Plant Physiol. (1970) 45, 70-75

Intermediates of Photosynthesis in Acetabularia mediterranieChloroplasts'

Received for publication July 28, 1969

R. G. S. BIDWELL,2 WENDY B. LEVIN, AND D. C. SHEPHARDDepartments of Biology and Anatomy, Case Western Reserve University, Cleveland, Ohio 44106

ABSTRACT

The chloroplast fraction isolated from Acetabularia medi-terranie was exposed to 14CO2 as NaH'4C03 in light and dark-ness, and soluble radioactive compounds were analyzed atfrequent intervals. The behavior of Calvin cycle intermedi-ates indicates that this cycle was responsible for much of thecarbon fixation in the chloroplasts. However, a substantialpart of recently fixed carbon was metabolized via glycolicand glyceric acids. Possible pathways for their metabolismare discussed. Some carboxylation of C3 acids was suggestedby the behavior of phosphoenolpyruvate and malate. Anumber of amino acids were formed. Small amounts of suchcompounds as citrate, succinate, and fumarate not usuallvassociated with photosynthesis might have been derivedfrom a low level ofmitochondrial contamination. About one-

third of the carbon fixed in light was present in acid-labileinsoluble compounds other than polysaccharides or pro-teins. Dark fixation of CO2 was very small compared withphotosynthesis.

A chloroplast preparation from Acetabularia mediterraniecarries on photosynthesis which appears similar to that of higherplants in many ways (6, 17). The rate ofCO2 fixation is essentiallythe same as that of intact cells, and it is maintained for prolongedperiods in vitro. C02 production occurring in light is similar tophotorespiration in higher plants (6). Phase contrast and electronmicroscopic examination showed that the preparation does notcontain peroxisomes and only a small contamination of cyto-plasm and mitochondria (8, 19). Mitochondrial metabolism couldnot be detected (17). This preparation should therefore provide a

useful system for the study of intermediary metabolism in photo-synthesis and photorespiration. This paper reports the prelimi-nary investigation of short term kinetics in light and darkness ofthe important intermediates and products of chloroplast photo-synthesis.

MATERIALS AND METHODS

Plant Material. Growth of cells and preparations of chloro-plasts have been described (6, 16). Experiments were conducted

I This work was supported by grants to R. G. S. Bidwell and D. C.Shephard from the National Science Foundation, and to R. G. S.Bidwell from the Canadian National Research Council, which aregratefully acknowledged.

I Present address: Department of Biology, Queen's University,Kingston, Ontario, Canada.

on the chloroplasts isolated from 2 to 3 g of cells, about 2 cmlong, each preparation containing 600 to 750 j,g of total choro-phyll (1). The chloroplasts were suspended in 5 ml of the Amedium described earlier (6), except that the pH was reduced to7.2 in order to reduce the equilibrium bicarbonate concentrationof the medium (15). As a result, the bicarbonate concentrationwas low enough that it probably became rate-limiting after about10 min of photosynthesis (6), but it ensured maximum specificactivity of fixed 4CO2 .

Experimental. Chloroplast preparations were illuminated byfluorescent lights (2500 ft-c), and 100 ,uc of NaH14CO3 (specificradioactivity about 40 mc/mmole) were added. Sampling beganimmediately. Samples of 25 Al were withdrawn with a SchwartzBiopette automatic pipette and were quickly applied to thecorner of 8-inch X 11-inch chromatography sheets (Whatman3MM). These were immediately placed in liquid nitrogen until theend of the experiment (30-60 min). A few chromatograms weremade with 50 or 100 ,ul. These did not have any spots which couldnot be seen on the 25 Al chromatograms, showing that all majorcompounds were being detected. With this technique, samplescould be taken at intervals as short as 8 sec, although the samplesize was apt to be erratic because of pipetting errors at this speed.Much of this problem was later eliminated by cutting the tips ofthe polyethylene pipettes at a sharp angle. Samples taken atlonger intervals were in close agreement. The time from with-drawal of the sample to the freezing of the chromatograms wasabout 4 sec. In the dark fixation experiment (Table I), all condi-tions and procedures were identical except that the experimentwas run in darkness.Chromatography and Radioautography. At the conclusion of

