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Plant Physiol. (1988) 87, 155-161 0032-0889/88/87/0155/07/$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. CANVIN Department 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 leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthet- ically active radiation). In the dark [1,4-"4C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO2. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after "4CO2 production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of "4CO2 over 2 hours indicates that, in the dark, the tricarboxylc acid cycle was the major route for succinate me- tabolism with CO2 as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate 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 were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in ad- dition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylc acid cycle operated in the light and that CO2 was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the 14CO2 release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark. (16). Some of these widely different estimates may result from differences in experimental material and in the protocol used (gas exchange versus tracer studies). Some gas exchange studies suggest that dark respiration continues in the light at a reduced level (3, 6). However, questions about exactly what reactions cause the CO2 evolution or 02 uptake and the degree of internal gas recycling within the leaf (14) make estimates of TCAC ac- tivity based solely on gas exchange measurements uncertain. The best evidence for the operation of the TCAC during photosyn- thesis was presented for the alga Scenedesmus (22). The specific activity and intramolecular label distribution in TCAC inter- mediates were measured after supplying ['4C]acetate or pyruvate and the authors concluded that the cycle operated at the same rate in the light and the dark. However, many higher plant tissues contain 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 can greatly confuse the interpretation of tracer experiments, and it has 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 were supplied to wheat leaf slices in the light or dark to determine carbon flow through the TCAC. It was presumed that essentially all the succinate that was utilized would be metabolized through the TCAC since there are few other biochemical reactions that use succinate as substrate. Acetate was used to determine carbon flow from 2-oxoglutarate to succinate. Considerable effort has been directed toward estimating dark respiration during photosynthesis because of the effect of this process on the carbon and energy economy of the plant. In spite of these efforts, the magnitude of mitochondrial ('dark') respi- ration in leaves of higher plants during photosynthesis is not known (16). Control of TCAC2 activity may occur in a number of ways (28, 29). Photosynthesis might affect this activity through changes in the amounts of various adenine or pyridine nucleotides (29) or triose phosphates (17). While measurements of cofactor levels in different compartments of isolated protoplasts in the light and dark have been made (18, 19, 27), such measurements only show how the various pathways might interact and do not provide any direct information on the effect of these altered cofactor levels on TCAC operation. Earlier data have been interpreted as indicating a reduction in 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 METHODS Plant and Growth Conditions. Winter wheat (Triticum aesti- vum L. cv Frederick) was grown in vermiculite and watered daily with tap water. Plants were maintained in a growth chamber with a 16 h day (25°C) and 8 h night (18°C). Light was provided by a mixture of cool-white fluorescent and incandescent bulbs in a ratio 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 night before harvest. Primary leaves, which had reached 70% of their maximum length, were cut transversely into 1 mm sections in 0.5 mM CaSO4. After rinsing and blotting the slices, 500 mg samples (about 450 ,ug of Chl [8]) were placed in 50 ml Erlen- meyer flasks. The harvest operation was conducted under dim light (<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) and 02 (21%) at 25°C and 1050 ,umol s '1m-2 PPFD. Leaf slice photosynthesis and dark respiration were measured in an oxygen electrode (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 of buffer solution (50 mm Mes, 0.5 mM CaSO4 [pH 5.0]) containing a radioactive substrate and incubated on a shaking water bath 155 www.plantphysiol.org on April 5, 2019 - Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved.

Dark Respiration during Photosynthesis in Wheat - Plant Physiology

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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. CANVIN Department 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 leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthet- ically active radiation). In the dark [1,4-"4C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO2. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after "4CO2 production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of "4CO2 over 2 hours indicates that, in the dark, the tricarboxylc acid cycle was the major route for succinate me- tabolism with CO2 as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate 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 were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in ad- dition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylc acid cycle operated in the light and that CO2 was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the 14CO2 release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.
