Photosynthetic Carbon Metabolism of Isolated Corn ... - NCBI

Plant Physiol. (1972) 49, 607-614

Photosynthetic Carbon Metabolism of Isolated Corn Cbloroplasts1 Received for publication September 16, 1971

DENNY O'NEAL,2 C. S. HEW,3 ERWIN LATZKO,4 AND MARTIN GIBBS Department of Biology, Brandeis University, Waltham, Massachusetts 02154

ABSTRACT Chloroplasts have been isolated from 4- to 6-day-old corn (Zea ,nays) leaves capable of assimilating 45 micromoles C02 per milligram chlorophyll per hour. The effects of various factors such as inorganic phosphate, reducing agents, inhibitors, intermediates of the photosynthetic carbon reduction cycle, organic acids, and oxygen on the photosynthetic rate and on the distribution of "4C within the products by these chloroplasts were determined. The photosynthetic carbon metabolism of the corn plastids appeared to be similar to that already observed in spinach and pea chloroplasts. It was concluded that the corn plastids can fix C02 at meaningful rates via the photosynthetic carbon reduction cycle of Calvin without the operation of a cycle involving the C-4 compounds, malate and aspartate.

The investigations of Kortschak et al. (16) and Hatch and Slack (11) resulted in the elucidation of a novel pathway for photosynthetic CO2 fixation in some species of higher plants. The data supporting the existence of this pathway (often referred to as the "C-4 pathway" or the "C-4 dicarboxylic acid cycle" and the plants as "C-4 plants") have been the subject of a recent review (12). The studies leading to the postulation of the C-4 pathway included in vivo pulse label experiments and enzymic studies with chloroplasts isolated in nonaqueous media. Another approach to the study of photosynthetic CO2 fixation is the study of the kinetics of "4CO2 fixation and product determination using chloroplasts isolated in aqueous media, as has been done successfully with spinach and pea chloroplasts (9, 24). Until recently it has not been possible to obtain chloroplasts from a C-4 plant (such as corn or sugarcane) which were capable of assimilating CO2 at reasonable rates. Some degree of success was recently achieved by isolating chloroplasts from young corn seedlings, and the results were described in a short communication (10). A more complete description of the carbon metabolism of these chloroplasts is the subject of this report. 1 This research was supported by grants from the National Science Foundation and the United States Atomic Energy Commis-

sion, AT (30-1)3447. 2 Present address: Department of Biology, Carleton University, Ottawa, Canada. 3 Present address: Department of Biology, Nanyang University, Singapore. IPresent address: Chemisches Institut, Technische Hochschule, Miinchen 805, Weihenstephan, Germany.

MATERIALS AND METHODS Zea mays seeds were obtained from Agway, Inc., Waltham, Massachusetts. They were germinated in vermiculite and grown at a light intensity of approximately 2000 ft-c in a controlled environment. The day temperature was 25 C, the night temperature was 20 C, and the photoperiod was 12 hr. Plants were generally harvested 2 to 5 hr after the lights came on and were used immediately to prepare chloroplasts. For most of the studies var. Early Fortune was used, although var. Golden Beauty was employed in a few studies with similar results. The seedlings used for chloroplast isolation were generally 4 to 6 days old. Ten to 25 g (fresh weight) of seedlings were ground in a stainless steel Waring microblender with 2 to 5 volumes of solution I containing 0.35 M sorbitol, 40 mM MES buffer, 5 mM MgCl2, 0.3 mM MnCl2, 0.25 mM KH2PO,, 3.0 mM Na2 EDTA, 40 mm sodium ascorbate, and 5% (w/v) polyvinylpyrrolidone 40, pharmaceutical grade (Sigma Chemical Co.). The pH was adjusted to 6.8 with NaOH. The seedlings were blended in ice-cold solution I twice or three times at the highest speed, each burst lasting about 3 sec. The mixture was filtered through Miracloth (Calbiochem) and centrifuged at 1250g for 2 min. The pellet was resuspended in approximately 5 ml of solution II containing 0.35 M sorbitol, 40 mM HEPES, 0.4 mM MgCl2, 0.2 mM KH,PO4, 1 mM Na2 EDTA, and 5 to 6 mm DTT.V The pH was adjusted to 7.8 with NaOH. It was found subsequently that the optimal molarity of sorbitol or mannitol in solution II (assay medium) was roughly 0.12 to 0.14 M and not 0.35 M. Therefore, some studies were performed using 0.14 M mannitol. The suspended chloroplasts were centrifuged for 2 min at 755g, and the pellet was suspended in 1 to 3 ml of solution II by means of the tip of a stirring rod covered with 4 layers of cheesecloth. Aliquots of these chloroplasts were added to tubes containing solution II plus 4 to 6 mm NaH"4CO3, specific radioactivity 10 to 20 ,uc/,umole. The chlorophyll concentration was generally 15 to 40 jug in a total volume of 0.75 to 1.5 ml. Between these concentrations, the rate of photosynthesis was linear with respect to chlorophyll. The assay tubes were placed in a water bath at 25 C and illuminated with spot lamps on both sides of the tubes. The light intensity was about 2500 ft-c. Nitrogen gas was bubbled slowly through the assay medium in most experiments, although 100% 02 was used in certain experiments. The chloroplasts were normally preincubated in the dark for 2 min prior to illumination. Aliquots were removed rapidly at intervals with a Pasteur pipette or a Schwarz Biopette (Schwarz/Mann Inc.) and added to tubes containing 0.01 ml of concentrated formic acid. Fifty-microliter aliquots were placed on aluminum planchets, dried under a spot lamp, and counted on a Nuclear Chicago gas flow counter. For determination of products, aliquots were spotted on Whatman No. I

