Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid ...

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Jan 14, 1980 - Pi concentrations, and the relatively low pH optimum for photosynthesis ... This work was supported by the College of Agricultural and Life.
Plant Physiol. (1980) 65, 1044 1048 0032-0889/80/65/1044/05/$00.50

Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum' Received for publication September 20, 1979 and in revised form January 14, 1980

MARTIN H. SPALDING' AND GERALD E. EDWARDS Horticulture Department, University of Wisconsin Madison, Wisconsin 53706 ABSTRACT Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praeakum D.C. Typical rates of CO2 fixation or C02-dependent 02 evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO2 fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millmolar) caused some inhibition. Both CO2 fixation and COrdependent 02 evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 miimolar Pi. The lag phase could be decreased by addition of dihydroxyacetonephosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator. A long lag period at optimum Pi concentration, no effect of high Pi levels on the lag phase, lack of severe inhibition of photosynthesis by high Pi concentrations, and the relatively low pH optimum for photosynthesis are characteristics unlike those previously reported with chloroplasts of C3 plants.

their chloroplasts. This may suggest some functional difference between CAM chloroplasts and C3 chloroplasts with respect to photosynthate partitioning. A method of isolating intact, functional chloroplasts from a CAM plant has been developed with S. praealtum by using the relatively gentle technique of chloroplast isolation from enzymically isolated protoplasts. The method of isolation and some characteristics of photosynthesis in these chloroplasts are reported here.

MATERIALS AND METHODS Plants and Reagents. Sedum praealtum D.C. plants were grown as described in Affon et aL (1). Young but fully expanded leaves (7th-14th leaf from apex) were utilized for protoplast isolation. Cellulysin was obtained from Calbiochem. Biochemicals and reagent grade enzymes were obtained from Sigma, and NaH'4CO3 was obtained from Amersham/Searle. Protoplast Isolation. Protoplasts were isolated and purified by a method similar to that described previously (16). Isolations were begun 2-4 h into the light period. Leaf slices were incubated 3-4 h in 2% Cellulysin, 300 mm sorbitol at pH 5.0. A crude protoplast pellet (I00g, 5 min) was resuspended in approximately 8 ml of a solutidn containing 20% (w/v) Ficoll, 300 mm sucrose, and 50 mM Hepes-KOH (pH 7.8). Layered on this to within 1 cm of the top in a 10-ml volumetric flask was a similar solution in which the Ficoll concentration was 10%o (w/v). The flask was filled with a CAM is one of the three major pathways for photosynthetic tird layer containing 300 mm sorbitol and 50 mm Hepes-KOH CO2 assimilation in higher plants (4). The C3 and C4 pathways (pH 7.8). The gradient was centrifuged in a clinical swinging have been extensively investigated through the use of isolated bucket centrifuge at approximately 100g for 10 min at room chloroplasts (5, 6, 9, 14, 17), but little has been published on the temperature. The intact floated to the interface bemetabolism of chloroplasts or other organelles isolated from CAM tween the 10%o Ficoll layerprotoplasts and the sorbitol solution. plants. Intact, well coupled mitochondria have recently been Chloroplast Isolation. The protoplasts were suspended in a isolated from the CAM species Sedum praealtum and studied with breaking medium (- 500 ,tg Chl/5 ml) containing 250 mm Hepesrespect to respiratory metabolism (1) and C4 acid decarboxylation KOH (pH 8.2), 150 mm sorbitol, 1% (w/v) soluble PVP-40, 2 mM capacity (15). The Hill reaction and photophosphorylation capac- EDTA, 1 mM MgCl2, I mm MnCl2, 0.1 mm K2HPO4, and 5 mM ities of isolated cactus chloroplasts (class II) have also been dithioerythritol (dithioerythritol deleted for 02 exchange experiinvestigated (8). Previous reports on carbon metabolism in isolated ments). The protoplasts were ruptured by drawing the suspension chloroplasts from CAM plants demonstrated only low rates of into and expelling it quickly from a 5-ml disposable syringe fitted photosynthetic CO2 fixation (10, 13). with a 25-gauge needle. This resulted in complete rupture of all Photosynthetically active chloroplasts isolated from CAM protoplasts. The suspension of ruptured protoplasts was centriplants would prove extremely useful in the study of the CAM fuged (400g, 3 min) quickly over a 2-ml cushion containing 5% pathway and its regulation at the subcellular level. Recent evi- (w/v) Ficoll, 300 mm sucrose, 50 mM Hepes-KOH (pH 7.6), 2 mM dence concerning the compartmentation of enzymes of the CAM EDTA, 1 mM MgCl2, and I mm MnCl2. The chloroplasts (more pathway in S. praealtum (16) suggests that CAM chloroplasts may than 50o of Chl) formed a pellet below the cushion and the have metabolic pathways and transport properties which combine remainder of the broken protoplast suspension remained above those of C3 and C4 mesophyll chloroplasts. the cushion. The chloroplast pellet was gently resuspended in 1-2 CAM plants also typically form a large amount of starch in ml of the resuspension medium (300 mm sorbitol, 50 mM HepesKOH, 2 mm EDTA, 1 mM MgCl2, 1 mm MnCl2 [pH 7.6]) using a ' This work was supported by the College of Agricultural and Life Pasteur pipette. Chloroplasts prepared by this method were rouSciences, University of Wisconsin, Madison, and National Science Foun- tinely found to be 80-99% intact based on ferricyanide-dependent dation Grant PCM 77-09384 to G. E. E. 02 evolution before and after osmotic shock (I 1). 2Current address: Department of Agronomy, University of Illinois, 14CO2 Fixation. Assays for CO2 fixation in chloroplasts were performed at 30 C in a medium (final volume 500 ,ul) containing Urbana, Illinois 61801. 1044

