RESULTS

Activity of Mitochondrial Enzymes in Leaves.

The activities
of several enzymes thought to be exclusively
associated with the mitochondria and the
tricarboxylic acid cycle were assayed
in leaves of A. hastata and Spinacia oleracea (C3
plants), and in mesophyll and bundle sheath cells of
A. spongiosa and Sorghum bicolor (C4 plants).
Expression of activities of these
enzymes on a Chl base provides a reasonable
assessment of the extent of mitochondrial carbon
metabolism in relation to pho-tosynthesis.
The data in Table I show that the activity of NAD
isocitrate dehydrogenase, fumarase, and citrate
synthase is commonly 5 to 10% of that of ribulose
diphosphate carboxylase, and about 5 to 10% of the
usual photosynthetic rate in leaves of
all four species (2-5 ,umoles C02/min mg Chl).
With minor
variations, these mitochondrial enzymes show
similar specific activity (Chl base) in leaves of C3
and C4 plants, and in meso-
phyll and bundle sheath cells of C4 plants.
Cytochrome c oxi-
 Table I. Specific Activity of Photosynthetic anid Respiratory Enzymes Extracted from Leaves of C3 anid
C4 Planzts

Plant Physiol. Vol. 53, 1974 LIGHT AND RESPIRATION IN LEAVES. III 895
dase activity was also similar in all extracts with the possible
exception of S. bicolor. In this case, high levels of mercapto- _ A.spongioso. .b)
ethanol used during extraction seemed to interfere with the vi
X
assay, even after treatment with Sephadex G-25. o 0
c 0o
Metabolism of Succinate-"C in the Light. Preliminary ex- .r
periments showed that when succinate-2, 3-"C was supplied to o a.,
A. spongiosa leaves in the light, malate and aspartate -
were
rapidly labeled and retained a high proportion of the radio-
activity absorbed. The proportion of label in sucrose increased 80
rapidly throughout the experiments and this transfer a
ceased OE °
-a
when leaves were darkened.
These observations were confirmed in leaf slice account for the labeling of photosynthetic products,
experiments using specifically labeled succinate. particularly in the case of succinate-2,3-"C. The
The photosynthetic metab-olism of leaf slices in inhibition of the labeling of citrate by light (Table
solution is closely comparable to that of intact II) argues against a stim-ulation of the rate of "CO2
leaves (19), yet the technique permits the rapid release in the light, and indicates that lable from
applica- succinate is diverted from the tricarboxylic acid
tion of labeled metabolites under controlled cycle prior to citrate, possibly as malate or
conditions. In oxaloacetate.
these experiments the rate of release of "CO2 from Effects of Light on Tricarboxylic Acid Cycle
succinate-1 ,4-"C in the dark greatly exceeded that Metabolism. The rapid, light-dependent transfer of
from succinate- "C from succinate-"C
2,3-w"C (Table II). This was consistent with the to photosynthetic products in leaves of C4 plants
removal, as
contrasts with observations in leaves of C, plants. In
CO2, of both labeled carbons in succinate-1,4-"C
during the the latter, the principal effect of illumination was a
first turn of the tricarboxylic acid cycle. Little "CO2 change in the distribu-
was re-
leased from either substrate in the light. However, Table II. Metabolism of Succinate-1,4-I4C and
Succinate-2,3-14C
succinate-1 , 4-"C and succinate-2, 3-`C were
in Slices of A. sponigiosa Leaf Tissue
rapidly converted to sucrose and other
photosynthetic products in the light at about equal 14C Incorporated
from:
rates (Table II). The conversion of succinate-"C to
photosyn-thetic products was considerably more Compound Treatment Succinate-
l,4-14C Succinate-2,3-14C
rapid than the rate of "CO2 release in the dark. Even
if "CO2 release continued un-abated in the light, 15 min| 30 min 15
min 30 min
refixation of CO2 during photosynthesis could not
 I.,,,' cal to that in mung beans (7). In leaves of C4 A.
013610 19 24 36 0 5 10
15 spongiosa, however, this interchange did not take
23
place, and both malate
Time (minutes).
and aspartate lost label immediately upon
FIG. 1. Transient changes in malate (A) and
illumination. This
aspartate (0) radio-
difference in response was observed in all
activity on illumination of leaves of Atriplex spp.
subsequent experi-ments. In these, the sum of
Excised leaves supplied with succinate-2,3-"C for 2
aspartate and malate is plotted for
hr in the dark + 15 min in H20 before illumination.
simplicity.