the experiment chromatograms were removed singly from theliquid nitrogen, and the origins were fixed by 1- to 2-min exposureto the hot vapors of vigorously boiling ethanol. This procedureavoids the problems of extraction and concentration and reducesthe nonbiological breakdown of labile intermediates to a mini-mum (5). Two-dimensional chromatograms were run in 80%phenol at pH 5.4 and butanol-acetic acid-water (12:3:5) andradioautographed. Representative radioautographs are shown inFigure 1. The spots were cut out and counted on both sides in anultrathin window gas flow Geiger-Mueller counter. The countsfrom both sides were averaged (4) and corrected for coincidence.All results are presented as counts per minute per sample asdetermined from a 25 MAl chromatogram, and they are strictlycomparable within each experiment. The sum of radioactivities ofall the compounds that moved from the origin was called "solubleradioactivity," and the radioactivity which did not move from theorigin was termed "insoluble." Loss of glycolate during chroma-tography was 5 to 7%, based on recovery of radioactivity beforeand after chromatography of glycolate solutions. No correctionwas applied.

Since neither the specific radioactivity of the added Na2'CO3nor the bicarbonate content of the medium was precisely known,it is not possible to calculate the results in terms of quantity of

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Plant Physiol. Vol. 45, 1970 PHOTOSYNTHETIC INTERMEDIATES IN CHLOROPLASTS

L

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FIG. 1. Radioautographs of chromatograms of chloroplast fractions after photosynthesis in 1'C-bicarbonate. AA: Aspartic acid; Ala: alanine;BAW: butanol-acetic acid-water, Cit: citrate; DP: sugar diphosphates; Fum: fumarate; GA: glutamnic acid; Gly: glycine; Glycer: glycerate;Glycol: glycolate; HMP: hexose monophosphates; Mal: malate; Or: origin ("insolubles"); PEP: phosphoenolpyruvate; PGA: phosphoglycerate;Ser: serine; Su: sucrose; Succ: succinate; TP: triosephosphate; X, Y, Z: unknowns.

E

E SOLUBLE

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-0 5 10 20TIA

FIG. 2. Total, soluble, and insoluble radioactivity (counts per minutlight and darkness.

carbon fixed. However, a rough estimate is possible. There were

approximately 3 to 4 ,ug of chlorophyll per 25-,al sample, and thespecific radioactivity of the bicarbonate in the medium was

roughly 30 mc/mmole. With appropriate corrections for self-absorption (4) and counter geometry in the determination of

in chloroplasts during the supply of 14C-bicarbonate in

radioactivity, the calculated rate of CO2 fixation is approximately15 ,umoles of CO2 per min per mg of chlorophyll, about whatwould be expected at the bicarbonate concentration of themedium at pH 7.2 (6, 15). A factor of 1O-4 Wil thus very roughlyconvert the recorded figures for counts per minute per sample to

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BIDWELL, LEVIN, AND SHEPHARD

TIME, minutes

FiG.13. Total radioactivity (counts per minute per sample) in chloroplasts during the supply of 14C-bicarbonate in light and darkness.

TIME, minutes

FIG. 4. Corrected total, soluble, and insoluble radioactivity (counts per minute per sample) in chloroplasts during the supply of 14C-bicar-bonate in light and darkness.

micromoles of CO2 per mg of chlorophyll (1 mg of chlorophyllrepresents about 109 chloroplasts, having a volume of 5 to 10 IAI).