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300 mm sorbitol, 50 mm Hepes-KOH (pH 7.6), 2 mm EDTA, 1 mM MgCl2, 1 mM MnCl2, 0.4 mM Pi, 10 mM NaH 4C03 (2-4 ,Ci/ ,umol), 200 units catalase, and 20-40 jig Chl with additions and deletions as indicated. Light was supplied by a General Electric Lucalox lamp providing a total quantum flux density of roughly 500 ,uE m-2 s' between 400 and 700 nm (found to be saturating for CO2 fixation; data not shown). The assays were run in 2.5-ml shell vials suspended in a temperature controlled water bath illuminated from below. Samples of 50 ,ul were withdrawn at time intervals (over approximately a 30-min time course), injected into 50 p1 20%1o (w/v) trichloroacetic acid, flushed with air, and acidstable radioactivity determined by liquid scintillation spectroscopy. When rates of fixation are reported, they refer to the linear rate obtained after the lag phase. 02 Exchange. 02 exchange in isolated chloroplasts was followed polarographically at 30 C using twin Clark-type electrodes (Rank Brothers, Bottisham, Cambridge, UK). The assay medium was identical to that for 14CO2 fixation with additions and deletions as indicated. In calculating rates of photosynthetic 02 evolution, a correction was made for the substantial dark 02 uptake observed in the preparations. Part of this dark 02 consumption was by the electrode, which was significant due to the small reaction volume (500 ,d). The light sources were 150 w General Electric projector flood lamps providing a quantum flux density of roughly 500 ILE m-2 s-l between 400 and 700 nm at the surface of the reaction vessel. Distribution of Labeled Products. The distribution of radioactively labeled, acid-stable products of 14CO2 fixation in isolated chloroplasts was investigated by separation of the chloroplasts from the medium rapidly in a Beckman microfuge. Samples of 100 or 200 ,u were withdrawn from l-ml assay volumes (composition as in normal "4CO2 fixation assays) and centrifuged for 10 s in 400-,tl microfuge tubes. The supematant (medium) and pellet (chloroplasts) were immediately separated and treated with an equal volume of 20%o (w/v) trichloroacetic acid and flushed with air. Acid-stable radioactivity was determined by liquid scintillation spectroscopy. Chl was determined according to Wintermans and De Mots (20). RESULTS AND DISCUSSION Rates of 14CO2 fixation and C02-dependent 02 evolution in the basal medium ranged from 10 to 50 ,umol CO2 mg- Chl h-I with typical rates of 20-30 ,umol CO2 mg-I Chl h-'. These rates are significantly higher than those previously reported for chloroplasts isolated from CAM plants (10, 13). The effect of pH on CO2 fixation is illustrated in Figure 1. When MoPS3 was used as buffer, there was substantial activity over the pH range of 7.0-7.6 (Fig. 1). With Hepes and Mes as buffers, there was also a relatively broad optimum from pH 6.6 to pH 7.6 (data not shown). This broad optimum was reproducible over several experiments whether the concentration of total inorganic carbon or the concentration of dissolved CO2 was held constant. Chloroplasts from C3 plants typically have a higher pH optimum (pH 7.6-8.0) with very little activity at lower pH (6, 19). The high activity at a relatively low pH may be important to CAM if the pH of the cytoplasm decreases significantly when malic acid is released from the vacuole in the morning. In subsequent experiments, pH 7.6 was used in order to better compare the properties of Sedum chloroplasts with those reported for C3 chloroplasts. During the time course for CO2 fixation (Fig. 2), there was a 3 Abbreviations: MOPS: 3-(N-morpholino)propanesulfonic acid; RSP: ribose 5-phosphate; DHAP: dihydroxyacetone phosphate; PGA: 3-phosphoglyceric acid; RuBP: ribulose 1,5-bisphosphate; PEP: phosphoenolpyr-