Solid bar represents darkness and open bar Figure 2a shows that in A. hastata, little
represents illumination. radioactivity was
transferred to sucrose on illumination, a response
tion of label between malate and aspartate and little
consistent with that in mung beans (6). The net loss
"C en-tered photosynthetic pools (6). The
of label from the malate + aspartate in the light
experiment shown in Ta-
reappeared in glutamate. Simi-lar changes were
ble II suggests that malate and aspartate, derived
noted in leaves of the F, and F, hybrids of A. rosea
from
succinate, may leave the tricarboxylic acid cycle for and A. patula spp. hastata (Fig. 2c), and very little
photosyn-thetic pools in A. spongiosa in the light. label was found in alanine, sugar phosphates, or
Comparative experi- sucrose in these plants. In contrast to this, in both A.
ments were therefore set up to examine the effects spongiosa and
of a light Sorghum bicolor, illumination resulted in a rapid
transient on the fate of malate and aspartate derived loss of label
from from malate + aspartate and its appearance in
succinate-2, 3-"C fed during a preceding dark photosynthetic
period. intermediates (Fig. 2, b and d). The rapid labeling of
Figure 1 shows the interchange of malate and alanine is
aspartate consistent with the labeling of pyruvate, presumably
label upon illumination of A. hastata leaves, a by de-
response identi-

4CO2 Light ucts was negligible.

Dark
Phosphorylated com- Light Effects of Malonate and Fluoroacetate on Light-dependent
pounds' Dark
Sugars Light
Dark Changes in Tricarboxylic Acid Intermediates. Leaves of A.
Glycine + serine Light spongiosa and Sorghum bicolor were supplied with succinate-
Dark 2,3-'4C in the dark and then treated with water, 50 mM malo-
Citrate Light nate (pH 4.0) or 100 mm fluoroacetate (pH 4.0) for a further
Dark
Malate + aspartate Light 30 min in the dark before illumination. Figure 3a shows results
Dark
1C absorbed Light for the light transient in A. spongiosa treated with malonate
Dark
and Figure 3b shows results for Sorghum bicolor with fluoro-
'Including 3-PGA and sugar
2 No sample. acetate. In both cases, these inhibitors drastically reduced the
periments and the transfer of carbon to photosynthetic prod- Sorghum bicolor. b)
transfer of label from malate + aspartate to sucrose. The
effects on the labeling of other photosynthetic intermediates boxylic acid cycle, rather than the refixation of
respiratory /
were comparable, and both inhibitors were effective when used '4CO2. This view is further supported by the
with leaves of either species. In most of the experiments when distribution of '4C
1
within the carbon atoms of glucose-14C prepared
malonate or fluoroacetate were used, label accumulated in 0 51015 23 0510 5 30
from the su-
either succinate or citrate respectively, suggesting that inhibi- Tim
crose-4C produced in these experiments.
e (minutes).
Sucrose-14C formed in the first minutes of
tion of the tricarboxylic acid cycle had, in fact, occurred. FIG
illumination of
Degradation of Sucrose Labeled via Tricarboxylic Acid . 3. Transient changes in the distribution of "4C in malate +
A. spongiosa and Sorghumn bicolor leaves
Cycle Intermediates. The above experiments show that in asparate (0) and sucrose (A) on illumination of leaves supplied
leaves of C4 plants in the light sucrose and other photosynthetic previously supplied in the dark as in Figure 2. (a) A. sponzgiosa
with succinate-2,3-'4C
leaves treated with 50 mm malonate
with succinate-2,3-'4C and (b)(as
in the dark S. bicolor leaves
in Fig.treated
2, b
and d) was converted to glucose-'4Cinhibitor) and degraded
intermediates are rapidly labeled, either from succinate-'4C fed with 100 mm fluoroacetate.
enzymically. Table III Controls ( ; treatments
to the leaves in the light, or from intermediates of the tricar- (---). Solid bar represents darkness and open bar represents illumi-
shows
boxylic acid cycle labeled in a previous dark period. The data that with the possible exception of A.
nation.
