Plant Physiol. (1974) 53, 879-885

The Effect of Light on the Tricarboxylic Acid Cyclein Green LeavesI. RELATIVE RATES OF THE CYCLE IN THE DARK AND THE LIGHT

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

E. A. CHAPMAN AND D. GRAHAMCommonwealth Scientific and Industrial Research Organization, Division of Food Research, and School ofBiological Sciences, Macquarie University, North Ryde 2113, Sydney, Australia

ABSTRACT

Excised green leaves of mung bean (Phaseolus aureus L. var.Mungo) were used to determine the effect of light on the rateof endogenous respiration via the tricarboxylic acid cycle. IL-lumination with white light at an intensity of 0.043 gram cal-ories cm'minn (approximately 8600 lux) of visible radiation(400-700 nm) gave a rate of apparent photosynthesis, mea-sured as net C02 uptake, of 21 mg CO2 dm-2hr-' which wasabout 11-fold greater than the rate of dark respiration. Thefeeding of '4CO2 or 14C-labeled acids of the tricarboxylic acidcycle in the dark for 2 hours was established as a suitablemethod for labeling mitochondrial pools of cycle intermediates.

At a concentration of 0.1 mM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, apparent photosynthesis was inhibited 82%,and the refixation of 14CO2 derived internally from endoge-nous respiration was largely prevented. In the presence of thisinhibitor endogenous respiration, measured as "CO2 evolution,continued in the light at a rate comparable to that in the dark.Consequently, under these conditions light-induced nonphoto-synthetic processes have no significant effect on endogenousdark respiration. Inhibitors of the tricarboxylic acid cycle,malonate and fluoroacetate, were used to determine the rela-tive rates of carbon flux through the cycle in the dark and inthe light by measuring the rate of accumulation of 4(C in eithersuccinate or citrate. Results were interpreted to indicate thatthe tricarboxylic acid cycle functions in the light at a rate simi-lar to that in the dark except for a brief initial inhibition ontransition from dark to light. Evidence was obtained that suc-cinate dehydrogenase as well as aconitase, was inhibited in thepresence of fluoroacetate.

Controversy has surrounded the question of the effect oflight on endogenous aerobic respiration via glycolysis and thetricarboxylic acid cycle in algae and green leaves of higherplants. Reports have included stimulation, inhibition, or noeffect of light on respiration. For example, physiological stud-ies, using the mass spectrometer to measure respiratory andphotosynthetic gas exchange simultaneously, showed that rela-tively low light intensities had little or no effect on rate ofrespiratory oxygen uptake in barley leaves or in suspensionsof the alga Chiorella (8). With the green alga Ankistrodesmusbraunii (9) or the algal flagellate Ochromonas malhamensis(52) it was found that respiratory CO2 evolution was almost

independent of light intensity, and that respiratory oxygenconsumption was not affected by low light although it wasenhanced at high light intensities. Hoch et al. (24) concludedfrom their results obtained with the mass spectrometer thatlow light intensities inhibited endogenous respiration in Ana-cystis, a blue-green alga, but not in Scenedesmus, a green alga,whereas higher light intensities promoted oxygen uptake inboth organisms. The total oxygen consumption or CO2 evolu-tion cannot be separated, however, into dark endogenous res-piratory and light-induced photorespiratory components, andthe metabolic system(s) responsible for each can only be in-ferred from physiological experiments (26, 33). In view ofthe complex interactions of photosynthesis, photorespiration,and endogenous respiration, an accurate assessment of en-dogenous respiration in the light is difficult to attain on thebasis of physiological measurements of gas exchange.

Early biochemical studies indicated that photosyntheticallyincorporated "CO2 (4, 7) did not readily enter the tricarboxylicacid cycle intermediates in the light, nor was pyruvate oxida-tion readily achieved in the light (18, 36). The interconver-sion of exogenously fed "C-labeled intermediates of the tri-carboxylic acid cycle was shown to occur both in the light andin the dark in leaves of mung bean, and it was suggested thatthe observed reversal by light of the ratio of "C in malate-aspartate in the dark was the result of differences in the ratioof NAD/NADH between light and dark (20). This postulatewas confirmed and correlated with changes of "C in inter-mediates of the tricarboxylic acid cycle during dark-lighttransition (19). It has been clearly demonstrated that light in-duces changes in the ratio of ATP/ADP (43) and in the ratioof oxidized-reduced nicotinamide adenine dinucleotides (19,22, 23, 37).

