Plant Physiol. (1974) 53, 893-898

The Effect of Light on the Tricarboxylic Acid Cycle in GreenLeavesIII. A COMPARISON BETWEEN SOME C3 AND C4 PLANTS

Received for publication January 31, 1973 and in revised form June 11, 1973

E. A. CHAPMAN1 AND C. B. OSMONDDepartment of Environmental Biology, Research School of Biological Sciences, Australian National University,Canberra, A. C. T. and Plant Physiology Unit, Commonwealth Scientific and Industrial Research Organization,Division ofFood Research, and School of Biological Sciences, Macquarie University, North Ryde 2113, Sydney,Australia

ABSTRACT

The chlorophyll-based specific activity of cytochrome oxidaseand three exclusively mitochondrial enzymes of the tricar-boxylic acid cycle showed little variation between leaves ofC3 and C4 plants or between mesophyll and bundle sheath cellsof Atriplex spongiosa and Sorghum bicolor. However, a large,light-dependent transfer of label from intermediates of thetricarboxylic acid cycle to photosynthetic products was afeature of leaves of C4 plants. This light-dependent transferof label was barely detectable in leaves of C3 plants and inleaves of F1 and F3 hybrids of Atriplex rosea (C4) and Atriplexpatula spp hastata (C3). The light-dependent transfer of labelto photosynthetic products in leaves of C4 plants was inhibitedby the tricarboxylic acid cycle inhibitors malonate and fluoro-acetate. The requirement for continued tricarboxylic acidcycle activity was also indicated in experiments with specifi-cally labeled succinate-'4C. These experiments, together withthe distribution of '4C in glucose prepared from sucrose-14Cformed during the metabolism of succinate-2,3-'4C, confirmedthat the photosynthetic metabolism of malate and aspartatederived from the tricarboxylic acid cycle, and not the refixationof respiratory C02, was the main path of carbon from thecycle to photosynthesis.

The continued operation of tricarboxylic acid cycle metabo-lism in illuminated green cells is now widely accepted (28). Inleaves of higher plants, this conclusion is supported by studieswith labeled tricarboxylic acid cycle intermediates (6, 7), andby the persistence in the light of 02 uptake at low concentra-tions of 0° known to inhibit glycolate synthesis and metabolism,or photorespiration (25, 30). The role of continued tricar-boxylic acid cycle respiration in the light in green cells is notclearly understood but it contributes to C02-generating reac-tions in these tissues (23, 30). In leaves of some C4 plants. mito-chondria appear to be particularly abundant in the bundle

'Permanent address: Plant Physiology Unit, Commonwealth Sci-entific and Industrial Research Organization Division of Food Re-search, and School of Biological Sciences, Macquarie University,North Ryde 2113, Sydney, Australia.

sheath cells (3, 9, 22). These mitochondria may have particularroles in relation to secretion of photosynthetic products (27),with the consumption of photosynthetic pyruvate (10) and,because the mitochondria are most abundant in plants of theaspartate donor type (16), they have been implicated in thetransamination and decarboxylation of photosynthetic aspar-tate (9). The experiments reported here were designed to ex-plore the activity of the tricarboxylic acid cycle in leaf cells ofC4 plants in the light. We were concerned with two questions:first, whether in these leaves the activity of the tricarboxylicacid cycle relative to photosynthesis was different from that inleaves of C3 plants and second, whether there were differencesin the participation of intermediates of the tricarboxylic acidcycle in photosynthetic metabolism between leaves of C3 andC4 plants. The experiments emphasized the rapid exchange ofmalate and aspartate between mitochondria and chloroplastsin leaves of C4 plants.

MATERIALS AND METHODSLeaf materials were obtained from plants grown in the

greenhouse. Mature leaves of Atriplex spongiosa FvM, andAtriplex hastata L., and young leaves of Spinacia oleracea L.and Sorghum bicolor L. var. Texas 610, were used. Leavesfrom two hybrids (Fl 8001/2 and F3 8004/3) of the crossA triplex rosea L. ( 8 ) and Atriplex patula spp. hastata Hall andChem. ( ) were provided by Dr. 0. Bjorkman. These hybrids,and all others examined to date, are incapable of normal C4photosynthesis even though many features of the C, parentare inherited (17).