(16). Some of these widely different estimates may result from differences in experimental material and in the protocol used (gas exchange versus tracer studies). Some gas exchange studies suggest that dark respiration continues in the light at a reduced level (3, 6). However, questions about exactly what reactions cause the CO2 evolution or 02 uptake and the degree of internal gas recycling within the leaf (14) make estimates of TCAC ac- tivity based solely on gas exchange measurements uncertain. The best evidence for the operation of the TCAC during photosyn- thesis was presented for the alga Scenedesmus (22). The specific activity and intramolecular label distribution in TCAC inter- mediates were measured after supplying ['4C]acetate or pyruvate and the authors concluded that the cycle operated at the same rate in the light and the dark. However, many higher plant tissues contain 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 can greatly confuse the interpretation of tracer experiments, and it has 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 were supplied to wheat leaf slices in the light or dark to determine carbon flow through the TCAC. It was presumed that essentially all the succinate that was utilized would be metabolized through the TCAC since there are few other biochemical reactions that use succinate as substrate. Acetate was used to determine carbon flow from 2-oxoglutarate to succinate.
Considerable effort has been directed toward estimating dark respiration during photosynthesis because of the effect of this process on the carbon and energy economy of the plant. In spite of these efforts, the magnitude of mitochondrial ('dark') respi- ration in leaves of higher plants during photosynthesis is not known (16).
Control of TCAC2 activity may occur in a number of ways (28, 29). Photosynthesis might affect this activity through changes in the amounts of various adenine or pyridine nucleotides (29) or triose phosphates (17). While measurements of cofactor levels in different compartments of isolated protoplasts in the light and dark have been made (18, 19, 27), such measurements only show how the various pathways might interact and do not provide any direct information on the effect of these altered cofactor levels on TCAC operation.
Earlier data have been interpreted as indicating a reduction in 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 METHODS Plant and Growth Conditions. Winter wheat (Triticum aesti-
vum L. cv Frederick) was grown in vermiculite and watered daily with tap water. Plants were maintained in a growth chamber with a 16 h day (25°C) and 8 h night (18°C). Light was provided by a mixture of cool-white fluorescent and incandescent bulbs in a ratio 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 night before harvest. Primary leaves, which had reached 70% of their maximum length, were cut transversely into 1 mm sections in 0.5 mM CaSO4. After rinsing and blotting the slices, 500 mg samples (about 450 ,ug of Chl [8]) were placed in 50 ml Erlen- meyer flasks. The harvest operation was conducted under dim light (<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) and 02 (21%) at 25°C and 1050 ,umol s '1m-2 PPFD. Leaf slice photosynthesis and dark respiration were measured in an oxygen electrode (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 of buffer solution (50 mm Mes, 0.5 mM CaSO4 [pH 5.0]) containing a 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 was continuously flushed (50 mlmin- l) through the sample flask into tubes containing 5% (w/v) NaOH solution to trap CO2. At the end of the feeding period, the buffer was recovered and samples were killed in liquid nitrogen and stored at - 20°C.
[1,4-14C]Succinate (152 nmol succinate, 2.93 x 106 dpm) was continuously supplied to darkened or illuminated slices. [2- 14C]Acetate (84 nmol acetate, 7.13 x 106 dpm) was supplied to samples for 20 min in the dark. After that time the feeding solution was removed, and the slices were rinsed with buffer solution and resuspended in 3 ml of fresh buffer for a chase period in the light or dark. During the chase period, the buffer was recovered, and the samples were killed in liquid nitrogen.
[1,4-14C]Succinate (8.8 mCi-mmol-1) and [2-14C]acetate (38.7 mCi mmol- 1) were purchased from New England Nuclear (Can- ada) Ltd. (Montreal, Que). Succinate was purified before use by 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 a glass tissue homogenizer. Insoluble material was removed by centrifugation and extracted a further four times with 2 ml of MCW. The combined supernatants were partitioned into a chlo- roform and an aqueous-methanol fraction. The latter was dried in a rotary evaporator at 35°C under vacuum. Aliquots from the chloroform 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) were prepared, and the extract was applied to the upper column (con- taining AG50 resin). The neutral fraction was eluted with 10 ml of water, the basic fraction (from the AG50 resin) with 10 ml of 2 N NH40H, and the acid I and acid II fractions (from the AG1 resin) 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 of formic acid was selected to elute trans-aconitate completely (and all other TCAC organic acids except cis-aconitate). cis-Aconitate and 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 10 min. Amino acids were separated using a Beckman model 120B analyzer (55 cm column, Wl resin eluted with lithium buffers). The column effluent was passed directly to a fraction collector and the radioactivity profile was determined. Peak fractions were combined, dried by rotary evaporation, and redissolved in water prior to scintillation counting. Amino acids were resolved up to alanine, and the others (including the basic amino acids) were recovered with 0.3 M LiOH and are referred to as residual amino acids.