'Abbreviation: DTT: dithiothreitol. 607

608

O'NEAL, HEW, LATZKO, AND GIBBS

chromatography paper and separated by either one dimensional chromatography in the "GW-3" solvent of Wood (27) or by two-dimensional chromatography in phenol-H,O containing 1.5 mm Na2 EDTA, and n-butyl alcohol-propionic acid-water. The radioactivity of the labeled compounds was measured either directly on the paper by a Nuclear Chicago end-window counter or by cutting out sections of the paper containing the compounds and counting them in scintillation vials containing toluene-PPO-POPOP. The chloroplasts remaining in the tube were spun down, mashed twice with water, and then reacted with 1 N HC1 for 10 min at 100 C. Following centrifugation, the supernatant fluid was analyzed by paper chromatography, and all the isotope was located in glucose. We, therefore, refer to this insoluble material as polyglucan. For electron microscopy the chloroplasts were fixed in 2% buffered glutaraldehyde, postfixed with 2% osmium tetroxide and the sections after stained with lead acetate. An RCA electron microscope was used for these studies.

Plant Physiol. Vol.

49, 1972

The concentration of the photosynthetic intermediates in the chloroplasts were determined by pipetting aliquots of the chloroplasts into 20% HClO4 at 0 C, such that the final HClO4 concentration was 5%. Following centrifugation for 5 min at 7000g, the supernatant fraction was titrated potentiometrically with 5 M K2CO3 to pH 5.5. The precipitate was removed by centrifugation at 3,000g for 3 min. Ribulose-1 , 5-diP, fructose1 , 6-diP, triose phosphates, (dihydroxyacetone-P and glyceraldehyde-3-P), and glycerate-3-P were determined enzymatically by the method of Latzko and Gibbs (18). Labeled glycerate was degraded by the method of Sakami (21). Chlorophyll was determined by the method of Arnon (1).

RESULTS Kinetics of '4CO2 Fixation; Products. The most active chloroplasts were obtained from plants 4 to 6 days of age and 2 to 4 cm tall. An electron micrograph of chloroplasts isolated from 4-day-old seedlings is shown in Figure 1. Monitored by

FIG. 1. Electron micrograph of chloroplasts isolated from 4-day-old seedlings. Final magnification. X 11,600.

Plant Physiol. Vol.

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CO2 FIXATION IN CORN CHLOROPLASTS

609

FIG. 2. Electron micrograph of chloroplasts isolated from 12-day-old corn seedlings. Final magnification, X 11,600.

a phase contrast microscope, about half of the chloroplasts appeared to be intact with their outer membranes present. If older plants (12 days) were used, a higher proportion of the chloroplasts were broken (Fig. 2). Grana seemed to be more developed and prevalent in the preparations of the older plants. Since there has been a report that photosystem II activity in the corn plant may be correlated with grana formation (26), the rates of 02 evolution by fragmented chloroplasts of the 4-day and 12-day plants fortified with spinach ferredoxin and TPN were compared and found to be roughly comparable (J. M. Robinson, unpublished data). Rates of 02 evolution in the order of 40 ,moles/ mg chl -hr were observed (see also ref.

14). The "CO2 fixation rate of the best chloroplast preparations was as high as 45 ,amoles/mg chl hr, although the more usual rates were 8 to 15 ymoles. Normally there was a white layer associated with the chloroplasts following centrifugation. This material was most likely starch. since it st?ined purple with

KI. There seemed to be a correlation between the presence of this material and lower rates of fixation. With respect to the time course of "CO2 fixation, there was often a slight lag following illumination, usually lasting only 1 to 2 min but as long as 4 min on a few occasions (see Fig. 3). When there was a lag, it was reduced, but not fully eliminated by the addition of dihydroxyacetone-P, glyceraldehyde-3-P, ribose-5-P, glycerate-3-P, sedoheptulose-7-P, and fructose-1 ,6diP at a concentration of 1 mm but not by fructose-6-P and glucose-6-P. The highest rate of fixation usually occurred between 4 to 8 min. When the experiments were carried out in the standard reaction mixture (solution II) under N2 and with a saturating concentration of bicarbonate, the products of 14CO2 fixation over a series of many incubations were as follows: glycerate3-P, 32 to 66%; dihydroxyacetone-P, 3 to 33%; glucose-6-P, 6 to 29%; fructose-6-P, 2 to 8%; fructose-1,6-diP, ribulose1,5-diP, and ribose-5-P, 3 to 11%; insoluble (polyglucan), 2

