The changing
Degradation of labelled product8 proportions of radioactivity in individual com-
Malic acid. Radioactivity in the C-1 plus C-4 and the C-2 pounds is shown in Fig. 2. Three trends are
plus C-3 of malate was determined by MnO2 oxidation apparent, the continued fall of the first-labelled
(Friedemann & Kendall, 1928). Samples of the C02 and products, malate and aspartate, the rise then fall of
acetaldehyde, trapped in sodium hydroxide and sodium 3-phosphoglycerate and hexose monophosphates,
bisulphite respectively, were counted in the liquid- and the steady increase of the percentage of the total
scintillation counter by using the procedure described for radioactivity in the end products sucrose and a
aqueous samples. The C-i of malate was determined by a glucan. The plot of total radioactivity in individual
modification of the Von Peckmann reaction described by compounds (Fig. 3) shows the asymptotic trend of
Racusen & Aronoff (1953). Trials with L-[U-14C]malate the early products and intermediates and the
established the exact conditions for the complete release of exponential rise in the radioactivity associated with
the C-1 as CO. Samples of malate containing at least
35,000 disintegrations/min. were dried in a combustion end products. When a similar experiment was
flask with L-malate carrier and heated at 800 for 45min. with carried out in the dark the rate of 14CO2 fixation
5ml. of 100% H2SO4 in the combustion apparatus for a was only 0-4% of that in the light. Even after
Nuclear-Chicago Dynacon electrometer. The CO released 5min. in 14C02 radioactivity was located almost
was then flushed into a 250ml. ion chamber and counted. entirely in aspartate and malate whereas no radio-
Similar samples were subjected to combustion with Van activity was detected in 3-phosphoglycerate or
Slyke reagent to determine the total radioactivity. hexose monophosphates. Our conditions of leaf
Aspartic acid. Radioactivity in the C-1 plus C-4 and the pretreatment and light-intensity differed from those
C-2 plus C-3 of aspartate was determined by treatment with used by Kortschak et al. (1965). Hlowever, our
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- 45r
activity in the C02, trapped in N-NaOH, and in the residual 5)
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 0
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 0
formic acid and the C-6 as formaldehyde (Aronoff, 1956,
p. 110). .0 :

RESULTS
IncorporaWon of radioactivity from 14CO2 a8 a 0 50 100 150
function of time. Experimental conditions were Time (sec.)
chosen to produce as nearly as possible a steady Fig. 1. Total 14C incorporated into leaf segments as a
state of photosynthesis under physiological condi- function of time. The radioactivity in the residue is included
tions of concentration of carbon dioxide and light- and the counts are for a 55% counting efficiency. Other
intensity. Studies by K. T. Glasziou & J. C. details are described in the Methods section.
Bioch. 1966, 101
106 M. D. HATCH AND C. R. SLACK 1966
results with the variety Pindar were qualitatively pounds was examined (Table 1). Results were very
similar to those obtained by these workers with the similar when leaves were killed either in boiling 80%
variety H37-1933. (v/v) ethanol or in the methanol-chloroform-formic
The effect of different killing procedures on the acid mixture at -80°. In these experiments leaf
distribution of radioactivity in individual com- segments were transferred from the chamber to the
killing mixture. To determine ifany modification of
the labelling pattern occurred during transfer, leaf
segments were exposed to 14CO2 in test tubes and

i
0
S

.4
0 ~ 44.