suggest that the transfer involves the photosynthetic metabo- spongiosa (5 min) the central carbons (3-C, 4-C) are
lism of malate, aspartate, or oxaloacetate labeled in the tricar- less heavily labeled than the more terminal carbons
Table 1II. Distr-ibiutioii of '4C in specific Carboni Atoms of
(1-C, 2-C, 5-C, 6-C). Furthermore, the label in the
Time (minutes)
FIG. 2. Transient changes in the distribution of central carbons declines with time, even though su-
14C in malate + aspartate (0 and * continuous dark crose label continues to increase (Fig. 2, b and d).
control), sucrose (A), gluta-mate (0), alanine (7) This distri-bution and trend is not consistent with
and sugar phosphates (0) in excised leaves supplied refixation of "4CO2
being the principal route of '4C to photosynthetic
with succinate-2,3-"C for 2 hr in the dark + 15 min products in these plants.
in H20 before illumination. Solid bar represents
darkness and open DISCUSSION
bar represents illumination. These experiments show that the activity of
several tri-
carboxylic acid cycle enzymes in leaves of C3 and
Co plants are 5 to 10% as active as ribulose-l , 5diP
carboxylase. The Chl-based specific activity of
fumarase, citrate synthase, and NAD
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Plant Physiol. Vol. 53, 1974 LIGHT AND RESPIRATION IN LEAVES. III
897
One important consequence of continued
isocitrate dehydrogenase is more or less constant in tricarboxylic acid
leaf cells of all species examined. Thus, although cycle activity in the light in leaves of C4 plants is
some cells, such as the bundle sheath cells of A. the rapid transfer of label from intermediates of the
spongiosa, seem to contain more mito- cycle to photosyn-
chondria than other cells (9) it seems likely that they thetic products. This process, which is clearly
also con- associated with a functional C4 pathway (cf. the A
tain more chloroplasts or at least more Chl (31). triplex hybrids) and which de-pends on continued
Few quanti- tricarboxylic acid cycle activity (cf. in-
tative examinations of electron micrographs have hibitor effects), was first noted by Karpilov (20) in
been made but these suggest only minor differences
comparative studies between Zea and Phaseolus.
in the ratio of chloro-plasts to mitochondria between
The effects of illumination
mesophyll and bundle sheath
cells of Zea and Sorghum, and between cells of C3 on the metabolism of tricarboxylic acid cycle acids
and C, in leaves
grasses (12). A substantially lower ratio was of C, mung beans and A. hastata is largely a
recorded in the bundle sheath cells of Chloris which response to the balance of reduced and oxidized
appears to resemble pub- pyridine nucleotides (6, 7, 14,
lished micrographs of A. spongiosa (9, 12). The 15), and very little carbon is transferred to
difficulties in making accurate estimates of this ratio photosynthetic
pools. The exchange of carbon between the
are considerable, and on
tricarboxylic acid
the evidence available there is little support for the cycle and photosynthesis in C, plants may occur via
proposition of unusual tricarboxylic acid cycle activity the refixa-
in relation to photo- tion of CO2 released during respiration or by the
synthetic metabolism in these cells. loss of inter-
The experiments are consistent with the
mediates from the tricarboxylic acid cycle.
continuation of tri-carboxylic acid cycle activity in Two observations reported here indicate that the
the light in leaves of C, plants, release and
refixation of respiratory CO2 is not the principal
but do not indicate the relative rate of turnover in
route of
light com-pared with that in the dark. In this respect carbon exchange between the tricarboxylic acid
leaves of C, plants cycle and
photosynthesis.
are similar to those of C, plants (6, 7, 14, 15, 23) 1. Release of "CO2 from succinate-1 ,4-"C during
and to algae
respira-tion greatly exceeds that released from
(24). We have assumed that the oxidation of
succinate is an ex- succinate-2, 3-14C. Both substrates are equally
clusive property of the mitochondria and that effective as a source of "C for photo-
malonate and fluoroacetate are inhibitors of this synthetic products, showing that the transfer is
process. We were unable largely inde-pendent of the rate of respiratory
however, to obtain satisfactory assays for succinate 14CO2 production. 2. If the
dehydro-genase in extracts or particles from leaf labeling of sucrose from succinate-2, 3-1C in the
tissue, using published light pro-ceeded by way of "4CO2, the central
methods. carbons of glucose-14C pre-
pared from labeled sucrose would be somewhat
more heavily labeled than the terminal carbons due
to the incorporation of
"CO2 into 3-phosphoglycerate. In the shortest times
used in the present experiments, carbons 1,2 and 5,6
were usually more
heavily labeled, consistent with the transfer of
2C,3C of suc-cinate via 2C, 3C of a labeled 3-
carbon compound, rather than
via "CO2. However, the possibility of dilution of
the activity in carbons 3,4 of glucose by "2CO2
fixation cannot be excluded in
these experiments.
