Dark Respiration during Photosynthesis in Wheat - Plant Physiology

Plant Physiol. (1988) 87, 155-1610032-0889/88/87/0155/07/$Ol .00/0

Dark Respiration during Photosynthesis in Wheat Leaf Slices'Received for publication October 26, 1987 and in revised form January 18, 1988

BARRY G. MCCASHIN*, EDWIN A. COSSINS, AND DAVID T. CANVINDepartment of Botany, University of Alberta, Edmonton, Alberta Canada T6G-2E9 (B.G.McC.,E.A.C.); and Department of Biology, Queen's University, Kingston, Ontario Canada K7L-3N6 (D.T.C.)

ABSTRACT

The metabolism of ["C]succinate and acetate was examined in leafslices of winter wheat (Triticum aestivum L. cv Frederick) in the dark andin the light (1000 micromoles per second per square meter photosynthet-ically active radiation). In the dark [1,4-"4C]succinate was rapidly takenup and metabolized into other organic acids, amino acids, and CO2. Anaccumulation of radioactivity in the tricarboxylic acid cycle intermediatesafter "4CO2 production became constant indicates that organic acid poolsoutside of the mitochondria were involved in the buildup of radioactivity.The continuous production of "4CO2 over 2 hours indicates that, in thedark, the tricarboxylc acid cycle was the major route for succinate me-tabolism with CO2 as the chief end product. In the light, under conditionsthat supported photorespiration, succinate uptake was 80% of the darkrate and large amounts of the label entered the organic and amino acids.While carbon dioxide contained much less radioactivity than in the dark,other products such as sugars, starch, glycerate, glycine, and serine weremuch more heavily labeled than in darkness. The fact that the sametricarboxylic acid cycle intermediates became labeled in the light in ad-dition to other products which can acquire label by carboxylation reactionsindicates that the tricarboxylc acid cycle operated in the light and thatCO2 was being released from the mitochondria and efficiently refixed.The amount of radioactivity accumulating in carboxylation products inthe light was about 80% of the 14CO2 release in the dark. This indicatesthat under these conditions, the tricarboxylic acid cycle in wheat leaf slicesoperates in the light at 80% of the rate occurring in the dark.

(16). Some of these widely different estimates may result fromdifferences in experimental material and in the protocol used(gas exchange versus tracer studies). Some gas exchange studiessuggest that dark respiration continues in the light at a reducedlevel (3, 6). However, questions about exactly what reactionscause the CO2 evolution or 02 uptake and the degree of internalgas recycling within the leaf (14) make estimates of TCAC ac-tivity based solely on gas exchange measurements uncertain. Thebest evidence for the operation of the TCAC during photosyn-thesis was presented for the alga Scenedesmus (22). The specificactivity and intramolecular label distribution in TCAC inter-mediates were measured after supplying ['4C]acetate or pyruvateand the authors concluded that the cycle operated at the samerate in the light and the dark. However, many higher plant tissuescontain large quantities of organic acids that are kinetically sep-arated from the turnover pools of the TCAC (21). The occur-rence of such pools which can interact with the TCAC pools cangreatly confuse the interpretation of tracer experiments, and ithas not been possible to make comparable specific activity meas-urements as were done with the algae.

In the present study [1,4-14C]succinate or 2-'4C]acetate weresupplied to wheat leaf slices in the light or dark to determinecarbon flow through the TCAC. It was presumed that essentiallyall the succinate that was utilized would be metabolized throughthe TCAC since there are few other biochemical reactions thatuse succinate as substrate. Acetate was used to determine carbonflow from 2-oxoglutarate to succinate.

Considerable effort has been directed toward estimating darkrespiration during photosynthesis because of the effect of thisprocess on the carbon and energy economy of the plant. In spiteof these efforts, the magnitude of mitochondrial ('dark') respi-ration in leaves of higher plants during photosynthesis is notknown (16).