0
Q.I-101
o
~io --
4-
to --

-4)o
P-4

0
Time (sec.)
0 20 40 60 80
Fig. 2. Proportion of the total radioactivity in individual Time (sec.)
compounds for periods in 14CO2 up to 15Osec. Data are
from the same experiment as described in Fig. 1. Details of Fig. 3. Time-course for the total radioactivity incorporated
procedures for indentifying and counting individual into individual compounds. Data are from the same experi-
compounds are described in the Methods section. 0, ment as described in Figs. 1 and 2. 0, Malate+ aspartate;
Malate+aspartate; *, 3-phosphoglycerate; A, hexose Cl, malate; *, 3-phosphoglycerate; A, hexose mono-
monophosphates; A, sucrose; *, glucan. 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.
10-6 x Total 14C Percentage of total 14C in individual compounds
incorporated
Expt. Time in (counts/min./ Killing 3-Phospho- Hexose mono-
no. 14CO2 (sec.) 100mg. of residue) method Malate Aspartate glycerate phosphates Sucrose Glucan
1 30 8-9 Ethanol 30 15 36 14-5 2-5 1-0
30 10.1 MCF 32 16 34 15 2-0 1-0
60 19-9 Ethanol 26 8-5 27 25 8-5 4-0
60 19-3 MCF 27 10 30 23 6-0 4.5
2 4 2-6 Ethanol 53 25 15 7 0 0
added to
tube
4 2-3 Leaf 56 25 14 5 0 0
removed
t ethanool
Vol. 101 PHOTOSYNTHESIS BY SUGAR-CANE LEAVES 107
then killed either by the direct addition of boiling open test tube (see the Methods section). A time-
80% (v/v) ethanol or by transferring them to the course study over a period from 4sec. to 90sec. in
same mixture. The two procedures gave almost 14CO2 gave results which were almost identical with
identical results (Table 1). those obtained with leaf sections. When an
To confirm that the results obtained were not due attached leaf was exposed to 14CO2 for 2sec. and
to an alteration of the normal photosynthetic killed by the direct addition of boiling 80% (v/v)
process caused by detaching and cutting the leaves ethanol the proportions of the incorporated radio-
experiments were conducted with leaves on the activity in malate, aspartate, 3-phosphoglycerate
plant. The apical 25 cm. of the youngest fully- and hexose monophosphates were 54%, 37%, 7%
expanded leaf of plants was exposed to 14CO2 in a and 2% respectively.
sealed chamber or, for short-term experiments, an 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-
oO hydrazine.

0
0
x IS Table 2. Radioactivity in oxaloacetate after
different killing procedures
P- 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
0 10 20 30 40 50 60 200 2,4-dinitrophenylhydrazone.
Time (sec.) Percentage of total 14C
Fig. 4. Changes in the distribution of radioactivity after
transfer of leaf segments from 14CO2 to carbon dioxide. With With Without'
After 15sec. in 14CO2 leaf segments were removed simul- reagent reagent reagent
taneously from the chamber to a stream of air without Compound at -80° at 820 at 820
changing the distance from the light source. At intervals Malate 54 57 47
individual segments were killed and analysed as described Aspartate 36 32 42
in the Methods section. 0, Malate+ aspartate; o], malate; Oxaloacetate 3.4 0-6 0
*, 3-phosphoglycerate; A, hexose monophosphates; 3-Phosphoglycerate 5-2 8-0 6-0
A, sucrose; *, glucan; +, total radioactivity. Other compounds 3*0 2-4 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 in individual compounds
Time in the dark and 3-Phospho- Hexose
carbon dioxide (sec.) Malate Aspartate glycerate monophosphates Sucrose Alanine Glucan
3 35 26 29 9 0 0 0-6
20 37 24 24 12 1 2 0-7
37 35 38 16 8 1 2-5 0-5
83 34 42 10 8 0 6 0-6
108 M. D. HATCH AND C. R. SLACK 1966
Changes in the dietribution of radioactivity after
tran8fer from 14CO2 to air. When leaf segments were
transferred from 14CO2 to air but maintained at the I

same light-intensity there was no loss of radio-

activity during the following 200sec. (Fig. 4). XO o
During the first 40sec. most of the radioactivity in 0

malate and aspartate was transferred to the hexose 04

monophosphate pool whereas the label in 3-phospho- o~

4-4
b X
glycerate increased only slightly. Subsequently D 0~~~C~1
radioactivity appeared in sucrose and a glucan at the 0 c;
C)

expense of hexose monophosphates and 3-phospho-

8
glycerate. The relatively constant amount of
radioactivity associated with malate and aspartate CB
.X
o eq
'o Pm
during the period from 40sec. to 200sec. was vfZI
probably due to label in the C-1, C-2 and C-3 (see set< o s-XD e
14)
|1 i-5 mc C 10
Table 4). To further test the possibility that q
14CO2 may be released and then re-fixed during 0)0)COC10 1*
these transformations a period in 14CO2 was fol- '0
Lx80
lowed by a period in 5% (v/v) unlabelled carbon
dioxide in air. Transformations similar to those ._4
t
shown in Fig. 4 were observed and there was no c0
v8°b
to
significant loss of radioactivity. ) - 0
When leaves were transferred from 14CO2 in the ,0
light to unlabelled carbon dioxide in the dark there II ~ oo~
000
was little change in distribution of radioactivity in eq

individual compounds during the first 20sec.