In Chlorella, a light-induced blockage of glycolysis at thetriose phosphate dehydrogenase step was proposed (27) whichis probably due to the changes in the ratio ATP/ADP (23,43). It is clear from these various studies that light has markedeffects upon the relative levels of cofactors of respiratorymetabolism and the pool sizes of respiratory intermediates.

In a different approach from those described above, thedetermination of the rates of equilibration of "C in the car-boxyl carbons of tricarboxylic acid cycle intermediates inScenedesmus (35) showed that there were no significant dif-ferences between those in the dark and in light saturating forphotosynthesis. The results were interpreted as showing nodifference in rate of the tricarboxylic acid cycle in the twoconditions.Many of the reported variations in the response of respira-

tion to light are doubtless due to the wide variation in experi-mental regimes, some of which included conditions suboptimal

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CHAPMAN AND GRAHAM

for photosynthesis. In the experiments to be described here,excised leaves of a higher plant have been used under relativelyhigh light intensities such that photosynthesis is operating ata rate some 10- to 12-fold greater than dark respiration.

In this paper the suitability of the experimental materialand experimental design, in particular the use of inhibitors,has been examined. An assessment of the relative fluxes ofcarbon in the tricarboxylic acid cycle in dark or light hasbeen made using inhibitors of photosynthesis (DCMU) and ofthe tricarboxylic acid cycle (malonate and fluoroacetate). Theresults indicate that, apart from an inhibition during the firstfew minutes of illumination, the tricarboxylic acid cycle op-erates in the light at a rate comparable to that in the dark.

MATERIALS AND METHODS

Growth of Plants. Mung beans (Phlaseolus aureus L. var.Mungo, Yates Seeds, Sydney, Australia) were grown from seedfor 7 days as described by Graham and Cooper (19).

Experimental Procedures. On the 8th day after planting,seedlings were placed in the dark for 2 hr and then the1stpair of leaves was harvested under a green safelight (54) byexcision at the petiole.

Leaves were fed radioactive isotope in the dark in the fol-lowing manner.

1.'CO.. Twenty leaves (total fresh weight 500 + 10 mg)were placed on moist filter paper in a 250-ml Petri dish inwhich "CO2 was liberated by injecting Na2,4CO3 into lacticacid. The initial concentrationof "'CO2 was about 0.2% (v/v).

2. "C-labeled tricarboxylic acid cycle acids. Leaves wereplaced in feeding trays as described previously (20) and 0.4 mlof radioactive isotope solution was fed to five leaves throughthe transpiration stream.At the end of 2 hr in the dark and before subsequent illumi-

nation, the leaves were removed from the "4C source andplaced in water in an air draught for 15 min.

Illumination. Leaf samples were illuminated by a 250 wtungsten-filament reflector spotlight placed at a distance of 35cm. The samples were maintained at room temperature (20-25 C) by using a water-cooled heat filter (53). Light intensitywas measured with a Kipp actinometer and was as follows.

1. Withi the Perspex lid in place. Total radiation was 0.089g cal cm-min-' (approximately 17,800 lux). Visible radiation(400-700 nm) was 0.043 g cal cm-2min-' (approximately 8,600lux).

2. Withlout Perspex lid. Total radiation was 0.095 g calcm2mirf' (approximately 19,000 lux). Visible radiation was0.046 g cal cMn2min` (approximately 9,200 lux).

Measuirement of CO2 Evolution. To collect 'CO2 or `CO2the feeding tray was covered with a gas-tight Perspex lid togive a vessel of 200-ml volume, and was connected to a supplyof compressed air flowing at the rate of 400 ± 5 ml min-1.The sample size was 10 leaves. 12CO, evolved was measuredusing a Beckman infrared gas analyzer (Model 315). "CO2was trapped in vials, each containing 5.5 ml of methanol-ethanolamine (4:1, v/v) (1), linked in series. At the end ofthe collection period, 10 ml of toluene scintillator were addedto each of these vials.