RESULTS

The first experiment ran 30 min in light, followed by 30 minin darkness, with samples being taken at intervals of 1 min orlonger. The total radioactivities are presented in Figure 2. Theinitially high rate of '4CO2 fixation fell off after about 6 min;this was probably due to the low bicarbonate content of themedium. A slow but steady loss of 'IC occurred in the dark,which supports the earlier conclusion that dark respiration ofthe chloroplast preparation occurs at the expense of recentphotosynthate (6). About one-third of the fixed 14C was in the

insoluble fraction, even in the earliest samples. The soluble andinsoluble fractions varied reciprocally during the dark period.After a brief interval during which soluble material was con-verted to insoluble there was a steady transfer of 14C from theinsoluble fraction to soluble compounds.The second experiment was run for 10.75 min in light, then

10.0 min in dark, followed by 5 min in light. Samples were takenat frequent intervals, usually in groups of two or three closetogether to check on the possibility of rapid oscillation in theconcentration of intermediates. The results for total radioactivityare presented in Figure 3. Owing to the hurry in sampling thepoints are somewhat scattered. For ease in comparison, a curvewas drawn to fit the data for total 14C, allowing for the fact that

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Plant Physiol. Vol. 45, 1970 PHOTOSYNTHETIC INTERMEDIATES IN CHLOROPLASTS

TIME, MINUTESFIG. 5. Corrected radioactivities (counts per minute per sample) in all soluble compounds of chloroplasts during the supply of 14C-bicarbonate

in light and darkness.

the pipette delivered smaller aliquots more frequently than largerbecause its tip sometimes touched the bottom of the container.All the data for each sample were then corrected by the factorrequired to put the total 14C content on the line as shown inFigure 3. The 14C content of the insoluble and total solublefractions corrected in this manner are presented in Figure 4.The results were essentially similar to those from the previousexperiment, except that no transient exchange between soluble

and insoluble fractions was evident immediately after darkening.In the second experiment the proportion of 14C entering the in-soluble fraction was lower and showed a lag phase of 1 or 2 minat the start.The distribution of 14C among photosynthetic intermediates

and products was essentially similar in both experiments; thedata from the second experiment are presented because the largernumber of samples gives a clearer picture of the labeling patterns.

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BIDWELL, LEVIN, AND SHEPHARD

The data for all the soluble compounds, corrected as describedabove, are summarized in Figure 5. Sucrose is evidently themain end product of photosynthesis. Sucrose attained the highestradioactivity, it was formed only in light (with a brief overshooton darkening), and it showed no sign of saturation during theexperiment. The intermediates of the Calvin cycle that weredetected-PGA,3 HMP, sugar diphosphates, and triose phos-phates-all behaved in the manner consistent with normaloperation of the cycle (2). They appeared after a time lag of afew seconds, rose rapidly to saturation, fell rapidly on darkening(except PGA, which rose initially as expected), and began toreturn to their previous saturation levels on reillumination. Itis likely, from its chromatographic position and its kineticbehavior, that the sugar diphosphate spot was mainly RuDP.The behavior of glycolate was unexpected, in that it acquired

lable rapidly at first, then saturated shortly after the Calvincycle intermediates, but at a much higher level. On darkening itfell abruptly at first, then more slowly to a low level, and on re-illumination it began to rise sharply again after a 2-min lag. Thisbehavior is characteristic of a compound which is a member of amajor pathway of metabolism with synthesis which continuesonly in light, but with catabolism which continues in darkness (2).Glyceric acid showed a pattern characteristic of a member of areaction mechanism with subsequent metabolism requiring light.It had, however, a lag period at the start and on reilluminationof 2 min. Serine, glycine, and aspartate all acquired radioac-tivity from near the beginning of the experiment, while glutamatehad a lag period of over 3 min. Alanine, aspartate, and glutamatecontinued to acquire radioactivity in the dark, while serine andglycine quickly approached a plateau. Malic acid acquired 14Cat a steady rate from near the start of the experiment, a char-acteristic of an end product of metabolism. However, it fellrapidly on darkening, suggesting that it was actively participat-ing in further reactions. PEP was present in small amounts, butit quickly reached saturation after an initial lag period of about2 min and as quickly fell to zero again when the chloroplastswere darkened. Succinic, citric, and fumaric acids and twounknowns, X and Z (Fig. 1), appeared late in the light and re-mained stable or increased slowly in darkness. A third unknown,Y (Fig. 1), appeared immediately on darkening and quicklystabilized, then fell off again on reillumination.Chromatographic analysis of the medium after the chloro-