(Table 3). Between 20sec. and 83sec. theproportion o o ^4 eq
r-i10
of radioactivity in 3-phosphoglycerate declined 10

from 24% to 10%. Accompanying this fall was an CO

s
increase in the radioactivity in aspartate and o .

C)
v m
Ca
alanine. The total radioactivity in the leaf segments O 1

V CIOV
C) - 0 0 10 CO
a:a
remained constant during the period in unlabelled Ca
O

carbon dioxide. 0

Radioactivity in individual carbon atorns of photo-

synthetic product8. The primary labelling of malic
acid occurred in the C-4 position (Table 4). After l CBC) I
20sec. the total radioactivity in this carbon became
relatively constant. At this time C-4 still contained el4 V
-4a 'o Q°a
80% of the total radioactivity, but label was not
detected in the C-2 and C-3 positions until 45sec.
The distribution of radioactivity in the C-1 plus
C-4 and the C-2 plus C-3 of aspartate was almost
identical with that of malate. At 3 sec. nearly all the t V
radioactivity in 3-phosphoglycerate was located in
the carboxyl carbon atom whereas the compound
was almost uniformly labelled at 150sec. It is
noteworthy that, with respect to the C-1, the C-2
e1 .z 0 ) E- C
plus C-3 of 3-phosphoglycerate became labelled e eq
eq_

much more rapidly than the C-2 plus C-3 of .; O02 o X 0 -0

0
malate. 00
co C> aq I'* 0 )10t
Glucose and fructose from hexose monophos- o -4

phates and sucrose were converted into their

common osazone and then partially degraded 900o.5
(Table 5). Almost half of the total radioactivity was
located in the C-1, C-2 plus C-3 positions after
20sec. and 45sec. The C-6 contained only a small
part of the radioactivity in C-4, C-5 plus C-6 at
20sec. but this proportion doubled by 45sec.
Vol. 101 PHOTOSYNTHESIS BY SUGAR-CANE LEAVES 109
Table 5. Di8tribution of radioactivity in the carbon atoms of hexose8 derived from
sucrose and hexo8e monopho8phates
Compounds were obtained from the same extracts used for the studies described in Figs. 1, 2 and 3 and Table 4.
Procedures used to isolate and degrade the hexoses are described in the Methods section.
Period C-1, C-2, C-3 C-6 (as % of C-4 plus C-5
in 14CO2 (as % of 14Cin (as % of 14C in
(sec.) Origin of hexose total 14C) C-4, C-5, C-6) C-4, C-5, C-6)
20 Sucrose 52 11 89
20 Hexose monophosphate 46 10 90
45 Sucrose 45 18 82
45 Hexose monophosphate 45 20 80