Extraction of Intermediary Metabolites. After light or darktreatment, intermediary metabolites were extracted from leavesby boiling in 80% aqueous ethanol (v/v) for 3 min followedby two extractions with boiling 20% aqueous ethanol (v/v)(5). The ethanolic extract was dried under reduced pressureat 30 to 35 C, resuspended in 1 ml of 50% ethanol, andwashed with 4 ml of chloroform which was separated to giveaqueous ethanol-soluble and chloroform-soluble fractions, re-spectively.

Chloroform-soluble Compounds. Chloroform fractions weredried and resuspended in 1 ml toluene and 0.2-ml sampleswere counted in toluene fluor (see below).

Ethanol-soluble Compounds. Samples of each fraction werechromatographed on Whatman No.1 paper using a 2-dimen-sional system; phenol-water (100 g/39.5 ml) in the first direc-tion and 1-butanol-acetic acid-water (74:19:45) in the seconddirection (6).

Insoluble Material. Residues of extracted leaves were solu-bilized by homogenizing them in 2 ml of NCS solubilizer(Nuclear Chicago Corporation, Des Plaines, Illinois, U. S. A.)contained in a 5-ml all-glass homogenizer with a motor-drivenplunger. The clear homogenate was made to5 ml with tolueneand 0.1 ml samples were counted in toluene fluor (see below).

Isolation of Intermediates. Chromatographs of ethanol-solu-ble compounds were radioautographed and preliminary identi-fications were made from published sources. All radioactiveareas were then cut from chromatographs, placed in toluenefluor, and counted. To confirm the identification of most com-pounds, papers were removed from the scintillant, washed withethyl ether, eluted with 50% ethanol, and chromatographed onWhatman No.1 paper with the appropriate known compoundsin at least two solvents as follows: amino acids: (a) 1-butanol-propionic acid-water (10:5:3) (6); (b) phenol-ammonia-water(160 g:0.5 ml:40 ml) (44). Organic acids: (a) 95% ethanol-ammonia-water (8:1:1) (41); (b) 1-butanol-90% formic acid-water (10:3:10) (41). Phosphate esters: (a) isobutyric acid-1N ammonia-0.1M EDTA (100:60:1.6) in the first direction,then 1-butanol-propionic acid-water (375:180:245) in thesecond direction (46), using Whatman No. 4 paper washedin aqueous EDTA (15): (b)1-propanol-ammonia-water (6:3:1)modified after Isherwood and Hanes (25); (c) ethyl acetate-acetic acid-water (3:3:1) (5).Compounds were detected on the chromatographs by spray-

ing with the following locating reagents: amino acids-nin-hydrin solution (30); organic acids-bromophenol blue solu-tion (44); phosphate esters-either (a) the reagent of Hanesand Isherwood (21) as modified by Bandurski and Axelrod(2), or (b) Rosenberg's reagent (42).Measurement of Radioactivity. Radioactivity was measured

in a Packard Tri-Carb liquid scintillation spectrometer, Model3375, after addition of 15 ml of toluene fluor (3 g of 2,5-diphenyloxazole and 0.2 g of p-bis-2,5-diphenyloxazole in 1litre of toluene) to a glass vial containing "C-labeled materialon paper or in methanol-ethanolamine mixture. Radioactivityin aqueous samples was measured by the addition of 10 ml ofmix I (toluene fluor-Triton X-100, 2:1) (38) to 1 ml of sam-ple. Where appropriate, corrections for quenching and effi-ciency were made.

Uptake of Dye. The rate at which water entered a cut leafin the dark or in the light was observed by placing the leafin 0.1 % aqueous ethanol containing a small quantity ofmethyl red, and following its path through the leaf under amicroscope.

Stomatal Count. Leaves previously fed "'C-acetate for 2 hrin the dark were excised, and casts were made of both sidesof the leaves (in the dark or after exposure to light for 30and 60 min) using clear nail polish. The numbers of open orclosed stomates were counted in the casts using a Reichert-Visopon projection light-microscope.