Control of TCAC2 activity may occur in a number of ways (28,29). Photosynthesis might affect this activity through changes inthe amounts of various adenine or pyridine nucleotides (29) ortriose phosphates (17). While measurements of cofactor levelsin different compartments of isolated protoplasts in the light anddark have been made (18, 19, 27), such measurements only showhow the various pathways might interact and do not provide anydirect information on the effect of these altered cofactor levelson TCAC operation.

Earlier data have been interpreted as indicating a reductionin dark respiration during photosynthesis ranging from 0 to 100%

1 Supported by the Natural Sciences and Engineering Research Coun-cil of Canada.

2Abbreviations: TCAC, tricarboxylic acid cycle; PPFD, photosyn-thetic photon flux density; MCW, methanol:chloroform:water.

MATERIALS AND METHODSPlant and Growth Conditions. Winter wheat (Triticum aesti-

vum L. cv Frederick) was grown in vermiculite and watered dailywith tap water. Plants were maintained in a growth chamber witha 16 h day (25°C) and 8 h night (18°C). Light was provided bya mixture of cool-white fluorescent and incandescent bulbs in aratio of 2.3:1 based on installed watts and adjusted to give 500,umols -m-2 (PPFD) at the surface of the vermiculite. Hu-midity was not regulated but ranged between 50 and 80%.

After 6 d of growth, the plants were subjected to a 14 h nightbefore harvest. Primary leaves, which had reached 70% of theirmaximum length, were cut transversely into 1 mm sections in0.5 mM CaSO4. After rinsing and blotting the slices, 500 mgsamples (about 450 ,ug of Chl [8]) were placed in 50 ml Erlen-meyer flasks. The harvest operation was conducted under dimlight (<3 ,umols- t*m- 2 PPFD).Gas exchange measurements on intact leaves were conducted

in an open system (23) with air levels of CO2 (340 ,ul L- 1) and02 (21%) at 25°C and 1050 ,umol s '1m-2 PPFD. Leaf slicephotosynthesis and dark respiration were measured in an oxygenelectrode (50 mm Mes [pH 5.0], 1 mm NaHCO3, 25°C, 1000,umolI --m-2 PPFD for photosynthesis).

Feeding Experiments. Leaf samples were suspended in 3 ml ofbuffer solution (50 mm Mes, 0.5 mM CaSO4 [pH 5.0]) containinga radioactive substrate and incubated on a shaking water bath

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Plant Physiol. Vol. 86, 1988

(25°C) in the light (1000 Amo[ s ' m -2 PPFD, incandescent light)or in the dark for up to 2 h. Humidified compressed air wascontinuously flushed (50 mlmin- l) through the sample flask intotubes containing 5% (w/v) NaOH solution to trap CO2. At theend of the feeding period, the buffer was recovered and sampleswere killed in liquid nitrogen and stored at - 20°C.

[1,4-14C]Succinate (152 nmol succinate, 2.93 x 106 dpm) wascontinuously supplied to darkened or illuminated slices. [2-14C]Acetate (84 nmol acetate, 7.13 x 106 dpm) was supplied tosamples for 20 min in the dark. After that time the feedingsolution was removed, and the slices were rinsed with buffersolution and resuspended in 3 ml of fresh buffer for a chaseperiod in the light or dark. During the chase period, the bufferwas recovered, and the samples were killed in liquid nitrogen.