Extraction and Assay of Enzymes. Leaves of C., plantsSpinacia oleracea and A triplex hastata were extracted in cold50 mm Bicine, 10 mm 2-mercaptoethanol, and 1.0% PolyclarAT, pH 7.6, (50 ml) for 30 sec in a Servall omnimixer, 80%line voltage, and filtered through Miracloth. The filtrate wascentrifuged for 10 min at 1OOOg and a portion of the super-natant was made to 0.2% Triton X-100. The Triton X-100-treated supernatant was then passed through a Sephadex G-25column equilibrated with 20 mM Bicine, pH 7.6, and elutedwith the same buffer. Extracts of leaves of C, plants were pre-pared by the differential grinding technique. A mesophyll cell-enriched extract was prepared by gently grinding 10 g of leavesin 50 ml of the above extracting buffer (5 sec, 10-15% linevoltage in Servall omnimixer). The residue from this extractwas returned to the blender and extracted in 50 ml of N2-satu-rated buffer with 0.3 M sucrose added. The blending conditions

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were 45 sec, 60% line voltage for A triplex spongiosa, and five 1-min periods, 80% line voltage with four 30-sec pauses forSorghum bicolor. These treatments removed adhering meso-phyll cells as judged by microscopic examination. The cleanbundle sheath strands were further extracted with glass beadsfor 1 min in 15 ml of buffer in a Janke Kunkel mill to yieldan extract enriched in the cell contents of bundle sheaths. Ex-tracts of both mesophyll and bundle sheaths were centrifuged,treated with Triton X-100, and passed through Sephadex G-25as described above.

Phosphoenolpyruvate carboxylase and ribulose diphosphatecarboxylase were assayed as described earlier (26). NAD iso-citrate dehydrogenase was assayed spectrophotometrically(340 nm) in 3 ml of a reaction mixture containing the follow-ing in /umoles, HEPES buffer pH 7.6, 150; MnSO4, 3; NAD,2; and sodium isocitrate, 12 (8). Fumarase was assayed at 250nm in 3 ml of a reaction mixture containing in ,.tmoles, sodiumphosphate buffer, pH 7.3, 150; and sodium malate, 150 (18).Citrate synthase was assayed at 412 nm in a 3-ml reaction mix-ture containing in ,umoles, tris HCl buffer, pH 8.0, 100; 5,5'-dithio-bis-(2-dinitrobenzoic acid), 2; acetyl coenzyme A, 0.01;and oxaloacetate, 5 (4). Cytochrome c oxidase was assayed at550 nm in a 3-ml reaction mixture containing in ,umoles, so-dium phosphate, pH 7.3, 150; and cytochrome c (reduced bydithionite), 0.5. Activity was expressed on a Chl base, Chlbeing determined on an aliquot of the crude homogenate (5).

Metabolism of Labeled Substrates. Slices of A. hastata andA. spongiosa leaves (0.5 mm wide) were cut with a slidingmicrotome, and were washed in aerated 50 mm phosphatebuffer, pH 6.0, containing 0.5 mm CaSO,, for 1 to 2 hr beforethe experiments. Labeled succinate was added to 2-ml solu-tions of phosphate buffer (50 mm, pH 6.0) containing 0.2 g ofleaf slices. "4CO2 evolved during experiments was collected onKOH-soaked discs of glass filter paper suspended above thereaction mixture. Experiments were done in a shaking waterbath at 25 to 27 C with illumination from a Philips HPLRlamp (1.5 X 105 ergs cm-2 sec', 400-700 nm). At the end ofthe experiment slices were thoroughly rinsed in distilled waterto remove label from the tissue free-space and the slices werekilled and extracted in 80% ethanol and water.

Experiments with intact leaves were conducted as describedearlier (6, 7). Leaves were placed in small vials containinglabeled substrates and allowed to absorb '4C for 2 hr in thedark. They were then transferred to a vial of water for a fur-ther 15 min in the dark before illumination. Leaves were sam-pled before illumination with a Philips Photolita lamp, approx-

imately 5 X 10' ergs cm-2 sec-1 full spectrum, again at intervalsduring the light period of 10 to 30 min, and again at intervalsafter darkening. Several control samples were kept in the darkthroughout. Previous experiments showed that this procedureestablished an approximate steady state of labeling in pools oftricarboxylic acid cycle acids in the dark, and dark controls inthe present experiments remained essentially unchanged dur-ing the time of the dark to light to dark transients. Proceduresfor extraction and chromatography of labeled compoundshave been described earlier (6, 26).