Plant

O'NEAL, HEW, LATZKO, AND GIBBS

610 7

,aO.2mM Pi+FDP

//

6-

x 0.2mM Pi

5re)

/

4-

/ 0u

/

3-

0/

2-

/x

0

2

2.5 mM Pi+ FDP

~~~0

// -

2.5mM Pi+F6P A

A

~~~~~~1 4

-

-, '0.5mM Pi+ R5P

A/

--~~~

~~~~

a /

/ K

'* 2.5m MP i

8 6 TIME (MIN.)

10

FIG. 3. Effect of inorganic phosphate on "CO2 assimilation in the presence and absence of sugar phosphates. The concentrations in the reaction mixture of 1.2 ml were: bicarbonate, 4.1 mM; DTT, 5 nM; mannitol, 0.12 M; HEPES, 40 mM; and chlorophyll, 55 lAg. The atmosphere was N2. Fifty-microliter portions of the reaction mixture were used to assay radioactivity in the acid-stable products. The fixation rate of the reaction mixture containing 0.2 mM Pi (minus sugar phosphates) was 6.5 ,umoles/mg chl -hr. Abbreviations: FDP: fructose 1, 6-diP; R5P: ribose 5-P; F6P: fructose 6-P. to 13%; glycerate, usually 1 to 2% but, on occasion, was 50 to 60%, and small amounts of glycolate, malate, aspartate, and maltose. We are unable to account for the variability in our

preparations. Nevertheless, the effects studied in this investigation within a single preparation were reproducible and, when necessary, were verified in a large number of separate experiments.

Effect of Inorganic Ions on Rate and Products. Sodium or potassium phosphate, if present at 0.1 to 0.25 mm, caused increases in the fixation rate up to 80%, but higher concentrations were inhibitory (Fig. 3). The concentration of Pi had a marked influence on the distribution of isotope (Table I). The most notable results of an inhibitory concentration of Pi were the sharp decrease in the insoluble fraction and the decline in the ratio of glycerate 3-P to triose phosphate. Sodium pyrophosphate, tested at concentrations of 0.1 to 10.0 mM, increased the rate between 2 and 50% (over a control lacking Pi) but on a molar basis was always less effective than Pi. However, pyrophosphate did not cause any inhibition even at a concentration of 10 mm, and, in contrast to Pi, pyrophosphate had little effect on the distribution of label. The inhibition caused by 2 to 2.5 mm Pi could be largely overcome by the addition of glycerate-3-P, fructose-1,6-diP, and ribose-5-P (Fig. 3 and Table I). Although 2 mm glycerate3-P caused little stimulation in the usual reaction mixture, it caused a 50% stimulation when Pi concentration was increased 10-fold. Phosphate concentrations above 0.1 mm are also known to inhibit CO2 fixation and oxygen evolution in spinach (22) and pea chloroplasts (5) and be reversed by some intermediates of the photosynthetic reductive pentose phosphate cycle of Calvin. Walker and Crofts (24) suggest the possibility that Pi inhibits by causing phosphorylated compounds of the photosynthetic carbon reduction cycle inside the chloroplast to leak out, thus

Physiol. Vol. 49, 1972

lowering their concentration resulting in the pronounced lag in fixation observed by Cockburn et al. (5) at high Pi concentrations. In contrast to the pea and spinach chloroplast, high Pi caused no pronounced lag in corn chloroplasts which may be the result of an abundance of carbon cycle intermediates. Phosphate inhibition of spinach ribulose-1,5-diP carboxylase (25) could be relevant here, but this alone would not explain all the pronounced shifts in distribution of isotope (Table I). Phosphate is also known to inhibit ADP glucose-pyrophosphorylase (20), which could account for the lower amount of insoluble polyglucan synthesized where Pi was over 0.2 mM. A possible explanation for the reversal of Pi inhibition by phosphate esters has been presented by Heldt and Rapley (13). They observed that in spinach chloroplasts Pi, glycerate-3-P, and triose phosphates compete with each other for entry into the chloroplast. Therefore, adding glycerate-3-P or triose phosphates to isolated chloroplasts should lead to decreased Pi uptake and may even displace Pi already inside the chloroplast. Their speculation is strengthened by our observation that the sugar phosphates not only overcame the inhibition by Pi but also restored the distribution of "4C found in the incubation medium containing 0.2 mm Pi (Table I). The fixation rate was not affected by the addition of Mge (0.1-4.0 mM) or Mn2' (0.1-0.6 mM) in the incubation medium, nor did NaCl have an effect at concentrations up to 20 mM. Effect of DTT and Reducing Agents. A reducing agent was required in the reaction mixture to attain high rates even when the incubations were carried out in an atmosphere of N2. DTT was the most effective of the several thiols tested, with the highest stimulation of fixation occurring at a concentration of 2.5 to 6 mm with Early Fortune or 10 mm with Golden Beauty (Table II). The percentage of stimulation varied widely between preparations, and occasionally DTT stimulated less than 10 or 15%, although in some preparation the dithiol caused a 5- to 10-fold increase. Sodium ascorbate (5-20 mM) caused at best a 5 to 35% stimulation, and 1 to 5 mM mercaptoTable