[1,4-14C]Succinate (8.8 mCi-mmol-1) and [2-14C]acetate (38.7mCi mmol- 1) were purchased from New England Nuclear (Can-ada) Ltd. (Montreal, Que). Succinate was purified before useby ion exchange chromatography as described below.Sample Extraction and Fractionation. The frozen slices were

extracted (13) in 2 ml of MCW (12:5:3, v/v/v) by grinding in aglass tissue homogenizer. Insoluble material was removed bycentrifugation and extracted a further four times with 2 ml ofMCW. The combined supernatants were partitioned into a chlo-roform and an aqueous-methanol fraction. The latter was driedin a rotary evaporator at 35°C under vacuum. Aliquots from thechloroform layer were dried in an air stream.The water-soluble extract was fractionated using ion exchange

resins (1). Polypropylene Econocolumns (Bio-Rad Laboratories,Richmond, CA) containing 1 cm3 of AG50-X8 (hydrogen form,100-200 mesh) and AG1-X8 (formate form, 100-200 mesh) wereprepared, and the extract was applied to the upper column (con-taining AG50 resin). The neutral fraction was eluted with 10 mlof water, the basic fraction (from the AG50 resin) with 10 ml of2 N NH40H, and the acid I and acid II fractions (from the AG1resin) with 10 ml of 11.7 N formic acid and 2 N HCl, respectively.All eluates were dried in an air stream at 35°C. The volume offormic acid was selected to elute trans-aconitate completely (andall other TCAC organic acids except cis-aconitate). cis-Aconitateand other more tightly bound compounds (e.g. sugar bisphos-phates) were eluted in the acid II fraction.The neutral fraction (mainly sugars) was analyzed by paper

chromatography (1). After autoradiography, sugars were local-ized on the chromatogram by spraying with 0.1 M o-anisidine,0.1 M phthalic acid in 95% ethanol and heating at 110°C for 10min. Amino acids were separated using a Beckman model 120Banalyzer (55 cm column, Wl resin eluted with lithium buffers).The column effluent was passed directly to a fraction collectorand the radioactivity profile was determined. Peak fractions werecombined, dried by rotary evaporation, and redissolved in waterprior to scintillation counting. Amino acids were resolved up toalanine, and the others (including the basic amino acids) wererecovered with 0.3 M LiOH and are referred to as residual aminoacids.

Organic acids in the acid I fraction were separated on an 11cm column (AG1-X8, formate form, 100-200 mesh) using a 50mm to 8 M formic acid gradient (2). Before starting the gradient,the column was washed with 30 ml of 50 mm formic acid toenhance the resolution of the early peaks. This method separatedglycerate, succinate, malate, isocitrate, citrate, fumarate, andtrans-aconitate. trans-Aconitate co-elutes with 2-oxoglutarate,but all the radioactivity emerging in this region was shown to beaconitate by paper chromatography and autoradiography. Themeans by which trans-aconitate becomes labeled is not known.It may arise non-enzymically from cis-aconitate during samplehandling, or it might be produced enzymically in the tissue sincesome grasses are known to accumulate this acid (5). No radio-active fumarate or isocitrate was detected in plant samples afterfeeding [14C]succinate or acetate. The identity of the labeled

organic acids was confirmed by paper chromatography (12) andautoradiography.

In the separation of the water soluble fraction into four com-ponents, the average recovery was 96.2 + 0.7% (SE). This wasfor 151 leaf samples that had been labeled with a variety oforganic acids, both 14C and 3H. For the ion exchange separationof amino acids, recovery of radioactivity was 96.4 + 1.4% (SE),(n = 14), and for the organic acids on the formic acid gradient,97.8 + 0.5% (SE), (n = 14).Malate and Glutamate Degradation. The intramolecular dis-

tribution of 14C in malate was determined according to Hatch(20), except that the pH was adjusted to 7.3 and the reactionwas run for 20 min at 23°C. Malic enzyme (chicken liver) andglutamic-pyruvic transaminase (porcine heart) were purchasedfrom Sigma Chemical Company (St. Louis, MO). The reactionwas terminated with 100 ,ul of 11.7 N formic acid and the sampleswere dried on an air stream at 35°C. Acid-stable 14C was meas-ured, and the radioactivity in C4 was determined by difference.A portion of the acid-stable product was applied to a 1 cm3column of AG50-X8 (H+), and alanine was eluted with 10 mlof 2 N NH40H. After drying, alanine was decarboxylated in a50 ml Erlenmeyer flask by reacting with 1 ml of ninhydrin reagent(containing 0.75 ml DMSO, 50mg ninhydrin, 7.5 mg hydrindatin,and 0.25 ml of citrate buffer) at 100°C for 20 min (25). Radio-active CO2 from Cl was trapped in a vial containing 0.5 ml ofphenethylamine over 15 h and was measured in a toluene:methylcellosolve-based cocktail. Activity in C2 + C3 was cal-culated by difference. Radioactivity in Cl of glutamate was alsodetermined by ninhydrin decarboxylation.