Degradation of Labeled Sucrose. A neutral fraction was iso-lated from leaf extracts by passage through Dowex-50-H+ andDowex-1-formate resins. Sucrose was separated from glucoseand fructose by descending chromatography on Whatman No.1 filter paper, using ethyl acetate-acetic acid-formic acid-water(18:3:1:4, v:v:v:v). Sucrose was eluted from the chromato-grams and converted to glucose and fructose by incubating 0.1ml of solution in 0.3 mm triethanolamine-HCl buffer, pH 7.4,with 0.2 ml of 1% invertase at 30 C for 3 hr. At the end of thisperiod glucose and fructose were degraded by enzymic meth-ods to obtain label in the individual carbon atoms (13, 29).The procedure was checked by the degradation of sucrose-U-'4C and glucose-1-"4C (Table III).

Radioactive compounds were obtained from the Radiochem-ical Centre, Amersham, England. Enzymes and substrateswere obtained from Boehringer, Calbiochem, and Sigma.

RESULTS

Activity of Mitochondrial Enzymes in Leaves. The activitiesof several enzymes thought to be exclusively associated withthe mitochondria and the tricarboxylic acid cycle were assayedin leaves of A. hastata and Spinacia oleracea (C3 plants), andin mesophyll and bundle sheath cells of A. spongiosa andSorghum bicolor (C4 plants). Expression of activities of theseenzymes on a Chl base provides a reasonable assessment of theextent 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 commonly5 to 10% of that of ribulose diphosphate carboxylase, andabout 5 to 10% of the usual photosynthetic rate in leaves ofall four species (2-5 ,umoles C02/min mg Chl). With minorvariations, these mitochondrial enzymes show similar specificactivity (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

RuDP carboxylasePEP carboxylaseNAD isocitrate dehydrogenaseFumaraseCitrate synthase

Cytochrome c oxidase

Number of experiments.2 SE of mean.

S. oleracea (4)1 A. hastata (4)A. spongiosa (3)

AMesophyll Bundle sheath

S. bicolor (2)

Afesophyll IBundle sheath

.smole suibstrate mi;i-r mg-' Chl2.340.730.140.160.09

+ 0.514 0.1+ 0.02i 0.03+ 0.01

15.5 ± 2.0

2.18 ± 0.70.32 i 0.050.15 i 0.09

0.070.10 i 0.04

1.0716.20.050.240.17

4 0.2i 0.14± 0.01± 0.04i 0.03

2.23 i 0.60.04

0.19 i 0.080.22 + 0.030.12 + 0.03

AA5,o mi,rI mg-' Chl18.3 i 2.2 10.9 ± 5.9 19.2 ± 4.8

13.50.270.110.06

0.290.150.050.05

7.6 5.0

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LIGHT AND RESPIRATION IN LEAVES. III

dase activity was also similar in all extracts with the possibleexception of S. bicolor. In this case, high levels of mercapto-ethanol used during extraction seemed to interfere with theassay, even after treatment with Sephadex G-25.

Metabolism of Succinate-"C in the Light. Preliminary ex-periments showed that when succinate-2, 3-"C was supplied toA. spongiosa leaves in the light, malate and aspartate wererapidly labeled and retained a high proportion of the radio-activity absorbed. The proportion of label in sucrose increasedrapidly throughout the experiments and this transfer ceasedwhen leaves were darkened.

These observations were confirmed in leaf slice experimentsusing specifically labeled succinate. The photosynthetic metab-olism of leaf slices in solution is closely comparable to that ofintact leaves (19), yet the technique permits the rapid applica-tion of labeled metabolites under controlled conditions. Inthese experiments the rate of release of "CO2 from succinate-1 ,4-"C in the dark greatly exceeded that from succinate-2,3-w"C (Table II). This was consistent with the removal, asCO2, of both labeled carbons in succinate-1,4-"C during thefirst turn of the tricarboxylic acid cycle. Little "CO2 was re-leased from either substrate in the light. However, succinate-1 , 4-"C and succinate-2, 3-`C were rapidly converted to sucroseand other photosynthetic products in the light at about equalrates (Table II). The conversion of succinate-"C to photosyn-thetic products was considerably more rapid than the rate of"CO2 release in the dark. Even if "CO2 release continued un-abated in the light, refixation of CO2 during photosynthesiscould not account for the labeling of photosynthetic products,particularly in the case of succinate-2,3-"C. The inhibition ofthe labeling of citrate by light (Table II) argues against a stim-ulation of the rate of "CO2 release in the light, and indicatesthat lable from succinate is diverted from the tricarboxylic acidcycle prior to citrate, possibly as malate or oxaloacetate.