plasts were removed by centrifugation showed that only about2% of the fixed radioactive carbon left the chloroplasts duringeither the light or the subsequent dark period. The rate of 14CO2production could not be determined. However, in an attemptto improve the carbon balance sheet, a comparable sample ofchloroplasts was allowed to fix "4CO2 in darkness. The results(Table I) show that the rate of dark fixation was about 4% of therate of photosynthesis and could have accounted only for theincrease observed in aspartic acid in the light-dark-light experi-ment.

Since a substantial amount of 14C entered the insoluble com-pounds at the origin, and some of this 14C was lost in darkness,the composition of this area of the chromatogram is of interest.Several such areas were cut out of chromatograms that had beenrun and were hydrolyzed with HCI under progressively more

severe conditions. Fifteen minutes at 1000 in 0.1 N HCI had littleeffect. During more severe hydrolysis (1 N HCI, 1000 for 1 hr)about 75% of the radioactivity was lost, and chromatography ofthe products of hydrolysis revealed radioactivity only in smallamounts in three unidentifiable spots together with some in-

3 Abbreviations: PGA: 3-phosphoglyceric acid; HMP: hexosemonophosphates; RuDP: ribulose diphosphate; PEP: phosphoenol-pyruvate.

Table I. Products of 10-min Dark Fixation of 14CO2 by aChloroplast Preparation

Results are comparable with those in Figure 5.

Compound Radioactivity

cpm/sampleOrigin area 15Hexose-monophosphate area 19Aspartic acid 276Glutamic acid 108Serine 17Glycine 7Alanine 42Citric acid 47Malic acid 112Succinic acid 4Fumaric acid 9Glyceric acid 10Glycolic acid 10Total 676

soluble radioactive "humin." Further hydrolysis (3 N HCI, 3hr) had no further effect. This suggests that the main insolublecompounds formed were neither polysaccharide nor peptide,but some moderately acid-labile substances yielding volatilecarbon on hydrolysis. The radioactivity so released was nottrapped in barium hydroxide solution, indicating that it is notin the form of CO2 .

DISCUSSION

The results indicate that the Calvin cycle of carbon reductionis operating in these chloroplasts. The intermediates that were

detected, RuDP, HMP, triose phosphate, and PGA all behavedin a similar manner to that reported by Bassham et al. (2, 13).This further supports observations from other plant sources

that chloroplasts fix CO2 by this pathway (9, 20). The productionof sucrose has not been routinely observed in chloroplasts (7,12), but in every experiment performed with Acetabularia chloro-plasts, including many not here reported, sucrose has beenthe major product of photosynthesis. Alanine, which was foundto accompany sucrose production in spinach chloroplasts (7),is always present in substantial amounts. In addition to the Calvincycle, the data for malate and PEP in Figure 5 suggests a ,-carboxylation system leading eventually to malate. The PEP-carboxylating enzyme evidently does not require light activation,as does the RuDP-carboxylating enzyme (13), since quantitiesof PEP quickly fell to zero on darkening. It is probable thatmalate itself is involved in further metabolism, perhaps of a light-requiring nature, since its quantity fell abruptly on darkening,then stabilized. The chloroplasts are able to synthesize severalamino acids and compounds associated with the Krebs cycle.Aspartate, alanine, and serine-glycine are all early products ofphotosynthesis, while glutamate, citrate, succinate, and fumarateare more rapidly formed in darkness. The possibility of theirformation by mitochondrial contamination cannot be excluded.The relatively large amount of carbon entering glycolate and