1.

Effect of Iniorganzic Phosphate antd Frlctose 1,6-diP

on

14C02 Fixation

The chloroplasts had photosynthesized for 9 min before stopping the reaction. Chromatography was carried out in Wood "GW-3" solvent. Following elution from the paper, radioactivity in the compounds was determined in a scintillation counter. Distribution of Isotope in the Presence of

Products 0.2 mm Pi

2.5

mm Pi

%c

Insoluble' Sugar diP2 and pentose-P3 Glucose-6-P Fructose-6-P Dihydroxyacetone-P Glycerate-3-P Relative fixation rate 1

in

1

2

5.4 2.0 12.5 6.9 21.5 49.1

1004

0.2 mm

2.5 mm

0.2 mt

0.2 mt

FDP

FDP

of total label

0.5 1.1 16.5 10.3 36.8 31.3 20

6.3 1.7

8.4 4.0

23.6 53.3 127

0.5 0 12.9 3.1 34.0 44.9 80

The insoluble material was a polyglucan. Boiling this material N HCl for 10 min yielded only "4C-glucose. Fructose 1,6-diP, with small amounts of ribulose 1,5-diP and

sedoheptulose 1,7-diP. 3 Ribose and ribulose 5-P. 4 Fixation rate was 6.5 ,moles CO2 'mg chl hr.

Plant

Physiol. Vol. 49, 1972

CO2 FIXATION IN CORN CHLOROPLASTS

Table II. Effect of Dithiothreitol oli 4CO2 Fixationz CO2 fixation was stopped after 15 min, and the products were chromatographed in Wood "GW-3" solvent. The compounds were counted in a scintillation counter. Distribution of Isotope in the Presence of

Products NoDTT

Insoluble Sugar diP, glucose-6-P,' and pentose-P' Fructose-6-P Dihydroxyacetone-P Glycerate-3-P Glycerate Relative fixation rate

0.5 10.4 6.2 8.7 62.4 1.2 1002

1mmDTT 2.5mmDTT10mmeDTT

%70 of total label 0.9 4.9 29.0 32.3 8.2 11.7 46.1 4.0 379

6.5 13.0 40.9 2.4 471

3.0 21.3 7.6 8.7 55.7 3.7 534

Glucose 6-P comprised the greatest percentage (over 60%) of the label in this group. 2 Fixation rate was 2.4 ,umoles, mg chl -hr.

ethanol had little effect. The presence of DTT in the reaction mixture brought about an increase of isotope in compounds at the carbohydrate level (data not shown). The requirement of the strong reducing agent in the incubation medium in order to attain relatively high rates of `4CO2 assimilation by corn chloroplasts is a major difference between the corn and spinach organelle. This difference may reflect the fact that intact spinach chloroplasts evolve 02 stoichiometrically to CO2 assimilated while we have not observed 02 evolution polarographically by our intact corn preparations. Indeed, the corn chloroplasts had a rapid 02 uptake which was accelerated during photosynthetic fixation of CO2. These 02-consuming reactions were eliminated during chloroplast lysis since TPN reduction by H20-washed fragments was accompanied by a stoichiometric release of 02 (J. M. Robinson, unpublished data, and ref. 14). It is possible that during isolation of the chloroplast, latent 02-consuming reactions were activated or absorbed onto the outer envelope of the organelle. Repeated washing in solution II did not decrease the rate of 02 uptake. This high endogenous 02 uptake observed in our corn preparations appears similar to that described by Baldry et al. (3). They demonstrated that 02 uptake in sugarcane chloroplasts was linked to the presence of polyphenols and phenoloxidase and was independent of light. Failure to demonstrate `4CO2 uptake in chloroplasts from older leaves may be partly due to high concentrations of these substances. Inhibitor Studies. Several kinds of inhibitors were tested and found to influence the rate of "4CO2 fixation. When chloroplasts were incubated in the usual reaction mixture but without DT1 or phosphate esters, 10 utM trifluorocarbonyl cyanide phenylhydrazone caused a 70% inhibition, 2 /uM DCMU brought about a 90% inhibition, and 5 tum sodium arsenite or iodoacetamide resulted in 50% inhibitions. Sodium fluoride at 10 mm produced a 40 to 50% inhibition together with a pronounced change in label distribution. The polyglucan fraction decreased 35 to 40%, glucose 6-P increased 35 to 80%, dihydroxyacetone-P increased by about 250%, and glycerate 3-P decreased by 40 to 45%. Antimycin A, at concentrations between 0.5 and 50 ,uM had no consistent effect on the fixation rate. This result contrasts with spinach chloroplasts which are stimulated several fold by low concentrations (2-10 ytM) of antimycin A (7, 22). In one exoeriment where the antibiotic inhibited fixation by 10%,