Effects of Light on Tricarboxylic Acid Cycle Metabolism.The rapid, light-dependent transfer of "C from succinate-"Cto photosynthetic products in leaves of C4 plants contrastswith observations in leaves of C, plants. In the latter, theprincipal effect of illumination was a change in the distribu-

Table II. Metabolism of Succinate-1,4-I4C and Succinate-2,3-14Cin Slices of A. sponigiosa Leaf Tissue

14C Incorporated from:

Compound Treatment Succinate-l,4-14C Succinate-2,3-14C

15 min| 30 min 15 min 30 min

cpm X 10-3 per g fresh wi

4CO2 Light 2.8 5.6 0.9 1.5Dark 36.5 122.2 1.8 8.3

Phosphorylated com- Light 166.8 286.6 123.2 211.9pounds' Dark 0 0 0 0

Sugars Light 42.7 117.9 57.6 139.0Dark 2 0 0 0

Glycine + serine Light 68.9 87.4 37.3 74.4Dark 0 0 0 0

Citrate Light 32.7 59.8 48.5 57.3Dark -' 353.4 382.0 739.3

Malate + aspartate Light 291.3 524.5 243.8 330.2Dark 2 825.0 464.4 1192.8

1C absorbed Light 1792 2516 949 2054Dark 1960 2992 2243 4000

'Including 3-PGA and sugar phosphates.2 No sample.

_ vi

o X0c 0o

.ro a.,

-

80a

°OE -a

013 6 10 19 24 36

Time (minutes).

I.,,,'0 5 10 15 23

FIG. 1. Transient changes in malate (A) and aspartate (0) radio-activity on illumination of leaves of Atriplex spp. Excised leavessupplied with succinate-2,3-"C for 2 hr in the dark + 15 min inH20 before illumination. Solid bar represents darkness and openbar represents illumination.

tion of label between malate and aspartate and little "C en-tered photosynthetic pools (6). The experiment shown in Ta-ble II suggests that malate and aspartate, derived fromsuccinate, may leave the tricarboxylic acid cycle for photosyn-thetic pools in A. spongiosa in the light. Comparative experi-ments were therefore set up to examine the effects of a lighttransient on the fate of malate and aspartate derived fromsuccinate-2, 3-"C fed during a preceding dark period.

Figure 1 shows the interchange of malate and aspartatelabel upon illumination of A. hastata leaves, a response identi-cal to that in mung beans (7). In leaves of C4 A. spongiosa,however, this interchange did not take place, and both malateand aspartate lost label immediately upon illumination. Thisdifference in response was observed in all subsequent experi-ments. In these, the sum of aspartate and malate is plotted forsimplicity.

Figure 2a shows that in A. hastata, little radioactivity wastransferred to sucrose on illumination, a response consistentwith that in mung beans (6). The net loss of label from themalate + aspartate in the light reappeared in glutamate. Simi-lar changes were noted in leaves of the F, and F, hybrids ofA. rosea and A. patula spp. hastata (Fig. 2c), and very littlelabel was found in alanine, sugar phosphates, or sucrose inthese plants. In contrast to this, in both A. spongiosa andSorghum bicolor, illumination resulted in a rapid loss of labelfrom malate + aspartate and its appearance in photosyntheticintermediates (Fig. 2, b and d). The rapid labeling of alanine isconsistent with the labeling of pyruvate, presumably by de-carboxylation of 2,3-w"C labeled malate, oxaloacetate, or as-partate. Label enters the sugar phosphate pool at about thesame time and is subsequently transferred to sucrose. Thesubstantial lag in appearance of label in sucrose probably re-flects the lag in photosynthetic capacity on illumination ofdarkened leaves. These transformations did not take place indark controls and, as shown in Figure 2, b and d, maintenanceof sucrose and sugar phosphate labeling was strictly light de-pendent. Glycine and serine were labeled only after illumina-tion.

Comparable results were obtained in experiments in whichfumarate-2,3-"C, acetate-2-"C, citrate-1,5-"C, or "CO2 wereused to label tricarboxylic acid cycle intermediates in the dark.In all cases, illumination of leaves of the C4 plants A. spongi-osa and Sorghum bicolor resulted in a rapid loss of label frommalate and aspartate and its reappearance in photosyntheticproducts. A. hastata leaves were used as controls in these ex-

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periments and the transfer of carbon to photosynthetic prod-ucts was negligible.