Organic acids in the acid I fraction were separated on an 11 cm column (AG1-X8, formate form, 100-200 mesh) using a 50 mm to 8 M formic acid gradient (2). Before starting the gradient, the column was washed with 30 ml of 50 mm formic acid to enhance the resolution of the early peaks. This method separated glycerate, succinate, malate, isocitrate, citrate, fumarate, and trans-aconitate. trans-Aconitate co-elutes with 2-oxoglutarate, but all the radioactivity emerging in this region was shown to be aconitate by paper chromatography and autoradiography. The means by which trans-aconitate becomes labeled is not known. It may arise non-enzymically from cis-aconitate during sample handling, or it might be produced enzymically in the tissue since some grasses are known to accumulate this acid (5). No radio- active fumarate or isocitrate was detected in plant samples after feeding [14C]succinate or acetate. The identity of the labeled
organic acids was confirmed by paper chromatography (12) and autoradiography.
In the separation of the water soluble fraction into four com- ponents, the average recovery was 96.2 + 0.7% (SE). This was for 151 leaf samples that had been labeled with a variety of organic acids, both 14C and 3H. For the ion exchange separation of 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 reaction was run for 20 min at 23°C. Malic enzyme (chicken liver) and glutamic-pyruvic transaminase (porcine heart) were purchased from Sigma Chemical Company (St. Louis, MO). The reaction was terminated with 100 ,ul of 11.7 N formic acid and the samples were 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 cm3 column of AG50-X8 (H+), and alanine was eluted with 10 ml of 2 N NH40H. After drying, alanine was decarboxylated in a 50 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 of phenethylamine 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 also determined 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 were obtained 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, 37 units) and a-amylase (type X-A, fungal, 67 units). The material remaining after the starch digestion (containing cellulose, hemi- cellulose, and lignin) was suspended in a gel of 4% (w/v) Cabosil in a dioxane-based cocktail before counting.
Radioactive CO2 was transferred to phenethylamine for count- ing in a toluene:methylcellosolve-based scintillation cocktail. Liquid scintillation cocktails were prepared (7), and two additional tol- uene-based solutions containing 33% (v/v) methylcellosolve or Triton X-100 were also used. Counting efficiency was measured by the external standard ratio or the channels ratio method, and data are reported as dpm.
RESULTS
Wheat seedlings showed no signs of stress when harvested. The photosynthetic rate of intact leaves in air was 240 ,umol C02 mg 'Chlh-1, and dark respiration was 18 ,umol C02 mg-l Chl h- 1 after 30 min in darkness. Photosynthesis of the leaf slices was 104 + 8 (SE) ,umol 02 mg- 1Chl h- , while respiration after 20 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 resistance to gaseous diffusion in solution. Since flasks were flushed with air during feeding experiments, the rate of photosynthetic CO2 fixation may have been less than the photosynthetic rate meas-
ured by the 02 electrode, and photorespiration may have been favored.
Succinate uptake by the wheat leaf slices in the dark was rapid and continued over the 2 h feeding (Fig. 1) so that up to 41% of the initial 14C was removed from the feed solution. Uptake in 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% of 1200 _ _ the total), while in the light, there was negligible release of '4CO.
(<2% after 2 h). In the light, there was a greater accumulation of 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 lesser E 800L 0 amounts in starch and the insoluble residue. co / ^/ ,o The organic acid fraction was rapidly labeled (Fig. 3) and Cl) / / ,,,- continued to accumulate 14C throughout the experiment. In the co / */ ,- '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 or E400 / Z _light (data not shown). Light stimulated incorporation into the X -:--° neutrals.