Insoluble Material and CO2. The MCW-insoluble material re-maining after the extraction was fractionated into protein, starch,and residual components (13). Enzymes for this procedure wereobtained from Sigma Chemical Company (St. Louis, MO). Pro-tein was solubilized with Protease (type 14, bacterial, 21 units)and starch was extracted with amyloglycosidase (Rhizopus, 37units) and a-amylase (type X-A, fungal, 67 units). The materialremaining after the starch digestion (containing cellulose, hemi-cellulose, and lignin) was suspended in a gel of 4% (w/v) Cabosilin a dioxane-based cocktail before counting.

Radioactive CO2 was transferred to phenethylamine for count-ing in a toluene:methylcellosolve-based scintillation cocktail. Liquidscintillation cocktails were prepared (7), and two additional tol-uene-based solutions containing 33% (v/v) methylcellosolve orTriton X-100 were also used. Counting efficiency was measuredby the external standard ratio or the channels ratio method, anddata are reported as dpm.

RESULTS

Wheat seedlings showed no signs of stress when harvested.The photosynthetic rate of intact leaves in air was 240 ,umolC02 mg 'Chlh-1, and dark respiration was 18 ,umol C02 mg-lChl h- 1 after 30 min in darkness. Photosynthesis of the leaf sliceswas 104 + 8 (SE) ,umol 02 mg- 1Chl h- , while respiration after20 min in the dark was 8.1 + 0.6 (SE) ,umol 02 mg- Chl-h-1.The lower values for slices may be due to the increased resistanceto gaseous diffusion in solution. Since flasks were flushed withair during feeding experiments, the rate of photosynthetic CO2fixation may have been less than the photosynthetic rate meas-

ured by the 02 electrode, and photorespiration may have beenfavored.

Succinate uptake by the wheat leaf slices in the dark was rapidand continued over the 2 h feeding (Fig. 1) so that up to 41%of the initial 14C was removed from the feed solution. Uptakein the light was about 80% of that observed in the dark. Radio-activity recovered from the tissue (including succinate and C02)was lower than that removed from the feeding solution (Fig. 1)and amounted to about 80% of the ['4C]succinate taken up by

156 McCASHIN ET AL.

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DARK RESPIRATION DURING PHOTOSYNTHESIS

l the large amount of 14CO0 released in the dark (up to 53% of1200 _ _ the total), while in the light, there was negligible release of '4CO.

(<2% after 2 h). In the light, there was a greater accumulationof 14C in the insoluble and organic solvent soluble fractions

1000 / _ amounting to 15% after 2 h compared to 2% in the dark (data(D / / 4 not shown). The insoluble fraction increased to 12% of the total__ ,S'radioactivity in the light, and most (80%) was protein with lesserE 800L 0 amounts in starch and the insoluble residue.co / ^/ ,o The organic acid fraction was rapidly labeled (Fig. 3) andCl) / / ,,,- continued to accumulate 14C throughout the experiment. In theco / */ ,- 'o dark, radioactivity in the amino acids approached that of the

600 u , o organic acids after 120 min. In the light, the amino acids were- / ,, less heavily labeled, and 14C in the organic acid fraction rose

X [ / sK ' | continuously. The acid II fraction contained little 14C in dark orE400 / Z _light (data not shown). Light stimulated incorporation into theX -:--° neutrals.