Effects of Malonate and Fluoroacetate on Light-dependentChanges in Tricarboxylic Acid Intermediates. Leaves of A.spongiosa and Sorghum bicolor were supplied with succinate-2,3-'4C in the dark and then treated with water, 50 mM malo-nate (pH 4.0) or 100 mm fluoroacetate (pH 4.0) for a further30 min in the dark before illumination. Figure 3a shows resultsfor the light transient in A. spongiosa treated with malonateand Figure 3b shows results for Sorghum bicolor with fluoro-acetate. In both cases, these inhibitors drastically reduced thetransfer of label from malate + aspartate to sucrose. Theeffects on the labeling of other photosynthetic intermediateswere comparable, and both inhibitors were effective when usedwith leaves of either species. In most of the experiments whenmalonate or fluoroacetate were used, label accumulated ineither succinate or citrate respectively, suggesting that inhibi-tion of the tricarboxylic acid cycle had, in fact, occurred.

Degradation of Sucrose Labeled via Tricarboxylic AcidCycle Intermediates. The above experiments show that inleaves of C4 plants in the light sucrose and other photosyntheticintermediates are rapidly labeled, either from succinate-'4C fedto the leaves in the light, or from intermediates of the tricar-boxylic acid cycle labeled in a previous dark period. The datasuggest that the transfer involves the photosynthetic metabo-lism of malate, aspartate, or oxaloacetate labeled in the tricar-

50

40

30 .

20

-,

0

-0

0

ca-E

79LI

CL)31

2(

0 5 10 15 23

Time (minutes)FIG. 2. Transient changes in the distribution of 14C in malate +

aspartate (0 and * continuous dark control), sucrose (A), gluta-mate (0), alanine (7) and sugar phosphates (0) in excised leavessupplied with succinate-2,3-"C for 2 hr in the dark + 15 min inH20 before illumination. Solid bar represents darkness and openbar represents illumination.

50

7

o mco.2 a: 0

-o

IM -2

o8 r-0 .c

0 5 10 15 23 0 5 10 15 30

Time (minutes).

FIG. 3. Transient changes in the distribution of "4C in malate +asparate (0) and sucrose (A) on illumination of leaves suppliedwith succinate-2,3-'4C in the dark as in Figure 2. (a) A. sponzgiosaleaves treated with 50 mm malonate and (b) S. bicolor leaves treatedwith 100 mm fluoroacetate. Controls ( ); inhibitor treatments(---). Solid bar represents darkness and open bar represents illumi-nation.

Table 1II. Distr-ibiutioii of '4C in specific Carboni Atoms ofGlhcose-' 4C

Glucose-14C was prepared from sucrose-'4C which was ex-

tracted after illumination of leaves of C4 plants fed succinate-2,3-14C in the dark.

C-i C-2 C-3 C-4 C-D C-6

A. spontgiosa, 5 min lightA. sponigiosa, 10 min lightS. bicolor, 6 min lightS. bicolor, 9 min light

Sucrose-U-' 4CGlucose-I -'IC

17.6 19.318.1 24.8

9.1 '34.519.9 28.5

16.6 17.499.9 0.1

/0

17.2 10.814.5 8.96.9 6.13.1i 3.1

17.0 16.30.0 0.0

19.5 16.417.4 15.524.7 1.8628.6 16.6

16.5 16.10.0 0.0

boxylic acid cycle, rather than the refixation of respiratory'4CO2. This view is further supported by the distribution of '4Cwithin the carbon atoms of glucose-14C prepared from the su-

crose-4C produced in these experiments.Sucrose-14C formed in the first minutes of illumination of

A. spongiosa and Sorghumn bicolor leaves previously suppliedwith succinate-2,3-'4C in the dark (as in Fig. 2, b and d) was

converted to glucose-'4C and degraded enzymically. Table IIIshows that with the possible exception of A. spongiosa (5 min)the central carbons (3-C, 4-C) are less heavily labeled than themore terminal carbons (1-C, 2-C, 5-C, 6-C). Furthermore, thelabel in the central carbons declines with time, even though su-

crose label continues to increase (Fig. 2, b and d). This distri-bution and trend is not consistent with refixation of "4CO2being the principal route of '4C to photosynthetic products inthese plants.

DISCUSSION

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

A.spongiosa.a)

Sorghum bicolor. b)

0

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o-,

--

/

A.spongiosa.