611

47%, and 93% at concentrations of 1.3 /.M, 13 ptM, and 65 /.cM, respectively, there was an increase in percentage of glycerate 3-P (from 43-63%) relative to the total amount of labeled products. Antimycin A causes a similar effect on the distribution of "4C in spinach preparations (23). Several known inhibitors of glyceraldehyde 3-P dehydrogenase including threose-2,4-diP and the pyridine nucleotide analogues, 3-acetylpyridine DPN, 3-acetylpyridine TPN, and 3-pyridine aldehyde DPN, did not affect "4CO2 fixation or distribution of isotope in corn chloroplasts when present in a final concentration ranging from 0.25 to 2.5 mm. In spinach chloroplasts, threose-2,4-diP and 3-pyridine aldehyde DPN at 0.25 to 1 mm caused a 50 to 90% inhibition, while 0.5 mM 3-acetyl pyridine DPN and 0.5 mm 3-acetyl pyridine TPN had no effect (data not shown). Effect of Phosphate Esters, Organic Acids, Free Sugars, and Nucleotides. A number of intermediates of the photosynthetic carbon reduction cycle stimulated `4CO2 fixation. Glyceraldehyde 3-P and dihydroxyacetone-P at 1 to 2 mm caused up to 100% stimulations (Fig. 4), closely followed by fructose-1,6diP and ribose-5-P. Sedoheptulose-7-P stimulated 20 to 40%, while P-enolpyruvate, glycerate-2-P, glucose-6-P, fructose-6-P, glucose-1-P, and gluconate 6-P usually were without effect. The results with glycerate-3-P were variable, with up to 40 to 50% stimulation in one experiment and little in others. Other compounds having no effect on "4CO2 fixation or isotope distribution included D-fructose, D-ribose, D-glucose, r-erythrose, dihydroxyacetone, DL-glyceraldehyde, glyoxylate, glycolate, Lmalate, aspartate, and oxaloacetate. These compounds were present at 1 to 2 mm concentrations. Glycolaldehyde and glycolaldehyde-P caused inhibition at 30 to 90% at concentrations between 0.3 and 1.0 mM. Inhibition by these 2-carbon 0

14.

i2S

5.2mM HC03 + DHAP

I0-

re)

5.2mM HCO3 x

8-

0

0~~~~~

6-

0

0.33mM

/

+ DHAP

4.

HCO3

A

/

0.33mM HC03

A

/

2A

_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2

4

6

8

10

TIME (MIN.)

FIG. 4. Effect of dihydroxyacetone-P (DHAP) on rate of 14C02 fixation under limiting (0.33 mM) and nonlimiting (5.2 mM) bicarbonate concentrations. In a total volume of 1.5 ml, the concentrations were as follows: sorbitol, 0.35 M; chlorophyll, 31.7 ,ug; DTT, 5 mM. Fifty-microliter aliquots were assayed. A: 0.33 mM bicarbonate; A\: 0.33 aM bicarbonate plus 1 mM DHAP; 0: 5.2 mM bicarbonate; 0: 5.2 mM bicarbonate plus 1 mM DHAP. Fixation rates at 5.2 mm bicarbonate and 0.3 mm bicarbonate were 14.7 and 8.8 Amoles/mg chl hr, respectively. -

O'NEAL, HEW, LA.TZKO, AND GIBBS

612

compounds were reported earlier for spinach chloroplasts (4) and may be due to the removal of dihydroxyacetone-P by an aldolase catalyzed reaction. The concentration of dihydroxyacetone-P, ribose 5-P, glycerate 3-P, and fructose 1,6-diP needed for approximately 50% of maximal stimulation were in the order of 10 to 20 p.M, whereas glyceraldehyde 3-P was at least 200 lzM. The stimulation by sugar phosphates was greatest if they 121

5mM HC03, N2A

10-

,

/5mM HCH3, 3

/02

8-

O.5mM HCO3,

2x6-

N2

A

(-)

4-

0

o 0

2

Physiol.

Vol.

49, 1972

present for 1 to 3 min before illumination, although they did increase fixation rate even if added 3 min after the onset of illumination. The label distribution was not influenced by time of addition of the sugar phosphates, however. Phosphate esters caused approximately the same degree of stimulation at both nonlimiting (5.2 mM) and limiting (0.33 mM) bicarbonate concentrations (Fig. 4). These effects of intermediates of the photosynthetic carbon reduction have been described in spinach (2) and pea (4) chloroplasts with similar findings. Oxidized and reduced pyridine nucleotides at 0.1 mm to 2 mm sometimes, but not always, caused up to a 25% stimulation, but were without much effect on the distribution of isotope. Spinach ferredoxin did not affect the rate or the products. Purine and pyrimidine nucleotides sometimes stimulated slightly (usually only 5-20%). ATP, and to a lesser extent GTP, UTP, and ADP (all at 0.5-3.0 mM) caused a decrease in percentage of label in the polyglucan (up to 85% decrease) and fructose 1 ,6-diP (up to 70% decrease) and an increase in triose phosphate (about a 40% increase). Effect of Nitrogen and Oxygen. In the presence of bicarbonate concentrations below 2 or 3 mm, 100% 02 often caused at least a 40% inhibition of CO2 fixation (Fig. 5), although the was degree of inhibition varied widely. The inhibition by generally greater as the bicarbonate concentration was decreased (Fig. 6). As was the case in an atmosphere of N,, several phosphate esters increased the fixation rate in 100% 02, but caused no more stimulation than that which occurred in N2. Sodium ascorbate (10 mM) and DTT (5 mM) did not alter the percentage of inhibition by 100% oxygen relative to the control. Similar results have been observed with spinach chlo-