Radiochemicals. "C-Labeled compounds were obtainedfrom the Radiochemical Centre, Amersham, England andhad the following specific activities: acetate-2-"C, 38 mc/mmole; "CO. as Na2"CO,. 56 mc/mmole; citrate-I ,5-14C,15.8 mc/mmole; fumarate-2,3-14C, 6.95 mc/mmole; malate-U-`C, 47.2 mc/nimole: and succinate-2,3-14C, 1.45 mc/mmole. One ,ic was used per leaf in all cases except "C-

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LIGHT AND RESPIRATION IN LEAVES. I

succinate when 0.13 ytc per leaf was used. All "C-substrateswith the exception of 'CO2 were checked for purity by 2-dimensional paper chromatography (6) and radioautography.

RESULTS AND DISCUSSION

Experimental Condition of Excised Leaves. The results re-ported in this paper indicate that excised leaves of mungbean are suitable material for investigating the influence oflight on endogenous respiratory metabolism via the tricar-boxylic acid cycle. All leaf cells showed substantial uptakeof the dye, methyl red, within 15 min of application. It islikely, therefore, that rapid distribution of solutes occursthroughout the leaf.

In the dark about 40% of the stomates were closed whereasafter illumination for 30 min under the usual experimentalconditions only 5% remained closed.When CO,-exchange was measured with an infrared gas

analyzer, the usual value for dark respiration was about 1.9mg dm-hr-' while after 30 min illumination the value forphotosynthesis was approximately 11-fold greater (21 mgdm-2hr-').

Uptake and Redistribution of Radioisotopes. The time re-quired for gross uptake by leaves of a radioactive substratewas determined by sampling the external solution during thefeeding period in the dark. By measuring "CO2 evolved fromthe leaves under similar conditions, the time course of me-

tabolism of a substrate was determined. "CO2 evolutionreached a maximum rate between 80 and 100 min and there-after declined very slowly, while uptake of "C-labeled solu-tion was essentially completed by 120 min. Therefore, inorder to achieve maximal uptake of "C and distribution toactive metabolic pools, a standard time of 2 hr was used forthe feeding of labeled substrates. Uptake of "C-substratesvaried with each leaf sample and accordingly, to enable com-

parisons to be made, data are generally expressed in this andthe subsequent papers (12, 13) as percentages of the "C inthe total ethanol-soluble fraction.The percentages of the fed substrates which were recovered

from the leaf samples unchanged (but including also "C me-

tabolized into the corresponding metabolic pool(s)) variedfrom 4.4% to 20.7% of that taken up. Because no markedutilization of this portion of the substrate occurred duringthe experimental period, it is concluded that such substratewas not readily available to the active metabolic pools. Fur-ther, the total amount of any of the "C-substrates fed duringthe 2-hr dark period was approximately equal to the total

pool size in the leaf sample (34). Because most of the fedsubstrate was metabolized during the dark period, it is con-sidered unlikely that the pattern of metabolism of the excisedleaves subsequently was greatly disturbed from normal by thefeeding of the substrates in the manner indicated.

Table I shows the gross distribution of "C among threemajor fractions, ethanol-solubles, chloroform-solubles, andinsolubles (for definition of these fractions see "Materialsand Methods"). Illumination resulted in relatively minorchanges in percentage distribution among the fractions exceptafter "C-succinate feeding, when a large increase of "C in theinsoluble fraction occurred.

Effect of DCMU. To determine whether or not respiratory"CO. production is influenced by photosynthesis or by someother light-induced process, photosynthetic electron transportwas inhibited by DCMU. The effectiveness of various con-

centrations of DCMU on "CO2 fixation in the light was de-termined. At 0.1 mM DCMU, photosynthesis (CO2 fixation)was inhibited 82% relative to the illuminated control. Thisconcentration of DCMU is high but the ability of the leavesto continue respiring at the same rate in the dark for at least6 hr indicated that the inhibitor was not deleteriously affect-ing the respiratory metabolism of the leaves.The effects of 0.1 mm DCMU on "CO2 evolution in the

dark and in the light when "CO2 or citrate-1 ,5-"C were fedto leaves in the dark are shown in Figure 1, a and b, respec-

tively. In leaves previously fed "CO2 in the dark, there is no

effect of the inhibitor on evolution of "CO2 in the dark nor

on the initial decrease in '"CO2 evolution in the first minutesof illumination. However, after about 5-min illumination, therate of "CO2 evolution returns to a level similar to that inthe previous dark period. Return to darkness after 30-minillumination gave an almost negligible "CO,-burst comparedwith the control.