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A.hastata. /

a)

o ./

m~ ~

0

3501 3 6 10 19 24

A-rosea x A.patulIa. c

,o 0

0/cn0_o_¢_^0

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LIGHT AND RESPIRATION IN LEAVES. III

isocitrate dehydrogenase is more or less constant in leaf cellsof all species examined. Thus, although some cells, such as thebundle sheath cells of A. spongiosa, seem to contain more mito-chondria than other cells (9) it seems likely that they also con-tain more chloroplasts or at least more Chl (31). Few quanti-tative examinations of electron micrographs have been madebut these suggest only minor differences in the ratio of chloro-plasts to mitochondria between mesophyll and bundle sheathcells of Zea and Sorghum, and between cells of C3 and C,grasses (12). A substantially lower ratio was recorded in thebundle sheath cells of Chloris which appears to resemble pub-lished micrographs of A. spongiosa (9, 12). The difficulties inmaking accurate estimates of this ratio are considerable, and onthe evidence available there is little support for the propositionof unusual tricarboxylic acid cycle activity in relation to photo-synthetic metabolism in these cells.The experiments are consistent with the continuation of tri-

carboxylic acid cycle activity in the light in leaves of C, plants,but do not indicate the relative rate of turnover in light com-pared with that in the dark. In this respect leaves of C, plantsare similar to those of C, plants (6, 7, 14, 15, 23) and to algae(24). We have assumed that the oxidation of succinate is an ex-clusive property of the mitochondria and that malonate andfluoroacetate are inhibitors of this process. We were unablehowever, to obtain satisfactory assays for succinate dehydro-genase in extracts or particles from leaf tissue, using publishedmethods.One important consequence of continued tricarboxylic acid

cycle activity in the light in leaves of C4 plants is the rapidtransfer of label from intermediates of the cycle to photosyn-thetic products. This process, which is clearly associated with afunctional C4 pathway (cf. the A triplex hybrids) and which de-pends on continued tricarboxylic acid cycle activity (cf. in-hibitor effects), was first noted by Karpilov (20) in comparativestudies between Zea and Phaseolus. The effects of illuminationon the metabolism of tricarboxylic acid cycle acids in leavesof C, mung beans and A. hastata is largely a response to thebalance of reduced and oxidized pyridine nucleotides (6, 7, 14,15), and very little carbon is transferred to photosyntheticpools. The exchange of carbon between the tricarboxylic acidcycle and photosynthesis in C, plants may occur via the refixa-tion of CO2 released during respiration or by the loss of inter-mediates from the tricarboxylic acid cycle.Two observations reported here indicate that the release and

refixation of respiratory CO2 is not the principal route ofcarbon exchange between the tricarboxylic acid cycle andphotosynthesis.

1. Release of "CO2 from succinate-1 ,4-"C during respira-tion greatly exceeds that released from succinate-2, 3-14C. Bothsubstrates are equally effective as a source of "C for photo-synthetic products, showing that the transfer is largely inde-pendent of the rate of respiratory 14CO2 production. 2. If thelabeling of sucrose from succinate-2, 3-1C in the light pro-ceeded by way of "4CO2, the central carbons of glucose-14C pre-pared from labeled sucrose would be somewhat more heavilylabeled than the terminal carbons due to the incorporation of"CO2 into 3-phosphoglycerate. In the shortest times used in thepresent experiments, carbons 1,2 and 5,6 were usually moreheavily labeled, consistent with the transfer of 2C,3C of suc-cinate via 2C, 3C of a labeled 3-carbon compound, rather thanvia "CO2. However, the possibility of dilution of the activity incarbons 3,4 of glucose by "2CO2 fixation cannot be excluded inthese experiments.

Leaves of many C4 plants contain high activities of malic en-zyme and P-enolpyruvate carboxykinase in bundle sheath cellswhich are believed to be involved in the decarboxylation of

malate and oxaloacetate during photosynthesis (1, 11). OtherC4 plants have high activities of aspartate amino transferasewhich may be involved in transamination of aspartate prior todecarboxylation (1). In each case the donor C4 acid yields CO2and a C, product, both of which may enter reactions of thecarbon reduction cycle. For example, in Sorghunm bicolor,labeled malic acid derived from succinate-1 ,4-"C would yield4CO2 and pyruvate-l-"C as a consequence of malic enzyme ac-tivity, and both labeled carbons may enter the carbon reductioncycle. If label proceeded as far as oxaloacetate, decarboxylationof 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-"Centers the carbon reduction cycle more rapidly than that ofthe remaining C, skeleton (16), the transfer of carbon fromsuccinate-I ,4-"C to photosynthesis should be initially morerapid than that from succinate-2, 3-`C. This was not observedin the present experiments, presumably because of the longtime scale of the comparative experiments.

This interpretation of the path of label from the tricarboxylicacid cycle to photosynthetic metabolism in C4 plants also ac-counts for the near absence of such a transfer in C, plantswhich contain much lower levels of malic enzyme. The ac-tivity of tricarboxylic acid cycle enzymes is similar in bothgroups of plants so release of CO2 from the tricarboxylic acidcycle and its refixation during photosynthesis is not likely toresult in the differences observed in Figure 2. We believe thatthey reflect the presence of a very active decarboxylation en-zyme for malate, oxaloacetate, or aspartate in C4 leaves whichis relatively inactive in C3leaves.