uvate.

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3i2 2

I 16. E

I8pl

a

6.0

6.4

6.8 pH

7.2

7.6

8.0

FIG. 1. Effect of varying pH on the maximum linear rate of CO2 fixation in isolated S. praealtum chloroplasts. The concentration of CO2 was held constant at approximately 0.6 mm over the entire range by varying the amount of NaHCO3 added. MOPS was used over the entire pH range. AA04mMPi ImM R5P

2r5

04mM Pi ImM R5P

10 00

d L

° 751

/

X

X

y

at 12 min

mM Pi< 0 ~~~~~~~~~~~~~~~~~~4

X

E

0

75

04 mM Pi 2.5~~~~

0

4

8

12

16 20 Time (min)

~3no Pi

24

28

32

FIG. 2. Time course of CO2 fixation in isolated S. praealtum chloroplasts under different conditions: (A) 0.4 mm Pi and I mM R5P from beginning; (A) 0.4 mM Pi from beginning, 1 mM R5P added at 12 min; (0) 0.4 mm Pi from beginiing; (0) no Pi initially, 0.4 mm Pi added at 12 min; (O) no Pi. All other conditions as described in Materials and Methods. Maximum rates in smol CO2 mg-' Chl h-' were: (A) 34, (A) 31, (0) 24, (0) 18, () 2.

fairly long lag phase (4-6 min; see Fig. 2) even at the optimal Pi concentration (Fig. 3). The lag was shortened by the addition of R5P or DHAP, but other Calvin cycle intermediates were less effective in this respect (Table I, Fig. 2). The lag phase is nearly eliminated in the absence of Pi and increases with increasing Pi up to the optimal concentration (Figs. 2 and 3). In this respect, the lag phase is similar to that seen in C3 chloroplasts (17). Unlike C3 chloroplasts the length of the lag phase is not increased as the Pi concentration is raised above the optimum (Fig. 3). The rate of CO2 fixation increases with increasing Pi concentration up to the optimum (0.4 mm Pi) and declines at higher Pi concentrations. However the inhibition at higher Pi concentrations is much less marked than that reported for C3 chloroplasts (6, 14, 17). The effect of Pi concentration on both the lag and final rate was similar when C02-dependent 02 evolution rather than CO2 fixation was monitored (data not shown). The effect of Pi on both the rate of CO2 fixation and the length of the lag phase suggests that the Pi translocator (9) is present and operative in these chloroplasts. However the relatively minor effect of greater than optimal Pi concentrations on CO2 fixation