These results are consistent with the inhibition of photo-synthesis by DCMU and with the partial cessation of theCO2-evolving mechanism (tricarboxylic acid cycle) in the firstminutes of illumination. Subsequently in the light, it is sug-

gested that the rate of respiratory production of "CO2, mainlyvia the tricarboxylic acid cycle, is similar to that in the dark.Presumably in the light, even when photosynthesis is largelyinhibited, a regulatory mechanism still operates to maintaina constant internal "CO2 level.When citrate-1,5-"C was fed in the dark, the effect of

DCMU on "CO. evolution in the light again supports theconclusion that the rate of endogenous respiration is similarto that in the dark. The initial rapid decrease in "CO, evolu-

Table 1. Distributiont of 14C amontg Extracted Fractions after Feedinig '4C-substrates to Leaves in the Dark

Extracted Fraction

Substrates Treatment Ethanol-soluble Chloroform-soluble Insoluble

cpm X 10-5 % of total cpm X 10-5 % of total cpm X 10-5 extralcpmX ~extracted cmX1' extracted cexi' % ftoatal14CO2 Dark 2.13 65.0 0.17 5.3 0.97 29.7

Dark plus 30 min light 2.71 61.0 0.01 0.3 1.50 38.7Acetate-2-1"C Dark 3.21 38.2 2.53 30.0 2.67 31.8

Dark plus 30 min light 7.41 45.3 3.88 23.7 5.08 31.0Succinate-2,3-"C Dark 4.82 80.8 0.28 4.7 0.86 14.5

Dark plus 30 min light 1.40 55.6 0.26 10.4 0.86 34.0Fumarate-2,3-lC Dark 4.19 72.4 0.45 7.7 0.12 19.9

Dark plus 10 min light 11.70 83.1 0.68 4.8 2.10 12.1Citrate-i,5-"C Dark 1.65 58.0 0.39 13.6 0.81 28.4

Dark plus 30 min light 3.00 69.6 0.47 11.0 0.84 19.4

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CHAPMAN AND GRAHAM

13

12

x a)

60 70 80 90 100 110 120 130 140MINUTES AFTER APPLICATION OF DCMU

FIG. 1. Effects of illumination on "'CO2 evolution by leaves inthe presence of 0.1 mM DCMU. Samples of 10 excised leaves werefed (a) `CO2 or (b) citrate-1, 5-`C for 2 hr in the dark then weretransferred to distilled water, and "CO2 evolution was measured asdescribed in "Materials and Methods." DCMU (in 1% v/v aqueousethanol) was present in the dark and during illumination. Controlswere in 1% (v/v) aqueous ethanol. Control: solid line; DCMU:broken line. Solid bar represents darkness and open bar repre-sents illumination.

tion in the first minutes of illumination is, however, not evi-dent, although the average evolution over the first 5-min pe-riod is slightly less than that in the dark or in the subsequentperiod in the light.

Continued evolution of "CO. in the light, in the presenceof DCMU at rates comparable with those in the dark, indi-cates that light-induced nonphotosynthetic processes, for ex-ample blue light effects (14, 29), have no significant effect onendogenous respiratory processes under the conditions ofthese experiments.

Determination of Carbon Fluxes in the Tricarboxylic AcidCycle in the Presence of Inhibitors. In order that the complexdata resulting from light-induced changes in intermediarymetabolites may be interpreted in terms of control points,recourse will be made in the following paper (12) to the useof the Chance crossover theorem (11). The application ofthis theorem requires that the relative flux of carbon be knownon transition from one condition to another. We postulatehere that a change in the rate of accumulation of carbon inan intermediate of the tricarboxylic acid cycle prior to ablock by an inhibitor would indicate a change in flux ofcarbon through the cycle. Accordingly, measurement of car-bon flux changes has been attempted in detached leaves ontransition from dark to light in the presence of inhibitors ofthe tricarboxylic acid cycle. The inhibitors chosen were malo-

nate to follow the accumulation of '4C in succinate, andfluoroacetate to follow the "4C accumulation in citrate.