There is no evidence in the present experiments of differ-ences between Sorghum bicolor and A. spongiosa in terms oftricarboxylic acid cycle metabolism in relation to photosynthe-sis. The activity of tricarboxylic acid cycle enzymes and therelatively slow movement of respiratory CO2 into photosyn-thates argue against an important role for the cycle in CO2generation for photosynthesis. It seems unlikely that mito-chondria in bundle sheath cells could respire a proportion ofphotosynthetic pyruvate as proposed elsewhere (10). In Sor-ghuin bicolor, malic enzyme activity in bundle sheath cells isadequate to sustain the generation of CO2 from malate at ratesdemanded 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 andsome other C, species are deficient in malic enzyme andP-enolpyruvate carboxykinase (1, 11). In addition there is goodevidence that aspartate is the source of CO2 for the carbon re-duction cycle in these plants (16). Leaf mitochondria show adegree 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 inleaf mitochondria, believed to be specifically related to themetabolism of photosynthetic glycine, is 10-fold greater thanthat in mitochondria of other plant tissues (2, 21). Thus, al-though mitochondria in bundle sheath cells of A. spongiosa arenot unusually active with respect to the tricarboxylic acid cycle,they may be involved in the decarboxylation of photosyntheticaspartate (9). The recent identification of a very active isoen-zyme of aspartate amino transferase specific to bundle sheathmitochondria 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 av isiting research worker in the Research School of Biological Sciences at theAustralian National University, Canberra. We are v-ery grateful to BronxvnWilliams for her skilled assistance.

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LITERATURE CITED

1. ANDREWS, T. J., H. S. JOHNSON, C. R. SLACK, AND MI. D. HATCH. 1971. Malic

enzyme and aminotransfer ases in relation to 3-phosphoglycerate formationin plants with the C4-dicarboxylic acid pathway of photosynthesis. Phyto-chemistry 10: 2005-2013.

2. BIRD, I. F., M. J. CORNELIUS, A. J. KEYS, AND C. P. WHITTINGHAM. 1972.

Oxidation and phosphorylation associated with the conversion of glycineto serine. Phytochemistry 11: 1587-1594.

3. BLACK, C. C. AND H. H. MOLLENHAUER. 1971. Structure and distribution ofchloroplasts and other organelles in leaves with various rates of photo-synthesis. Plant Physiol. 47: 15-23.

4. BOGIN, E. AND A. WALLACE. 1969. Citrate synthase from lemon fruit.Methods Enzymol. 13: 19-22.

5. BRUINSMA, J. 1963. The quantitative analysis of chlorophylls a and b inplant extracts. Photochem. Photobiol. 2: 241-249.

6. CHAPMAN, E. A. AND D. GRAHAM. 1974. The effect of light on the tri-carboxylic acid cycle in green leaves. I. Relative rates of the cycle in thedark and the light. Plant Physiol. 53: 879-885.

7. CHAPMAN, E. A. AND D. GRAHAM. 1974. The effect of light on the tri-carboxylic acid cycle in green leaves. II. Intermediary metabolism and thelocation of control points. Plant Physiol. 53: 886-892.

8. Cox, G. F. 1969. Isocitrate dehydrogenase (NAD-specific) from pea mito-chondria. Methods Enzymol. 13: 47-51.

9. DOWNTON, W. J. S. 1971. The chloroplasts and mitochondria of bundlesheath cells in relation to C4 photosynthesis. In: M. D. Hatch, C. B.Osmond, and R. 0. Slatyer, eds., Photosynthesis and Photorespiration.Wiley-Interscience, New York. pp. 419-425.

10. EDWARDS, G. E. AND C. C. BLACK. 1971. Photosynthesis in mesophyll cellsand bundle sheath cells isolated from Digitaria sanguanalis (L.) Scop.leaves. In: M. D. Hatch, C. B. Osmond, and R. 0. Slatyer, eds., Photo-synthesis and Photorespiration. Wiley-Interscience, New York. pp. 153-168.

11. EDWARDS, G. E., R. KANAI, AND C. C. BLACK. 1971. Phosphoenolpyruvatecarboxykinase in leaves of certain plants which fix CO2 by the C4 di-carboxylic acid cycle of photosynthesis. Biochem. Biophys. Res. Commun.45: 278-285.