the light the radioactivity in malate and aspartate

DISCUSSION moved into other pools in the sequence already
Our aim was to examine the radioactive products suggested. In contrast radioactivity in the C4
formed from 14CO2 under steady-state conditions dicarboxylic acid pool increased significantly in
for photosynthesis and at a light-intensity and the dark and there was no movement of 14C into
concentration of carbon dioxide as close to physio- hexose monophosphates or sucrose. The increase
logical conditions as possible. Under these condi- of radioactivity in the dicarboxylic acid pool and
tions we assumed that only physiologically signi- the appearance of label in alanine occurred at
ficant carboxylation reactions were operative. For the expense of 3-phosphoglycerate. It appears
determining the sequence of chemical events a unlikely that the transfer of carbon from the
number of other assumptions are necessary. The dicarboxylic acids to 3-phosphoglycerate in the
major assumptions are that pool sizes remain light proceeds by a process involving decarboxyla-
constant and that the absolute pool size of inter- tion and re-fixation of carbon dioxide.
mediates is sufficiently large to allow the radio- The feature of photosynthesis is a net gain of
activity to be detected. Other factors which must carbon. This would be achieved only if the transfer
be considered are labelling of compounds by side of carbon from the C4 dicarboxylic acids to 3-
reactions from the main path, secondary labelling phosphoglycerate proceeded without the release
of intermediates by cycling, and labelling of and loss of carbon dioxide. Known pathways for
independent second pools. this transformation involve the decarboxylation of
With these points in mind we interpret the time- oxaloacetate or malate with the appearance of their
studies to indicate that the first product of the C-1, C-2 and C-3, as the C-1, C-2 and C-3 respectively,
major carboxylation reaction is either malate or of 3-phosphoglycerate. Besides the problem of loss
oxaloacetate and that aspartate is probably formed of carbon dioxide, the operation of this pathway is
by a side reaction from these products. The 14C inconsistent with the fact that the labelling of the
then moves from the C4 dicarboxylic acid pool to C-2 and C-3, relative to the C-1, is much more
3-phosphoglycerate and subsequently to sucrose and rapid for 3-phosphoglycerate than for malate.
a glucan via the hexose phosphates. Since the major Rather, our results indicate that the C-4 of the C4
products of 14C02 fixation in the dark are malate dicarboxylic acids is the source of the C-1 of 3-
and aspartate the same carboxylation reaction may phosphoglycerate. The earliest labelling of malate
be operating for both processes. In the dark the rate occurs in the C-4 and the total radioactivity in this
of fixation of carbon dioxide is only 0-4% the rate carbon atom approaches an optimum value more
in light and no radioactive phosphorylated com- rapidly than the radioactivity in the C-1 of 3-
pounds were detected. Hence light not only phosphoglycerate. The simplest interpretation is
produces a 250-fold increase in the rate of fixation that the C-4 carboxyl group of a C4 dicarboxylic
of carbon dioxide but is also necessary for the acid is transferred by a transcarboxylation reaction
transfer of radioactivity from the C4 dicarboxylic to an acceptor substance, the latter providing the
acid pool into 3-phosphoglycerate. C-2 and C-3 of 3-phosphoglycerate. The carbon
Support for this interpretation was obtained from dioxide-acceptor for the initial carboxylationr
experiments in which a period in 14CO2 in the light reaction could be regenerated from the remaining
was followed by periods in unlabelled carbon di- C3 compound.
oxide in either light or dark. In both cases the total A definitive conclusion about the nature of the
radioactivity in leaf segments remained constant primary carboxylation reaction cannot be derived
during the period in unlabelled carbon dioxide. In from the available data. Enzymeswhich carboxylate
 110 M. D. HATCH AND C. R. SLACK 1966
pyruvate to give malate, and phosphopyruvate Sucrose
to give oxaloacetate, are known to occur in leaves.
They are malate dehydrogenase (decarboxylating)
(EC 1.1.1.40) ('malic enzyme') and phosphopyruv- Hexose phosphates
ate carboxylase (EC 4.1.1.31). The operation of (Ribulose diphosphate?)
another enzyme, ATP-oxaloacetate carboxy-lyase Carboxyl acceptor
(Mazelis & Vennesland, 1957) would not be favoured
by the high ATP/ADP ratio which should prevail 3-Phospho. .
in the light. However, our data indicate that the glycerate 2/