0 /sz The distribution of 14C in individual components of the water 200 soluble fraction is shown in Figure 4. Incorporation into the
succinate pool of the leaf slices was high soon after the feeding
40 j.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 the Time (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 and incorporation (C) into stable products (plus C02) in the dark (closed dramatic effect on these two compounds, as has previously been symbols) 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 chloroplast malate dehydrogenase by light (9) and the increase in the amount of reduced pyridine nucleotides. It is, however, thought that the operation 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 labeled the same intermediates that were found after feeding succinate
o / or acetate. This suggests that the fumarase reaction of the TCAC X _ wo / - was active in these leaf slices.
CL 200 -
E 0 - -----------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 80 100 120 Analysis of metabolic products (Fig. 2) showed that the water Time (m in)
soluble fraction contained most of the label throughout the 2 h feeding period. Initially 98% of the 14C was in water soluble FIG. 3. Incorporation of radioactivity from [1,414C]succinate into water compounds, 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
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FIG.4. Incorporation of radioactivity from [1,4- 14C]succinate into various organic and amino acids in the dark (closed symbols) and light (open sym
bols).
a w
Time (min)
SQ~~~- -~ - -
- 1
Time (min)
There was no apparent effect of light on the accumulation of 14C 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 is consistent with a reincorporation of 14CO2 via photosynthetic and photorespiratory 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 residual amino 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 and Cl with slightly more occurring in C4. The average distribution of 14C in C4 was 52.7 + 0.5% (SE), (n = 7), which suggests that a small amount of 8-carboxylation may have occurred in the dark. Light caused greater incorporation into malate carbons. Labeling of Cl increased more rapidly and reached a higher level than was observed in the dark. However, while Cl accumulation started to plateau by 100 min, label in C4 continued to rise up to 120 min. This is consistent with the incorporation of 14CO2 by p-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). The difference between C4 and C1 suggested that much of the malate with 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 could not be derived from carboxyl-labeled succinate metabolized through the TCAC, they must arise from three carbon acceptors for 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 the other hand, if malate containing 14C in C2 + C3 enters the TCAC, one expects glutamate to be labeled in carbons 2 and 3. In the dark, 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, labeling was available for each sample, and although there is some var-
I
20 _ GLYCINE o
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Time (min)
FIG. 5. Distribution of radioactivity from [1,414C]succinate in malate in 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 increase in the incorporation of '4C into carbons 2 to 5 of glutamate in the light. This suggests that glutamate was derived largely from succinate metabolism in the TCAC, and there was not a large amount of malate labeled in C2 + C3 entering the TCAC in the light.
This experiment with [1,4-14C]succinate demonstrated that, in the light, labeling of the TCAC intermediates around to 2-oxo- glutarate was detected (except for fumarate and isocitrate as discussed above), but it did not clearly show that reactions be- tween 2-oxoglutarate and succinate were operative. In order to examine this section of the cycle, [2-14C]acetate was supplied to leaf slices. In this experiment (Fig. 7), a 20 min pulse of [2-14C]acetate in the dark was followed by a chase period (up to 120 min) in either dark or light. At pH 5.0, the rate of acetate uptake into the tissue was sevenfold higher than the rate of incorporation, and after removal of the feed solution, acetate within the tissue continued to be fixed into acid-stable products. In the light, radioactivity from acetate continued to accumulate in 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 effect of light on malate dehydrogenase. This is thought to occur out- side of the mitochondria and does not directly reflect on TCAC activity. The 14C that did accumulate in aspartate in the light samples was much less than in the dark and quickly reached a constant 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 compounds and into CO2 (Fig. 1) which was released from the tissue in the dark and apparently refixed in photosynthesis in the light (Fig.