12. FREDERICK, S. E. AND E. H. NENwCOMB. 1971. Ultrastructure and distributionof microbodies in leaves of grasses with and without CO2 photorespiration.Planta 96: 152-174.

13. GENOVESE, J., K. SCHMIIDT, AND J. KATZ. 1970. Enzymic degradation of iso-topically labeled compounds. I. Degradation of 14C-labeled glycerol. Anal.Biochem. 34: 161-169.

14. GRAHAM, D. AND J. E. COOPER, 1967. Changes in levels of nicotinamide adeninenucleotides and Krebs cycle intermediates in mung leaves after illumina-tion. Aust. J. Biol. Sci. 20: 319-327.

15. GRAHAM, D. AND D. A. WALKER. 1962. Some effects of light on the intercon-version of metabolites in green leaves. Biochem. J. 82: 554-560.

N4D OSMOND Plant Physiol. Vol. 53, 1974

16. HATCH, M. D. 1971. The C4 pathway of photosynthesis. Evidence for an in-termediate pool of carbon dioxide and the identity of the donor C4 di-carboxylic acid. Biochem. J. 125: 425-432.

17. HATCH, M. D., C. B. OSMOND, J. H. TROUGHTON, AND 0. BJ6RKMAN. 1972.Physiological and biochemical characteristics of C3 and C4 Atriplex speciesand hybrids in relation to the evolution of the C4 pathway. Carnegie Inst.Wash. Yearbook, 1971. pp. 135-141.

18. HILL, R. L. AND R. A. BRADSHAW. 1969. Fumarase. Metliods Enzymol. 13:91-99.

19. JONES, H. G. AND C. B. OSMOND. 1973. Photosynthesis of thin leaf slices insolution. I. Teclniques. Aust. J. Biol. Sci. 26: 15-24.

20. KARPILOV, Y. S. 1970. Photorespiration in the leaves of maize. Proc. MoldavianInst. Irrig. Veg. Res. 2: 46-64.

21. KISAKI, T., N. YOSHIDA, AND A. IMAI. 1971. Glycine decarboxylase and serineformation in spinach leaf mitochondrial preparation with reference tophotorespiration. Plant Cell Physiol. 12: 275-288.

22. LAETSCH, W. M. 1971. Chloroplast structural relationships in leaves of Csplants. In: M. D. Hatch, C. B. Osmond, and R. 0. Slatyer, eds., Photo-synthesis and Photorespiration. Wiley-Interscience, New York. pp. 323-349.

23. LUDWIG, L. J. AND D. T. CANVIN. 1971. The rate of photorespiration duringphotosynthesis and the relationship of the substrate of light respiration tothe products of photosynthesis in sunflower leaves. Plant Physiol. 48: 712-719.

24. MARSH, H. V., J. M. GALMICHE, AND M. GIBBS. 1965. Effect of light on thetricarboxylic acid cycle in Scenedesmus. Plant Physiol. 40: 1013-1022.

25. MULCHI, C. L., R. J. VOLK, AND W. A. JACKSON. 1971. Oxygen exchange ofilluminated leaves at carbon dioxide compensation. In: M. D. Hatch, C.B. Osmond, and R. 0. Slatyer, eds., Photosynthesis and Photorespiration.Wiley-Interscience, New York. pp. 35-50.

26. OSMOND, C. B. A-ND B. HARRIS. 1971. Photorespiration during C4 photo-synthesis. Biochim. Biophys. Acta 234: 270-282.

27. OSMOND, C. B., J. H. TROUGHTON, AND D. J. GOODCHILD. 1969. Physiological,biochemical, and structural studies of photosynthesis and photorespirationin two species of Atriplex. Z. Pflanzenphysiol. 61: 218-237.

28. RAVEN, J. A. 1972. Endogenous inorganic carbon sources in plant photo-synthesis. I. Occurrence of the dark respiratory pathways in illuminatedgreen cells. New Phytol. 71: 227-247.

29. SCHMIDT, K., J. GENOVESE, AND J. KATZ. 1970. Enzymic degradation of iso-topically labeled compounds. II. Glucose labeled with 14C and tritium. Anal.Biochem. 34: 170-179.

30. VOLK, R. J. AND W. A. JACKSON. 1972. Photorespiratory phenomena in maize.Oxygen uptake, isotope discrimination, and carbon efflux. Plant Physiol.49: 218-223.

31. Woo, K. C., N. A. PYLIOTIS, AND W. J. S. DOWNTON. 1971. Thylakoid aggre-gation and chlorophyll alchlorophyll b ratio in C4 plants. Z. Pflanzenphys-iol. 64: 400-413.

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