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T

15 E

0O

0

EoAA

o

50

10

0 0

1

2

3

4

5

mM Pi

FIG. 3. Effect of varying Pi concentration on the final linear rate (0) of and lag phase duration (A) for CO2 fixation in isolated S. praealtum

0

2

6 8 10 20 mM Pi FIG. 4. Effect of Pi concentration on the final linear rate of PGAdependent 02 evolution at a subsaturating PGA concentration in isolated S. praealtum chloroplasts. Rates of 02 evolution were corrected for a dark 02 consumption of approximately 5 umol mg-' Chl h-'. 4

chloroplasts. Table I. Effects of Various Metabolites on the Rate of CO2 Fixation and the Length of the Lag Phase in Isolated Chloroplasts of S. praealtum See reference 17 for calculation of lag time. Condition CO2 Fixation Control Lag

Mg-' ~Imol Chl h-' Control + I mM RuBP + 1 mM RSP + I mM Fructose-1,6-bisP + I mM Fructose-6-P + I mM DHAP + 0.2 mm PGA + 2,M DCMU + 10 mM Glyceraldehyde Dark Dark + I mM PEP

20.1 22.6 26.2 28.1 26.0 25.6 22.8 2.2 0.9 0.3 1.1

%

min

100

4.2 3.9 2.7 3.8 4.6 2.3 3.7

112 130 139 129 127 113 11 5 2 6

12o

0

mM PGA

FIG. 5. Effect of PGA concentration on the final linear rate of (0) CO2 indicate the characteristics of the Pi translocator in Sedum chloroplasts are in some way different from those in C3 chloro- fixation or (0) PGA-dependent 02 evolution (no NaHCO3 added) and on plasts. Alternatively, these data may reflect a difference between the length of the lag in (A) CO2 fixation or (A) PGA-dependent 02 the level of exchangeable metabolites in Sedum chloroplasts and evolution in isolated S. praealtum chloroplasts. Rates of 02 evolution were that in C3 chloroplasts. Further evidence for an operational Pi corrected for a dark 02 consumption of approximately 4 ,umol mg-' Chl translocator in the Sedum chloroplasts is shown in Figure 4 in h-'. which the effect of Pi concentration on the rate of PGA-dependent added, with the exception of RuBP, are known to penetrate 02 evolution at a subsaturating PGA concentration (see Fig. 5) is illustrated. There is an optimal Pi concentration above which Pi spinach chloroplasts at least slowly (9). The stimulation by R5P is inhibitory. This suggests that Pi is competing with PGA for the was similar even when added after the chloroplasts had attained Pi translocator as is seen in C3 chloroplasts (9). a linear rate of CO2 fixation (Fig. 2). This suggests that the steadyIt is notable that a substantial lag occurs if Pi is added to the state concentration of RuBP in the light might be subsaturating chloroplasts following a period of illumination in the absence of for RuBP carboxylase. The fact that several other Calvin cycle Pi (see Fig. 2). Spinach and wheat (C3) chloroplasts respond intermediates also stimulate the rate of CO2 fixation at the optimal immediately (with no lag) to addition of Pi under similar condi- Pi concentration supports this suggestion. tions (6, 18). This may indicate that the lag phase observed in S. Incorporation of CO2 was almost totally light dependent and praealtum chloroplasts is not due only to light activation of was inhibited 89% by 2 ,UM DCMU (Table I). The CO2 fixation enzymes and the build up of cycle intermediates as suggested for was also inhibited 97% by the Calvin cycle inhibitor, glyceraldethe induction phase in C3 photosynthesis (17). If this were true, hyde, and I mm PEP stimulated CO2 fixation in the dark only light activation of enzymes and the build up of cycle intermediates slightly. These data indicate that the observed CO2 fixation was might occur in the absence of Pi, and the addition of Pi would due to photosynthetic CO2 assimilation via RuBP carboxylase and then result in rapid stimulation by allowing export of excess triose- that involvement of PEP carboxylase was minimal. PEP carboxP. ylase was previously demonstrated to be extrachloroplastic in S. CO2 fixation at the optimal Pi concentration was stimulated by praealtum (16). the addition of Calvin cycle intermediates (Table I), with RuBP The effect of varying PGA concentrations on PGA-dependent and PGA being less effective than others tested. All intermediates 02 evolution (no CO2 added) and on 14CO2 fixation (10 mM may

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0

6

E

4~~~~~~~

/

2

100~~~~~~ B

80c

0

0.