Malonate, at the concentrations used in the present experi-ments, is a relatively specific inhibitor for succinate dehy-drogenase (51). The implication that malonate is an inhibitorof photosynthesis has no sound foundation. Both the findingsof Bassham et al. (3) showing inhibition of 14CO2 entry intomalate, then thought to be a primary product of photosynthe-sis, and the observation of Kortschak et al. (28) that malo-nate inhibits malate formation in sugar-cane leaves may beexplained on the basis of an inhibition of the tricarboxylicacid cycle; e.g., see Chapman and Osmond (13).

Fluoroacetate is a competitive inhibitor of mitochondrialaconitase. In plants however, it is not as potent an inhibitoras it is in animals. There are at least three possible reasonsfor this: (a) insensitivity of plant aconitase to fluorocitrate(31, 32, 45, 49, 50); (b) inability of either the thiokinase orcitrate synthase to accept the fluoroderivative (16); and (c)defluorination of fluoroacetate (39, 40, 49, 50). Plants, how-ever, are capable of forming fluorocitrate, (32, 49, 50, 55)and citrate does accumulate though to a lesser extent than inanimal tissues (16, 49, and this paper). The concentrations offluoroacetate necessary to demonstrate aconitase inhibitionwill vary considerably depending upon the detoxificationmechanism in operation. Although fluorocitrate is thought tobe specific for mitochondrial aconitase, inhibition of succinatedehydrogenase has been reported (10, 17) and we also found evi-dence for this (Table II and Fig. 3b). Vickery and Vickery (47)have suggested that fluoroacetate biosynthesis in Dichapetalum

Table II. Effect of Either Fluoroacetate or Maloniate onz Light-iniduced Changes in '4C-Labeled Initermediates of the

Tr-icarboxylic Acid CycleAfter 2 hr feeding of 14CO in the dark each sample of 5 leaves

was removed and placed either in water (control) or in 0.1 Mfluoroacetate at pH 4.0 for 10 min in the dark or in 0.01 M malonatefor 80 min in the dark. Values represent percentage of distributionof 14C in total ethanol-soluble compounds. Values in bracketsrepresent the difference between light and dark samples.

atI M lC Fluoroacetate 0.01 i Malonate

Intermediate

Dark 20 min Dark 20 min Dark 20 minlight light light

Aspartate 18.3 4.5 22.4 14. 1 12.9 8.2(-13.8) (-8.3) (-4.7)

Citrate 14.6 14.0 15.4 19.5 20.3 7.2(-0.6) (+4.1) (-13.1)

Fumarate trace trace 1.4 2.4 3.7 4.7(+1.0) (+1.0)

Malate 39.1 53.3 17.0 19.8 13.0 20.5(+14.2) (+2.8) (+7.5)

Succinate 2.0 1.7 20.2 18.3 24.9 32.7(-0.3) (-1.9) (+7.8)

Glutamate 16.8 11.5 15.6 16.5 15.2 10.1(-5.3) (+0.9) (-5.1)

Other organic 8.0 9.6 7.2 8.1 9.4 12.0acids and amino (+1.6) (+0.9) (+3.6)acids

Sugar phosphates 0 0.9 0 0.3 0 0.7(+0.9) (+0.3) (+0.7)

Sucrose 1.2 4.5 0.8 1.0 0.6 3.8(+3.3) (+0.2) (+3.2)

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LIGHT AND RESPIRATION.IN LEAVES. I

is intimately associated with photosynthesis but there seemsto be no evidence to support this. In the present experimentswith mung bean leaves neither CO2 nor `CO2 fixation was in-hibited by 0.1M fluoroacetate, compared with controls over aperiod of 2 hr though in this time "C accumulated in citrate."CO, evolution in the dark and the accumulation of either