were

X~~~~~~ 00.5mM HC0-, 02 ~~Z.l A~~~~~~~ 0~~~~~

2-~~~~~~~~~~ 2-1

Plant

4

6 TIME (MIN.)

10

8

FIG. 5. Effect of oxygen and nitrogen on '4CO2 fixation at varying bicarbonate concentrations. In a total volume of 1.50 ml, the osmotic pressure was maintained by 0.22 M mannitol. Chlorophyll was 68 ,ug. Dithiothreitol was omitted. Fifty-microliter aliquots were assayed. *: 0.5 mm bicarbonate, 02; 0; 0.5 mM bicarbonate, N2; A: 5 mM bicarbonate, N2. The fixation rate at 5 mM bicarbonate under N2 was 15.3 ,umoles/mg chl -hr.

roplasts (7). With respect to label distribution, the effect of 02 was striking, as shown in Table III. The increase in dihydroxyacetone-P and decrease in polyglucan were pronounced, as was the appearance of glycolate under low bicarbonate and high 02. However, even when the concentration of bicarbonate was between

0.25 and 0.75 mm (under 100% 0,), less than 15% of the label generally appeared in glycolate, unlike the case in spinach where up to 60% of the label appears as glycolate under similar conditions (8, 19). The data presented in Table III were also selected to illusTable III. Effect of Oxygeni, Nitrogeli, alid Bicarboniate onl 14C02 Fixatioln The chloroplasts had photosynthesized for 10 min either in 100% N2 or 100V( 02 before ending the reaction with formic acid. The products were chromatographed in Wood "GW-3" solvent, and the compounds were counted in a scintillation counter. Distribution of

Productsa

Isotope

5mm

5mM

0.5mmM

0.5mm

HC03-

HCO3and 02

and N2

HCO3-

HCO,and 02

and N2

% of total

0.20

0.30

MANNITOL (MOLARITY) FIG. 6. Effect of mannitol concentration on the rate of CO2 fixation. Chloroplasts were isolated in the usual reaction mixture, suspended following centrifugation in 0.18 M mannitol and finally suspended in the desired mannitol concentration after the second centrifugation. Components other than mannitol in the reaction mixtures (total volume of 1.16 ml) were 40 mM HEPES, 0.4 M MgCl2, 0.2 mM MgCl2, 0.2 M KH2PO4, 1 mM Na2 EDTA, 5 mM DTT, and 5 mM bicarbonate. The chlorophyll concentration was 38 ,>g and the atmosphere was air. The highest rate (0.12 M mannitol) was 13.1 umoles/mg chl [hr.

Insoluble

in the Presence of

label

Sugar diP, pentose-P

8.7 4.6

4.2 4.3

8.1 5.3

0.7 1.7

Glucose-6-P Fructose-6-P

6.2 2.5

11.4 4.3

8.9 2.8

16.4 6.8

Unknown

6.9

7.6

7.4

8.5

Relative fixation rate

6.6 2.9 59.3 2.3 1001

14.2 6.4 41.2 6.4 79

14.5 20.2 31.6 1.2 63

16.3 8.8 15.5 23.3 29

Fixation rate

10.2 ,umoles/mg chl - hr.

Dihydroxyacetone-P Glycerate-3-P Glycerate Glycolate was

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C02 FIXATION IN CORN CHLOROPLASTS

trate that, on occasion, the bulk of the isotope was found in glycerate rather than in glycerate 3-P. No attempt was made to investigate the factors involved. Other Factors. The optimal concentration of sorbitol or mannitol for `CO2 fixation was about 0.12 to 0.14 M (Fig. 6). The concentration of mannitol had some effect on the label distribution of isotope, but the effect was variable and no consistent trends were apparent. In contrast, to maintain the osmotic pressure for high rates of CO2 fixation in pea and spinach chloroplasts, sugars, sodium chloride, or sugar alcohols such as sorbitol or mannitol at concentrations of 0.33 to 0.4 M are commonly used. It is well known that the ability of chloroplasts to catalyze the reduction of CO2 to carbohydrate is related to the intactness of the double outer envelope. Therefore, it would appear that the corn organelle isolated from young seedlings is able to maintain its integrity at much lower osmotic pressures than that usually employed with other chloroplasts isolated from fully expanded leaves. The concentration of "C-bicarbonate giving approximately 50% maximal velocity varied between 0.4 to 0.9 mm in three experiments. As bicarbonate was increased from 0.25 mm to 8.0 mm, the percentage of "C in dihydroxyacetone-P and glucose 6-P fell, while glycerate 3-P and polyglucan increased. Similar findings have been reported for other chloroplasts (9, 24). The rate of "CO2 fixation was not affected when the temperature was increased from 20 C to 30 C, but fell about 50% when