0 /sz The distribution of 14C in individual components of the water200 soluble fraction is shown in Figure 4. Incorporation into the

succinate pool of the leaf slices was high soon after the feeding

40j.fi .

began, and the level dropped slightly over the 2 h experiment.0 ) 0 140 60 80 100 10 A There was little difference between light and dark samples. Much020 40 60 80 100 120 radioactivity accumulated in malate and aspartate during theTime (min) experiment. In the dark, both increased continuously but as-

FIG. 1. Uptake (Ii of [1,4-'4C]succinate by wheat leaf slices and total partate was greater than malate. Light had an immediate andincorporation (C) into stable products (plus C02) in the dark (closed dramatic effect on these two compounds, as has previously beensymbols) and the light (open symbols). reported (15), such that much less '4C appeared in aspartate than

in malate. This is consistent with the activation of the chloroplastmalate dehydrogenase by light (9) and the increase in the amountof reduced pyridine nucleotides. It is, however, thought that theoperation of the mitochondrial malate dehydrogenase is not af-fected by these changes.

Label from ['4C]succinate and acetate was not detected in,,-fumarate or isocitrate. This suggests that the pools of these acids

600 are small or that label from the mitochondrial turnover pool does,, not accumulate in a storage pool. MacLennan et al. (21) showed

that the content of TCAC acids varies widely in plant organs,_L PI and the degree of exchange between turnover and other pools

E ,' differs for individual acids. These workers did not detect isoci-trate in wheat leaves. In another experiment (not presented here),

` 400_we found that ['4C]fumarate supplied to wheat leaf slices labeledthe same intermediates that were found after feeding succinate

o / or acetate. This suggests that the fumarase reaction of the TCACX _ wo / - was active in these leaf slices.

CL 200 -

p/>/ q Q ~~~~~~~~400-,"'

E0 - -----------C 300-- O_

0 20 40 60 80 100 120 1 'O°l 200

Time (min) X 011 /FIG. 2. Incorporation of radioactivity from [1 ,4-14C]succinate into the 2 100 _'

water soluble fraction (C) and CO2 (I) in the dark (closed symbols) 0_and light (open symbols). a

the slices in both light and dark. 0 20 40 60 80 100 120Analysis of metabolic products (Fig. 2) showed that the water Time (m in)

soluble fraction contained most of the label throughout the 2 hfeeding period. Initially 98% of the 14C was in water soluble FIG. 3. Incorporation of radioactivity from [1,414C]succinate into watercompounds, but this dropped to 45% in the dark and to 84% in soluble components: organic acid (O), amino acid (A), and neutral (j)the light. The major difference between the two treatments was fractions in the dark (closed symbols) and light (open symbols).

157




FIG.4. Incorporation of radioactivity from [1,4-14C]succinate into various organic and amino acidsin the dark (closed symbols) and light (open sym

bols).

QEcocn

C,)

0a-

-

C]

20

10

0

10

THREONINE

0 --~~~~~~O---10 _

Residual Amino ----Acids b--oQ---

aw

0 30 60 90 120

Time (min)

100

50

0

50 I _GLUTAMATE

0 _ o -r __---I^. I,50-SERINE O >

SQ~~~- -~ --

- 1

10ASPARAGINE

0

10

GLUTAMINE[-<i

0

10 _ -

-

0

0 30 60 90 120

Time (min)

There was no apparent effect of light on the accumulation of14C in citrate, trans-aconitate, asparagine, glutamine, and alanine(Fig. 4). Light did cause increased accumulation in glycerate,glycine, serine, threonine, and the residual amino acids. This isconsistent with a reincorporation of 14CO2 via photosynthetic andphotorespiratory metabolism. Light is known to stimulate as-

partate metabolism to other amino acids (24) and could be re-

sponsible for the increased labeling of threonine and the residualamino acids (e.g. possibly lysine, isoleucine, methionine).The intramolecular distribution of radioactivity in malate was

determined (Fig. 5). In the dark, 14C increased in both C4 andCl with slightly more occurring in C4. The average distributionof 14C in C4 was 52.7 + 0.5% (SE), (n = 7), which suggests thata small amount of 8-carboxylation may have occurred in thedark. Light caused greater incorporation into malate carbons.Labeling of Cl increased more rapidly and reached a higher levelthan was observed in the dark. However, while Cl accumulationstarted to plateau by 100 min, label in C4 continued to rise upto 120 min. This is consistent with the incorporation of 14CO2 byp-carboxylation into the C4 position. The discrepancy between