C4 dicarboxylation acids are in rapid equilibrium so j
that the primary carbon dioxide-fixation product \
need not be the acid involved in the subsequent Aspartate
transcarboxylation reaction. Phosphopyruvate 30 4Ci
Oxaloacetate is known to participate in several
trans-carboxylation reactions (Wood & Stjern- 1 2C+C*Os 30
7\e < I II1 Oxal aetate
holm, 1961; Hlsmann, 1962) and therefore appears Pyruvate 10 11.
the more likely carboxyl donor. The carboxyl
acceptor may be either a C2 compound such as {3.te
glycolaldehyde phosphate or the C5 compound of the
Calvin cycle, ribulose 1,5-diphosphate. The latter Scheme 1. Proposed pathway for photosynthetic fixation
possibility is appealing since the subsequent steps of carbon dioxide in sugar-cane leaves. The broken arrow
may then be identical with those of the Calvin cycle indicates a minor pathway.
(Calvin & Bassham, 1962). The rates of labelling of
3-phosphoglycerate and hexose monophosphates,
and the distribution of radioactivity in their
individual carbon atoms, are consistent with this 3-phosphoglycerate as the first detectable radio-
possibility. active product.
The above interpretations are summarized in
Scheme 1, which shows two cyclic processes linked
by a transcarboxylation reaction. The role of one
cycle is to fix carbon dioxide and to provide carboxyl REFERENCES
groups to react with a carboxyl acceptor in the other
cycle. The latter cycle operates to regenerate the Aronoff, S. (1956). Techniquee of Radiochemistry. Ames:
carboxyl acceptor for the transcarboxylation Iowa State College Press.
Bandurski, R. S. & Axelrod, B. (1951). J. biol. Chem. 193,
reaction. The relative rates of labelling of the C-2 405.
and C-3, compared with the C-1, for malate and 3- Bassham, J. A. (1964). Annu. Rev. Plant Phy8iol. 15, 101.
phosphoglycerate is consistent with some of the Benson, A. A., Bassham, J. A., Calvin, M., Goodale, T. C.,
latter compound being converted into the C3 carbon Haas, V. A. & Stepka, W. (1950). J. Amer. chem. Soc. 72,
dioxide-acceptor. While this movement of carbon 1710.
is not a basically essential part of the scheme it may Bieleski, R. L. & Young, R. E. (1963). Analyt. Biochem. 6,
serve to replace carbon lost from the first cycle by 54.
side-reactions. Block, R. J., Durram, E. L. & Zweig, G. (1958). Paper
The possibility that the pathway outlined in Chromatography and Paper Electrophore8i8, p. 239. New
York: Academic Press Inc.
Scheme 1 is peculiar to sugar cane appears remote. Calvin, M. & Bassham, J. A. (1962). The Photo8ynthe8is of
Hence the question why similar labelling patterns Carbon Compound,s, p. 10. New York: W. A. Benjamin
have not been observed during studies on other Inc.
species arises. If malate and aspartate are not Friedemann, T. E. & Kendall, A. I. (1928). J. biol. Chem. 82,
essential intermediates they may be labelled only if 23.
sufficient malate dehydrogenase and transaminase Greenberg, D. M. & Rothstein, M. (1957). In Methods8 in
are present in the chloroplasts. Detection of radio- Enzymology, vol. 4, p. 713. Ed. by Colowick, S. P. &
active oxaloacetate would depend upon an adequate Kaplan, N. 0. New York: Academic Press Inc.
pool size and preventing its breakdown during H*ilsmann, W. C. (1962). Biochim. biophys. Acta, 62, 620.
extraction. The only procedure we found satis- Jermyn, M. A. (1955). In Modern Methods of Plant Analyai8,
vol. 2, p. 205. Ed. by Peach, K. & Tracey, M. V. Berlin:
factory for measuring the radioactivity in oxalo- Springer-Verlag.
acetate involved killing off leaves at -80° in the Kortschak, H. P., Hartt, C. E. & Burr, G. 0. (1965). Plant
presence of 2,4-dinitrophenylhydrazine. Hence, if Physiol. 40, 209.
standard killing procedures are used, the operation Mazelis, M. & Vennesland, B. (1957). Plant Physiol. 32,
of the pathway described in Scheme 1 could give 591.
Vol. 101 PHOTOSYNTHESIS BY SUGAR-CANE LEAVES 111
Peterkofsky, A. & Racker, E. (1961). Plant Physiol. 86,409. White, L. M. & Secor, G. E. (1953). Arch. Biochem. Biophy8.
Pirie, N. W. (1955). In Modern Methods of Plant Analysis, 43, 60.
vol. 4, p. 30. Ed. by Peach, K. & Tracey, M. V. Berlin: Wood, H. G. & Stjernholm, R. (1961). Proc. nat. Acad. Sci.,
Springer-Verlag. Wa8h., 47, 289.
Racker, E. (1957). Arch. Biochem. Biophys. 69, 300. Zelitch, I. (1965). J. biol. Chem. 240, 1869.
Racusen, D. W. & Aronoff, S. (1953). Arch. Biochem. Ziegler, C. A., Chleck, D. J. & Brinkerhoff, J. (1957). In
Biophys. 42, 25. Liquid Scintillation Counting, p. 158. Ed. by Bell, G. C. &
Stiller, M. (1962). Annu. Rev. Plant Physiol. 13, 151. Hayes, F. N. Oxford: Pergamon Press Ltd.