the temperature was 10.8 C. Pea chloroplasts respond similarly to temperature (4). Malate and aspartate did not appear to be metabolized by these chloroplasts. When 0.1 mM "C-L-malate (uniformly labeled) or 2.7 MM "C-L-aspartate (/3-carboxyl labeled) was incubated with chloroplasts (14 ,.tg chlorophyll) for 30 min in the presence of 0.5 mM bicarbonate, no label was found in other compounds, even when the chloroplasts were supplemented with the supernatant solution following centrifugation in solution I, 0.5 mM ATP, or 1.0 mm a-ketoglutarate. That 0.5 mm bicarbonate diluted out the "CO2 released from the labeled malate can be discounted, since chromatography of the reaction mixture revealed only one radioactive compound, namely malate. In addition, unlabeled L-malate did not inhibit the photosynthetic rate in the presence of "CO2. Interestingly, these results were observed even though malic enzyme is present in these chloroplast preparations (10). There is the possibility, however, that the malic enzyme was inhibited by the high concentrations of bicarbonate used in these experiments. Degradation of Glycerate 3-Phosphate. To be sure that the complete photosynthetic carbon reduction cycle was functioning in these chloroplasts, the glycerate 3-P that had accumulated after 8 min of photosynthesis in saturating bicarbonate and without a phosphorylated primer such as fructose-1,6diP, was enzymically converted to glycerate and degraded. In one experiment where the fixation rate was 9 tsmoles/mg chlhr, the percentage of the label in C-1 (COOH-carbon) was 72%, and 28% was found in C-2 and C-3. In another experiment, in the presence of 3 mm ribose-5-P and with a photosynthetic rate of 20 ,umoles/mg chl hr, 75% of the label was in the carboxyl-carbon. We interpret these data to indicate that our corn chloroplasts possess a complete photosynthetic carbon reduction cycle. Levels of Intermediates. The total concentration of several intermediates of the carbon reduction cycle obtained after incubating the chloroplasts for 10 min in the light was determined (data not shown). When illumination was interrupted, the changes in the levels of these intermediates was similar to what one finds with spinach chloroplasts (18), where fructose

1,6-diP. ribulose-1 , 5-diP, and triose phosphate drop rapidly

with a corresponding rise in glycerate-3-P. The concentration of these compounds was about equal to that in detached spinach chloroplasts (18).

CONCLUSION The photosynthetic carbon metabolism of plastids isolated from young corn seedlings appears to be rather similar to that observed in isolated spinach and pea chloroplasts (9, 24). Although the fixation rate in corn was lower, most of the differences are quantitative rather than qualitative, such as percentage of stimulation by sugar phosphates, percentage of inhibition by oxygen and product distribution. Perhaps the most important results of the present study lie in their relationship to a better understanding of the nature of the CO2 fixation pathway in C-4 plants. The corn plastids characterized in these studies can fix CO2 at meaningful rates via the photosynthetic reductive pentose phosphate cycle of Calvin without the necessity of a functional C-4 cycle or a decarboxylation of malate or aspartate. It is possible, of course, that these plastids are atypical owing to their origin in very young seedlings. Apparently they have not fully differentiated into typical mesophyll and bundle sheath chloroplasts (Fig. 1), for few of them possessed well developed grana, whereas fully developed corn mesophyll chloroplasts are known to possess well defined grana similar to sugarcane (17). However, the intact seedlings used in this study for chloroplast isolation do have the same CO2 fixation patterns, i.e., high amounts of isotope in malate and aspartate after a short period of photosynthesis, as do older plants (L. J. Laber, unpublished data). This is not to suggest that malate and aspartate play no important roles in CO2 fixation in the corn plant. These compounds may serve as means for storing CO2 that is subsequently released within the cell and reduced in the Calvin cycle. If this speculation is correct, then the metabolism of CO2 by the corn plant would be comparable to that described for the crassulacean type plants. The observations that the corn chloroplasts used in this study appeared to have a functional Calvin cycle are consistent with the data provided by Edwards et al. (6), which imply that isolated bundle sheath cells of the C-4 plant Digitaria sanguinalis L. fixed CO2 directly by the Calvin cycle, and the data of Kortschak and Nickell (15) in which the major products of CO2 fixation in parenchyma callus tissue of sugarcane were phosphorylated compounds of the Calvin cycle, rather than the C-4 compounds, malate, and aspartate. Acknowledgment-We are deeply indebted to Dr. Shimon Klein of the Hebrew University for doing the electron microscopy. LITERATURE CITED 1. ARN-ON-, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24: 1-15. 