C4 and Cl became noticeable after about 20 min. At this time,14C02 began to be released in the dark samples (Fig. 2). Thedifference between C4 and C1 suggested that much of the malatewith increased labeling in C4 does not have ready access to fu-marase (i.e. it is located outside of the mitochondria).The internal carbons of malate also accumulated considerable

radioactivity in the light after 40 min. Since these carbons couldnot be derived from carboxyl-labeled succinate metabolizedthrough the TCAC, they must arise from three carbon acceptorsfor 8-carboxylation which have been generated by photosyn-thetic fixation of released 14CO2.

Glutamate was degraded to determine the intramolecular dis-tribution of 14C. Glutamate formed from carboxyl-labeled suc-

cinate via the TCAC should be labeled exclusively in C. On theother hand, if malate containing 14C in C2 + C3 enters the TCAC,one expects glutamate to be labeled in carbons 2 and 3. In thedark, the average recovery of 14C in Cl was 86 + 3% (SE),(n = 7), and in the light, C, contained 78 + 3% (SE), (n = 6)(Fig. 6). Only a single determination of glutamate C, labelingwas available for each sample, and although there is some var-

I

_ 0 SUCCINATE -

0-

-- - - - CITRATE_ -

I_I

trans-ACONITATE0 _-0o ----

,- °

P,

U * r * 7r0 30 60 90 120

20 _GLYCINE o

10 - ,o'

L la

° -AC . * 1I* a@--T

158 McCASHIN ET AL.




140

120

a)

Eco

co',)

Q-C

100

80

60

40

20 4'

A~~~~~~~~ II-Ar0 AI I

0 20 40 60 80 100 120

Time (min)

FIG. 5. Distribution of radioactivity from [1,414C]succinate in malatein the dark (closed symbols) and light (open symbols): C4 (c), Cl (O),and C2+3 (A).

iability in the data, there did not appear to be a major increasein the incorporation of '4C into carbons 2 to 5 of glutamate inthe light. This suggests that glutamate was derived largely fromsuccinate metabolism in the TCAC, and there was not a largeamount of malate labeled in C2 + C3 entering the TCAC in thelight.

This experiment with [1,4-14C]succinate demonstrated that, inthe light, labeling of the TCAC intermediates around to 2-oxo-glutarate was detected (except for fumarate and isocitrate asdiscussed above), but it did not clearly show that reactions be-tween 2-oxoglutarate and succinate were operative. In order toexamine this section of the cycle, [2-14C]acetate was supplied toleaf slices. In this experiment (Fig. 7), a 20 min pulse of[2-14C]acetate in the dark was followed by a chase period (up to120 min) in either dark or light. At pH 5.0, the rate of acetateuptake into the tissue was sevenfold higher than the rate ofincorporation, and after removal of the feed solution, acetatewithin the tissue continued to be fixed into acid-stable products.In the light, radioactivity from acetate continued to accumulatein succinate, malate, and citrate (Fig. 7) indicating that this por-tion of the TCAC was operational during illumination. The ab-sence of 14C buildup in aspartate probably resulted from an effectof light on malate dehydrogenase. This is thought to occur out-side of the mitochondria and does not directly reflect on TCACactivity. The 14C that did accumulate in aspartate in the lightsamples was much less than in the dark and quickly reached aconstant level suggestive of a relatively small pool that equili-brated rapidly.