"C-succinate or "C-citrate were used to determine the lowestconcentrations of the two inhibitors effective as partial in-hibitors of respiration. When "'CO, was the labeled substrate,"CO2 evolution in the dark was inhibitod 50% by 0.O1Mmalonate and 19% by 0.1M fluoroacetate. "C-succinate ac-cumulated in the presence of malonate and "C-citrate accu-mulated in the presence of fluoroacetate. Lower concentra-tions, e.g., 5mM malonate or 50mM fluoroacetate, were in-effective even after prolonged periods of exposure. It shouldbe noted that inhibition of the tricarboxylic acid cycle, mea-sured as "CO2 evolution, may well be higher than 50% and19% since MacLennan et al. (34) have shown that at least50% of the CO2 evolved from some plant tissues arises fromreactions outside the tricarboxylic acid cycle.

Under similar experimental conditions, when acetate-2-"4Cor fumarate-2, 3-"4C were fed a stimulation of "CO2 evolutionoccurred in the presence of either malonate or fluoroacetate.Stimulation was greatest at the lowest concentrations of in-hibitors. Stimulatory effects of fluoroacetate on respirationhave also been reported for lettuce leaves (48) but no satis-factory explanation of these findings can be offered.

Using "CO2 as the labeled substrate and 0.O1M malonate or0.1M fluoroacetate as the inhibitors, the relative rates of ac-cumulation of 14C in either succinate in the presence of 0.O1Mmalonate, or citrate in the presence of 0.1M fluoroacetatewere determined in the dark and in the light.The results of a representative experiment with malonate

are shown in Figure 2. In the presence of malonate in thedark, a considerable accumulation of "C-succinate was foundand accumulation continued during the time course of theexperiment (compare with Table I in ref. 12). This result isconsistent with inhibition of succinate dehydrogenase in thetricarboxylic acid cycle. Illumination of the leaves resulted ina small but reproducibly consistent decline in the accumula-tion of succinate during the first 5 min of illumination, which

4

zC-LA0I. a

z

230ocn

zo

_ Zrz '

a.

zF 0

I.-

80 90 100 110TIME (MIN )

FIG. 2. Effects of illumination on th"C in "C-succinate in the presence of"CO2 was fed in the dark to samples ofleaves were transferred to the inhibitcthe dark was begun after 80 min and ,

illuminated from 120 min. Dark: solid li

Un0z

g2

0

-wmJ0U'

0 1z4I

J

0

z0

z0

00

z

w 2

Wr

a

10 20 30 40 50

TIME ( MIN )60 70

FIG. 3. Effects of illumination on the percentage distribution of"C in "C-citrate and "C-succinate in the presence of 0.1 M fluoro-acetate at pH 4.0. Conditions were similar to those described inFigure 2 but sampling began after 10 min and illumination beganat 45 min: a: "C-citrate; b: "C-succinate. Dark: solid line; light:broken line.

is interpreted as a decline in carbon flux through the cycle.Subsequently in the light, accumulation of "C-succinate re-sumed at a rate which was as high or higher than that ob-served in the dark.

This initial decline in carbon flow through the tricarboxylic,S0 acid cycle, although apparently small, was obtained consist-

0/ ently. It was obtained for "CO, evolution in the presence of/ DCMU in two separate duplicated experiments with "CO2 as/y -O substrate (Fig. 1 a), and one duplicated experiment with "C-

citrate as substrate (Fig. ib). The decline in the percentage of< / 4"C in succinate was obtained in three separate experiments

with malonate as inhibitor (Fig. 2), and in two experimentsout of three when fluoroacetate was the inhibitor (Table IIand Fig. 3).

Changes in the percentage of "C in citrate in the lightT ON paralleled those in the dark control in two experiments (Fig.