the large light-dark transient changes of glycolate and glycerateindicate that these two compounds are part of a major pathwayof photosynthetic carbon metabolism. Glycolate may be a

byproduct from Calvin cycle intermediates (3, 14). The lagperiod of 3 to 5 min before it reaches maximum accumulationrate at the start and on reillumination suggests that it is formedfrom some intermediate which takes this time to saturate. Thebehavior of HMP or RuDP is appropriate, and they are likely

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Plant Physiol. Vol. 45, 1970 PHOTOSYNTHETIC INTERMEDIATES IN CHLOROPLASTS

precursors of glycolate by diversion of the C2 fragment from thetransketolase reaction. On the other hand, the decline of glycolateon darkening is greater and more abrupt than that of HMPand RuDP, which is not what would be expected if it werederived from them. As a possible alternative, glycolate might bederived by a suggested direct synthesis from CO2 (7, 18, 21).

It is evident (Fig. 5) that glycolate undergoes substantialturnover, its rapid catabolism continuing for a short time ondarkening. There is strong evidence for the participation ofglycolate in photorespiration (14, 19, 21, 22), and these chloro-plasts do photorespire, although the measured rates are ratherlow (6). However, the data in Figures 2 and 3 show no indicationof the sudden net loss on darkening of over 2000 cpm which wouldhave occurred if all the glycolate which disappeared had beenconverted to CO2. It is therefore necessary to consider alterna-tive fates for glycolate carbon. Among the compounds whichunderwent substantial change on darkening (Fig. 5), the gainsin the 3-carbon compounds alanine, PGA, and glycerate wereoffset by losses in HMP, RuDP, and glycolate. It therefore seemsprobable that glycolate was converted to a 3-carbon compound.The initial reciprocal gain in glyceric acid followed by its paralleldecline with glycolate suggests that carbon from glycolate wasrapidly converted to glycerate, which was being more slowlyconverted to other compounds.Three possible alternatives for the conversion of glycolate to

glycerate may be considered. First, interconversion by theglycolate pathway via glycine and serine (10) seems unlikelybecause the production of serine-glycine did not continue at therequired rate after darkening, and no transients were observed.The initial slope and starting points of the glycolate and serine-glycine curves suggest either interconversion or a common pre-cursor, but not a precursor-product relationship. Also peroxi-somes, in which several steps of the glycolate pathway to serinetake place (11, 19), are absent from this preparation (6). Second,glycolate or a derivative of it might condense with glyceralde-hyde-3-p to form pentose phosphate which could yield PGAby carboxylation. However, this would require that the con-centration of RuDP stay at or near its light level until the disap-pearance of glycolate slowed down, a period of 2 min afterdarkening. Instead it fell most rapidly during this time, whichmakes this reaction sequence unlikely. The third alternative is amore direct conversion of glycolate to glycerate via a hypotheticalreaction sequence requiring light which could continue brieflyin darkness. No clues to the identity of such a reaction can befound from the data in Figure 5. It is evident that further investi-gation of photosynthetic carbon fixation is required to reveal thepathways of synthesis and breakdown of glycolate in Acetabulariachloroplasts.

LITERATURE CITED

1. ARNON, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24: 1-5.

2. BASSHAM, J. A. AND M. CALVIN. 1957. The Path of Carbon in Photosynthesis.Prentice-Hall, Englewood Cliffs, N.J.

3. BASSHAM, J. A. AND M. KIRK. 1962. The effect of 02 on the reduction of C02to glycolic acid and other products during photosynthesis by Chlorella. Biochem.Biophys. Res. Comnun. 9: 376-380.