DISCUSSION

The wheat leaf slices metabolized up to 80% of the absorbed[14C]-succinate into a variety of stable water soluble compoundsand into CO2 (Fig. 1) which was released from the tissue in thedark and apparently refixed in photosynthesis in the light (Fig.

EcoCD,

Cf)0

X1

0-c]

0 20 40 60 80 100 120

Time (min)FIG. 6. Distribution of radioactivity from [1,4-14C]succinate in glu-

tamate in the dark (closed symbols) and light (open symbols): total 14C(ED), C1 (E[), and C2_s (A)-

2). Tricarboxylic acid cycle intermediates quickly acquired label(Fig. 4) indicating a rapid flux of '4C through the cycle. Thelinearity of 14C02 release in the dark after 40 min (Fig. 2) suggestsa steady state metabolism of succinate. In the dark, 94% of theradioactivity was present in compounds (Figs. 2 and 4) directlyrelated to the TCAC (CO2, organic acids, and amino acids) overthe 2 h experiment. In the light, little 14CO2 was released fromthe slices (Fig. 2), but a variety of products (Fig. 4) were labeled,presumably by the photosynthetic refixation of 14CO2 (glycerate,sugars, starch, sugar bisphosphates), by photorespiration (gly-cine, serine), and by 3-carboxylation (malate). These compoundsaccumulated 40% of the total label after 2 h, and about 84% ofthe 14C metabolized in the light occurred in products directlyrelated to CO2 refixation or in TCAC intermediates. These re-sults confirm our original assumption that essentially all the ab-sorbed succinate is metabolized by the TCAC and not by otherreactions.Continued accumulation of label in organic and amino acids

beyond 40 min indicates that this buildup occurred in pools out-side of the TCAC turnover pools (presumably outside the mi-tochondria). If this were the case, one cannot gauge the rate ofTCAC activity simply by monitoring accumulation of '4C in theseextra-mitochondrial sites, since differences in the amount of 14Cmay simply reflect an alteration in the rate of influx or effluxfrom the large pools (21), and any changes in the activity of theTCAC pools would go undetected. But TCAC operation couldbe assessed by measuring a product that is unlikely to accumulatein the tissue, namely,14CO2.

This approach was somewhat complicated by the fact that,

159



McCASHIN ET AL.