3a) with fluoroacetate, while in the third experiment (in which40% of "C accumulated in citrate), "C accumulated more

EI rapidly in the light for 4 min and then paralleled the dark120 130 140 150 control. The explanation of the "C-citrate data is necessarily

complicated because of the position of citrate at the entry tothe cycle and its relation to controls in the cycle. If citrate

0e01 M malonate at pH4ri.b synthase is inhibited in the light then carbon flow into citrateEfive leaves for 2 hr and the will be slowed. If, however, isocitrate dehydrogenase is also)r in the dark. Sampling in inhibited in the light then this will tend to inhibit carbon flowa series of leaf samples was out of citrate. The result will depend upon the degree toine; light: broken line. which citrate synthase and isocitrate dehydrogenase are in-

a)

!0- o--O~---0

10 CCITRATE

IiLIGHT ON

c 1

b)

SUCCINATE

' 0 -O,

10

1iLIGHT ON

- SUCCINATE

1. LIGH

I I I

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CHAPMAN AND GRAHAM

hibited and upon the temporal relationship of the control bythese enzymes. Hence it is likely that when aconitase is onlypartially inhibited, as in two of our experiments, then thecontrols at citrate synthase and isocitrate dehydrogenase mayresult in an imperceptible change in '4C in citrate. Whenaconitase is more fully inhibited, citrate-'4C accumulates fasterin the light than in the dark for the first 4 min while at thesame time '4C-succinate declines. Such a result is consistentwith a slower flow of carbon out of citrate.

Likewise the initial decline in "4C-succinate may be inter-preted either as a decreased synthesis or an increased utiliza-tion of succinate. The ratio of NAD/NADH is known to de-crease in the light (19) thereby restricting the oxidation ofisocitrate and malate. Such a situation favors a slower entryof "4C into succinate rather than a faster removal from suc-cinate. In addition, no evidence was obtained of an increasedsynthesis in the light of Chl, an alternative product of succinylCoA metabolism.

Therefore, taken together with the results of the experi-ments with DCMU (Figs. la and lb), it is reasonable to in-terpret the changes observed on illumination in the presenceof malonate (Fig. 2) or fluoroacetate (Fig. 3) as an initial de-cline in flow of carbon through the tricarboxylic acid cycle,followed by a return to a rate which is about the same orsomewhat greater than that occurring in the dark.

In the light, malonate and fluoroacetate markedly inhibitedthe increase in labeled malate and the decrease in labeledaspartate compared with a control (Table II). Because malo-nate and fluoroacetate are mitochondrial enzyme inhibitors,such a finding indicates that the labeled malate and aspartatepools are either predominantly mitochondrial or in rapidequilibrium with the mitochondrial pool.

CONCLUSIONS

It has been established that excised mung bean leaves aresuitable material in which to measure the effects of moderatelyhigh light intensity, which is sufficient to nearly saturatephotosynthesis, on endogenous respiratory processes. Use ofmitochondrial enzyme inhibitors has established that tricar-boxylic acid cycle intermediates labeled with 'C in the darkare in mitochondrial pools or in pools in rapid equilibriumwith them. Operation of the tricarboxylic acid cycle wasdemonstrated both in the dark and the light by the use ofinhibitors. Light-induced nonphotosynthetic processes appearnot to affect endogenous respiratory metabolism under theseconditions.The relative rates of the tricarboxylic acid cycle during

dark-light transition and during the illumination period havebeen established. The results indicate that for about the first5 min of illumination there is a decrease in carbon flowthrough the tricarboxylic acid cycle but that during most ofa 30 min period of illumination the rate of carbon flow in thetricarboxylic acid cycle is at least as high as that in the dark(abotut 1.9 mg CO2 dm-hr-') or somewhat higher. This in-formation will be applied in the following paper to the de-termination of the control points in the tricarboxylic acidcycle under the conditions of these experiments.

Acknowledgments-The work reportecd in this paper ai(I the following paperwas carried out in the laboratories of the CSIRO Division of Food ResearchPlant Physiology Unit in the School of Biological Sciences, University of Sydney,Sydney, Australia. We are grateful to Professor M. G. Pitman ancl the staff ofthe Botany Department for their generous provision of services and facilities. Inparticular we wisli to thank Dr. P. Martin for the use of his infrared gas ana-lyzer and advice on its operation. We wish also to thank Dr. R. J. Mead of theUniversity of Western Australia for his very helpful discussion on the utilizationof fluioroacetate by plants.

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