Leaves of many C4 plants contain high activities
of malic en-zyme and P-enolpyruvate
carboxykinase in bundle sheath cells
which are believed to be involved in the
decarboxylation of
malate and oxaloacetate during photosynthesis (1,
11). Other
C4 plants have high activities of aspartate amino
transferase
which may be involved in transamination of
aspartate prior to decarboxylation (1). In each case
the donor C4 acid yields CO2
and a C, product, both of which may enter reactions
of the carbon reduction cycle. For example, in
Sorghunm bicolor, labeled malic acid derived from
succinate-1 ,4-"C would yield
4CO2 and pyruvate-l-"C as a consequence of malic
enzyme ac-tivity, and both labeled carbons may
enter the carbon reduction cycle. If label proceeded
as far as oxaloacetate, decarboxylation
of oxaloacetate to pyruvate and CO2 would yield
similar results.
Malate-2, 3-"C, on the other hand, yields only
pyruvate-2, 3-"C. Because the carbon from the C-4
carboxyl of malate-1 ,4-"C enters the carbon
reduction cycle more rapidly than that of the
remaining C, skeleton (16), the transfer of carbon
from succinate-I , 4-"C to photosynthesis should be
initially more rapid than that from succinate-2, 3-`C.
This was not observed
in the present experiments, presumably because of
the long
time scale of the comparative experiments.
This interpretation of the path of label from the
tricarboxylic
acid cycle to photosynthetic metabolism in C4
plants also ac-
counts for the near absence of such a transfer in C,
plants which contain much lower levels of malic
enzyme. The ac-tivity of tricarboxylic acid cycle
enzymes is similar in both groups of plants so
release of CO2 from the tricarboxylic acid
cycle and its refixation during photosynthesis is not
likely to result in the differences observed in Figure
2. We believe that they reflect the presence of a very
active decarboxylation en-
zyme for malate, oxaloacetate, or aspartate in C4
leaves which
is relatively inactive in C3leaves.
There is no evidence in the present experiments
of differ-ences between Sorghum bicolor and A.
spongiosa in terms of tricarboxylic acid cycle
metabolism in relation to photosynthe-sis. The
activity of tricarboxylic acid cycle enzymes and the
relatively slow movement of respiratory CO2 into
photosyn-thates argue against an important role for
the cycle in CO2
generation for photosynthesis. It seems unlikely that
mito-
chondria in bundle sheath cells could respire a
proportion of
photosynthetic pyruvate as proposed elsewhere
(10). In Sor-ghuin bicolor, malic enzyme activity in
bundle sheath cells is adequate to sustain the
generation of CO2 from malate at rates
demanded by photosynthesis (1). Any contribution
from mito-chondrial respiration is probably trivial
by comparison.
In A. spongiosa the situation is less clear, in that
this and some other C, species are deficient in malic
enzyme and
P-enolpyruvate carboxykinase (1, 11). In addition
there is good
evidence that aspartate is the source of CO2 for the
carbon re-duction cycle in these plants (16). Leaf
mitochondria show a
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degree of specialization with respect to other
decarboxylase en-zymes which may be involved in
photosynthetic carbon me-
tabolism. For example, the activity of glycine
decarboxylase in leaf mitochondria, believed to be
specifically related to the metabolism of
photosynthetic glycine, is 10-fold greater than
that in mitochondria of other plant tissues (2, 21).
Thus, al-though mitochondria in bundle sheath cells
of A. spongiosa are
not unusually active with respect to the tricarboxylic
acid cycle,
they may be involved in the decarboxylation of
photosynthetic aspartate (9). The recent
identification of a very active isoen-zyme of
aspartate amino transferase specific to bundle sheath
mitochondria of these plants (M. D. Hatch, personal
communi-cation) is consistent with this possibility.
Acknowledgmnents-'Most of this work was
carried out while E. A. C. was a
visiting research worker in the Research School of
Biological Sciences at the
Australian National University, Canberra. We are v-
ery grateful to Bronxvn
Williams for her skilled assistance.