4. BIDWELL, R. G. S. 1961. Determination of radioactivity on paper chroma-tograms. Can. J. Bot. 39: 607-610.

5. BIDWELL, R. G. S. 1962. Direct paper chromatography of soluble compoundsin small samples of tissue adhering to the paper. Can. J. Biochem. Physiol.40: 757-761.

6. BIDWELL, R. G. S., W. B. LEVN, AND D. C. SHEPHARD. 1969. Photosynthesis,photorespiration and respiration of chloroplasts from Acetabularia mediterranea.Plant Physiol. 44- 746-756.

7. EVERSON, R. G., W. COCKBURN, AND M. GIBBS. 1967. Sucrose as a product ofphotosynthesis in isolated spinach chloroplasts. Plant Physiol. 42: 840-844.

8. FREDERICK, S. E. AND E. H. NEWCOMB. 1969. Microbody-like organelles in leafcells. Science 163: 1353-1355.

9. HAVIR, E. A. AND M. GIBBS. 1963. Studies on the reductive pentose phosphatecycle in intact and reconstituted chloroplast systems. J. Biol. Chem. 283: 3183-3187.

10. KEARNEY, P. C. AND N. E. TOLBERT. 1962. Appearance of glycolate and re-lated products of photosynthesis outside of chloroplasts. Arch. Biochem.Biophys. 98: 164-171.

11. KISAKI, T. AND N. E. TOLBERT. 1969. Glycolate and glyoxylate metabolism byisolated peroxisomes or chloroplasts. Plant Physiol. 44: 242-250.

12. LATZKO, E. AND M. GaBBs. 1969. Level of photosynthetic intermediates inisolated spinach chloroplasts. Plant Physiol. 44: 396-402.

13. PEDERSON, T. A., M. KIRK, AND J. A. BASSHAM. 1966. Light-dark transients inlevels of intermediate compounds during photosynthesis in air-adapted Chlorella.Physiol. Plant. 19: 219-231.

14. PRrTcHARD, G. G., W. J. GRIFFIN, AND C. P. WHT-INGHAM. 1962. The effect ofcarbon dioxide concentration, light intensity and isonicotinyl hydrazide on thephotosynthetic production of glycollic acid by Chlorella. J. Exp. Bot. 13: 176-184.

15. SEVERINGHAUs, J. W. 1965. Blood gas concentrations. In: W. 0. Fenn andH. Rahn, eds., Handbook of Physiology, Sect. B, Vol. II. American Physiologi-cal Society, Washington, D.C. pp. 1475-1487.

16. SHEPHARD, D. C. 1970. Axenic culture of Acetabularia in synthetic media.In: D. Prescott, ed., Methods in Cell Physiology, Vol. IV. Academic Press,New York. In press.

17. SHEPHARD, D. C., W. B. LEVIN, AND R. G. S. BIDwE.L. 1968. Normal photo-synthesis by isolated chloroplasts. Biochem. Biophys. Res. Commun. 32: 413-420.

18. STILLER, M. 1962. The path of carbon in photosynthesis. Ann. Rev. PlantPhysiol. 13: 151-170.

19. TOLBERT, N. E., A. OEsTER, T. KISAKx, R. H. HAGEMAN, AND R. K. YAMAzAKi.1968. Peroxisomes from spinach leaves containing enzymes related to glycolatemetabolism. J. Biol. Chem. 243: 5179-5184.

20. WALKER, D. A., W. COCKBURN, AND C. W. BALDRY. 1967. Photosyntheticoxygen evolution by isolated chloroplasts in the presence of carbon cycle inter-mediates. Nature 216: 597-599.

21. ZELITCH, I. 1965. The relation of glycolic acid synthesis to the primary photo-synthetic carboxylation reaction in leaves. J. Biol. Chem. 240: 1869-1875.

22. ZELITCH, I. 1966. Increased rate of net photosynthetic carbon dioxide uptakecaused by the inhibition of glycolate oxidase. Plant Physiol. 41: 1623-1631.

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