CITRATE

0

SUCCINATE

----------

- ~~~~0

-I,-S

0 30 60 90 120


0 30 60 90 120

Time (min)

FIG. 7. Incorporation of radioactivity from [2-14C]acetate into organic and amino acids in wheat leaf slices in the dark (closed symbols) and light

(open symbols).

Table I. Incorporation of Radioactivity from [1,4-14C]Succinate into 14C02 or Products Formed from 14CO2by Fixation in the Light

Results are expressed as a percent of total 14C recovered in the samples (not including succinate). Regardingthe malate fractions, malate labeled in carbons 2 and 3 must be produced by ,3-carboxylation of a C3 acceptorformed from photosynthetic fixation of 14CO2. The data (Fig. 5) show 9.7% of the 14C in malate to be presentin carbons 2 and 3 after 2 h in the light. If carbons 2 and 3 are equally labeled (4.8% each) at least an

equivalent amount of radioactivity must be present in carbon 1. Carbon 1 contains 36.4% of the total malatelabel after 2 h, thus 31.6% (36.4-4.8) of the Cl label must be derived directly from I1,4-'4Clsuccinate. Withan equivalent amount of label in carbon 4, 63.2% of [14C]malate would be derived from the TCAC, and theremainder (36.8%) would be from photosynthetic or,t3-carboxylation fixation of "4CO2. At 2 h in the light,malate contains 36.3% of the sample radioactivity, thus the additional 14C in carbons 1, 2, and 3 is 5.2% ofthe total counts, and in C4, 8. 1% of the total counts. A similar calculation yields the results for the 1 h sample.Regarding the protein fraction, after 2 h, 39% of the '4C in amino acids is in glycine and serine. If incorporationof all radioactive amino acids into protein is assumed to be equal, the calculated values show the amount ofprotein radioactivity that would be derived from amino acids produced from photosynthetic fixation of 14CO2.The value for 1 h is derived in a similar manner.

Time (min)Fraction 60 120

Dark Light Dark Light

% total 14C recoveredCO2 36.3 1.0 52.5 1.6Neutral 1.2 6.3 0.3 8.6Acid II 0.4 0.9 0.4 1.2Glycerate 0.6 2.6 0.3 2.3Glycine 0.0 2.6 0.0 2.5Serine 0.1 5.5 0.1 5.4Starch 0.1 1.0 0.1 1.6Malate C4 0.0 7.2 0.0 8.1Malate-C,12+3 0.0 3.4 0.0 5.2Protein 0.0 2.6 0.0 3.5

Total 38.7 33.1 53.7 40.0

160

150

loo0

50

0)

EcocoX 200

0

. 150

0.

1001

50

o

I

trans-ACONITATE

'-U .--------------y i

GLUTAMATE

-0~~~~-----0-

ASPARTATE

0O----a----0-- -

1.




although the same proportion was metabolized, the uptake of[14C]succinate in the light was only about 80% of that absorbedin the dark (Fig. 1). In the acetate feeding experiment (Fig. 7),there was a greater accumulation of 14C in succinate in the lightcompared to the dark. This has been observed previously (10)and could occur if the activity of succinate dehydrogenase was

less in the light. If this resulted in decreased metabolism of suc-

cinate and if uptake were linked to utilization, then uptake wouldbe reduced. But we do not know the reason for the lower ab-sorption or whether it is important. The reduced absorption didmean that we could not directly compare total radioactivity invarious compounds between the light and the dark, but ratherthat we had to compare the percent distribution of 14C amongthe different products.

Light stimulated 14C incorporation into malate (Fig. 5), whichprobably accumulated (4) outside the mitochondria (18). Radio-activity in C2 and C3 can only be derived from a C3 compoundthat is labeled by the photosynthetic incorporation of '4CO2 (26).The extra label in C4 compared to Cl in the light (Fig. 5) probablyarose from B-carboxylation, as it is known that ,3-carboxylationis stimulated in the light (22). This increased radioactivity incarbons 2, 3, and 4 of malate in the light suggests that 14CO2released from the TCAC was refixed by ribulose bisphosphatecarboxylase and phosphoenolpyruvate carboxylase (4).

In the dark, 38.7% of the 14C from succinate was released as

14C02 after 1 h, and 53.7% was released after 2 h (Table I). Inthe light, label from [14C]succinate was detected in most TCACintermediates (Fig. 4) indicating that the TCAC was active inilluminated leaf tissue. The results with [2-14C]acetate (Fig. 7)indicated that the conversion of 2-oxoglutarate to succinate oc-curred in both the light and dark. If 14C entered products likeglycerate, sugar, starch, glycine, seine, and malate in the lightby refixation of '4CO2 released during the metabolism of succi-nate in the TCAC, then we can estimate the relative rate of theTCAC activity by comparing the total 14C accumulation in thesecarboxylation products during illumination with the rate of 14CO2release in the dark. The sum of 14CO2 release and the carbox-ylation products shows that 14CO2 production from succinate inthe light was 33.1% of the metabolized succinate after 1 h and40.0% after 2 h (Table I). These rates of 14CO2 release areconsistent with the continued metabolism of succinate in illu-minated leaf slices and indicate that under these conditions, 'dark'respiration continues in the light at 75 to 85% of the rate in thedark. The situation occurring under conditions that favor pho-tosynthesis over photorespiration remains to be examined sinceTCAC activity might be influenced by photorespiratory glycineproduction (11).

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Dark Respiration during Photosynthesis in Wheat - Plant Physiology