E co CD,
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. The linearity of 14C02 release in the dark after 40 min (Fig. 2) suggests a steady state metabolism of succinate. In the dark, 94% of the radioactivity was present in compounds (Figs. 2 and 4) directly related to the TCAC (CO2, organic acids, and amino acids) over the 2 h experiment. In the light, little 14CO2 was released from the 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 compounds accumulated 40% of the total label after 2 h, and about 84% of the 14C metabolized in the light occurred in products directly related 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 other reactions. 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 of TCAC activity simply by monitoring accumulation of '4C in these extra-mitochondrial sites, since differences in the amount of 14C may simply reflect an alteration in the rate of influx or efflux from the large pools (21), and any changes in the activity of the TCAC pools would go undetected. But TCAC operation could be assessed by measuring a product that is unlikely to accumulate in the tissue, namely,14CO2.
This approach was somewhat complicated by the fact that,
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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 14CO2 by Fixation in the Light
Results are expressed as a percent of total 14C recovered in the samples (not including succinate). Regarding the malate fractions, malate labeled in carbons 2 and 3 must be produced by ,3-carboxylation of a C3 acceptor formed from photosynthetic fixation of 14CO2. The data (Fig. 5) show 9.7% of the 14C in malate to be present in 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 malate label after 2 h, thus 31.6% (36.4-4.8) of the Cl label must be derived directly from I1,4-'4Clsuccinate. With an equivalent amount of label in carbon 4, 63.2% of [14C]malate would be derived from the TCAC, and the remainder (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% of the 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 incorporation of all radioactive amino acids into protein is assumed to be equal, the calculated values show the amount of protein 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 recovered CO2 36.3 1.0 52.5 1.6 Neutral 1.2 6.3 0.3 8.6 Acid II 0.4 0.9 0.4 1.2 Glycerate 0.6 2.6 0.3 2.3 Glycine 0.0 2.6 0.0 2.5 Serine 0.1 5.5 0.1 5.4 Starch 0.1 1.0 0.1 1.6 Malate C4 0.0 7.2 0.0 8.1 Malate-C,12+3 0.0 3.4 0.0 5.2 Protein 0.0 2.6 0.0 3.5
Total 38.7 33.1 53.7 40.0
160
150
loo0
50
0)
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DARK RESPIRATION DURING PHOTOSYNTHESIS
although the same proportion was metabolized, the uptake of [14C]succinate in the light was only about 80% of that absorbed in the dark (Fig. 1). In the acetate feeding experiment (Fig. 7), there was a greater accumulation of 14C in succinate in the light compared 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 would be reduced. But we do not know the reason for the lower ab- sorption or whether it is important. The reduced absorption did mean that we could not directly compare total radioactivity in various compounds between the light and the dark, but rather that we had to compare the percent distribution of 14C among the different products.
Light stimulated 14C incorporation into malate (Fig. 5), which probably accumulated (4) outside the mitochondria (18). Radio- activity in C2 and C3 can only be derived from a C3 compound that is labeled by the photosynthetic incorporation of '4CO2 (26). The extra label in C4 compared to Cl in the light (Fig. 5) probably arose from B-carboxylation, as it is known that ,3-carboxylation is stimulated in the light (22). This increased radioactivity in carbons 2, 3, and 4 of malate in the light suggests that 14CO2 released from the TCAC was refixed by ribulose bisphosphate carboxylase 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). In the light, label from [14C]succinate was detected in most TCAC intermediates (Fig. 4) indicating that the TCAC was active in illuminated 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 like glycerate, sugar, starch, glycine, seine, and malate in the light by refixation of '4CO2 released during the metabolism of succi- nate in the TCAC, then we can estimate the relative rate of the TCAC activity by comparing the total 14C accumulation in these carboxylation products during illumination with the rate of 14CO2 release in the dark. The sum of 14CO2 release and the carbox- ylation products shows that 14CO2 production from succinate in the light was 33.1% of the metabolized succinate after 1 h and 40.0% after 2 h (Table I). These rates of 14CO2 release are consistent 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 the dark. The situation occurring under conditions that favor pho- tosynthesis over photorespiration remains to be examined since TCAC activity might be influenced by photorespiratory glycine production (11).
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