2. BAMBERGER, E. S. AND M. GiBBs. 1965. Effect of phosphorylated compounds and inhibitors on C02 fixation by intact spinach chloroplasts. Plant Physiol. 40: 919-926. 3. BALDRY, C. W., C. BUCKE, J. COOMBS, AND D. GROSS. 1970. Phenols, phenoloxidase, and photosynthetic activity of chloroplasts isolated from sugarcane and spinach. Planta 94: 107-123. 4. BALDRY, C. W., C. BUCKE, AND D. A. WALKER. 1966. Temperature and photosynthesis. I. Some effects of temperature on carbon dioxide fixation by isolated chloroplasts. Biochimn. Biophys. Acta 126: 207-213. 5. COCKBURN, W., D. A. WALKER, AND C. W. BALDRY. 1968. Photosynthesis by isolated chloroplasts. Reversal of orthophosphate inhibition by Calvin cycle intermediates. Biochem. J. 107: 89-95. 6. EDWARDS, G. E., S. S. LEE, T. M. CHEN., AN-D C. C. BLACK, JR. 1970. Carboxylation reactions and photosynthesis of carbon compounds in isolated mesophyll and bundle sheath cells of Digitaria sanguinalis (L.) Scop. Biochem. Biophys. Res. Commun. 39: 389-395. 7. ELLYARD, P. W. 1968. The Warburg effect: investigations using isolated spinach chloroplasts. Ph.D. thesis. Cornell University, Ithaca. 8. ELLYARD, P. W. AND M. GIBBS. 1969. Inhibition of photosynthesis by oxygen in isolated spinach chloroplasts. Plant Physiol. 44: 1115-1121.

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9. GIBBS, M. 1967. Photosynthesis. Annu. Rev. Biochem. 36: 757-784. 10. GIBBS, M., E. LATZKO, D. O'NEAL, AND C. S. HEW. 1970. Photosynthetic carbon fixation by isolated maize chloroplasts. Biochem. Biophys. Res. Commun. 40: 1356-1361. 11. HATCH, M. D. AND C. R. SLACK. 1966. Plhotosyntlhesis by sugar cane leaves. A new carboxylation and the pathway of sugar formation. Biochem. J. 101: 103-111. 12. HATCH, 'M. D. AND C. R. SLACK. 1970. Photosynthetic C02-fixation pathways. Annu. Rev. Plant Physiol. 20: 89-116. 13. HELDT, H. W. AND L. RAPLEY. 1970. Specific transport of inorganic plhosphate, 3-phosphoglycerate, dihydroxyacetone phosphate, and of dicarboxylates across the inner membrane of spinach chloroplasts. FEBS Lett. 10: 143-148. 14. IIEW, C. S. AND M. GIBBS. 1970. Light induced 02 evolution, triphosphopyridine nucleotide reduction and phosphorylation by chloroplasts of maize, sugarcane, and sorghum. Can. J. Bot. 48: 1265-1269. 15. KORTSCHAK, H. P. AND L. G. NICKELL. 1970. Calvin-type carbon dioxide fixation in sugarcane stalk parenchyma tissue. Plant Physiol. 45: 515-516. 16. KORTSCHAK, H. P., C. E. HARTT, AND G. 0. BURR. 1965. Carbon dioxide fixation in sugarcane leaves. Plant Physiol. 40: 209-213. 17. LAETSCH, W. M. AND I. PRICE. 1969. Development of dimorphic clhloroplasts of sugar cane. Amer. J. Bot. 56: 77-87. 18. LATZKO, E. AN-D NM. GIBBS. 1969. Level of photosynthetic intermediates in isolated spinach chloroplasts. Plant Physiol. 44: 396-402.

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19. PLAUT, Z. AND M. GIBBS. 1970. Glycolate formation in intact spinach chloroplasts. Plant Physiol. 45: 470-474. 20. PREISS, J. AN-D T. KOSUGE. 1970. Regulation of enzyme activity in photosynthetic systems. Annu. Rev. Plant Physiol. 21: 433-466. 21. SAKANII, W. 1950. Formation of formate and labile methyl grouips from acetone in the intact rat. J. Biol. Chem. 187: 369-378. 22. SCHACTER, B. 1970. Studies on the control of photosynthesis in isolated chloroplasts. Ph.D. thesis. Brandeis University, Waltham. 23. SCHACTER, B. Z., AM. GIBBS, AND M. L. CHAMPIGNY. 1971. Effect of antimycin A on photosynthesis by intact spinach chloroplasts. Plant Physiol. 48: 443446. 24. WALKER, D. A. AND A. R. CRoFrs. 1970. Photosynthesis. Annu. Rev. Biochem. 39: 389-428. 25. WEISSBACH, A., B. L. HORECKER, AND J. HURWITZ. 1956. The enzymatic formation of phosphoglyceric acid from ribulose diphosphate and carbon dioxide. J. Biol. Chem. 218: 795-810. 26. Woo, K. C., J. M. ANDERSON', N. K. BOARDMAN, W. J. S. DOWNTON, C. B. OSMOND, AND S. W. THORPE. 1970. Deficient photosystem II in agranal bundle sheath chloroplasts of C4 plants. Proc. Nat. Acad. Sci. U.S.A. 67: 18-25. 27. W,VooD, T. 1961. A procedure for the analysis of acicl-soluble phosphorus compounds and related stubstances in muscle and otlier tissues. J. Chromatog. 6: 142-154.