, 1CL

60

0~~~~~~~~~~~~~~~~~~~~~~~~~0 00~~

0

40v

0

° 20_

01 0

8

4

12

16 Time (min)

20

24

28

32

FIG. 6. A: time course for total "CO2 fLxation (0, 0) or label remaining in the chloroplast (A, A) either in the absence of added Pi (0, A) or in the presence of 0.4 mM Pi (0, A) in isolated S. praealtum chloroplasts. B: per cent of total label incorporated during "4CO2 fixation in isolated S. praealtum chloroplasts which is exported to the medium either in the absence of added Pi (0) or the presence of 0.4 mM Pi (0). Maximum rates or total 14CO2 fixation in ,umol CO2 mg-' Chl h-' were 11 in the absence of Pi and 29 in the presence of 0.4 mm Pi. Table II. Effect of Preincubation with Pyridoxal-S'-P on the Distribution of CO2 Fixation Products between the Medium and the Chloroplasts in Isolated S. praealtum Chloroplasts

Medium

Chloroplasts

[Pi] +PLPa mM

0 0.4

cpm at

5,586 6,402

-PLP 20 min 5,242

6,556

change %

7

2

+PLP

-PLP 20 min 7,180 9,240 23,606 47,202 cpm at

change %

22 50

a Pyridoxal-5'-P.

NaHCO3) are illustrated in Figure 5. The rate of 02 evolution was considerably higher than that of CO2 fixation with concentrations of PGA higher than 0.1 mM. This suggests that external PGA may be exchanging for internal triose-P (if analogous to the Pi translocator in C3 chloroplasts) resulting in PGA-dependent 02 evolution but no CO2 fixation. Since PGA also stimulated CO2 fixation, the PGA must also exchange for a compound (probably Pi) other than triose-P. This conclusion is supported by the Pi requirement for maximum PGA-dependent 02 evolution (Fig. 4), since a PGAPi exchange would tend to deplete the chloroplast of Pi. It appears that PGA may enter the chloroplast in exchange for a metabolite which is needed to overcome the lag period, since the length of the lag phase for CO2 fixation increases with increasing PGA concentration (Fig. 5). The effect of PGA on the lag phase might

also be explained if a high internal concentration of PGA competes strongly with phosphoribulokinase for ATP. Spinach chloroplasts isolated by the method of Lilley and Walker (12) and assayed in the presence of 0.4 mm Pi were similar to the Sedum chloroplasts in the response of CO2 fixation and PGA-dependent 02 evolution to varying PGA concentrations, with the exception that some inhibition (10-15%) of the maximum rate of CO2 fixation was observed at the highest PGA concentration (4 mM). These data with both Sedum and spinach chloroplasts are contrary to the strong inhibition of CO2 fixation by PGA reported previously for spinach chloroplasts (2, 3). Such inhibition could occur where the PGA/Pi ratio in the medium is relatively high. Alternatively, since PGA increases the lag, it may appear to inhibit CO2 fixation if measurements are made only during the first few minutes of illumination or with only one sampling time. The effect of Pi on CO2 fixation was investigated further by determining its influence on the distribution of acid-stable radioactivity between the chloroplasts and the medium. Figure 6A illustrates the data obtained from a time course of CO2 fixation at either optimal Pi (0.4 mM) or in its absence. Note that the label in the chloroplasts is virtually identical under both conditions throughout most of the time course. The major difference between the two is in the amount of label exported to the medium (Fig. 6B). During the lag phase there is little difference between the two Pi concentrations, either in the amount of label in the medium or in the total label incorporated (see also Fig. 2). However, between 7 and 10 min after illumination, the preparation containing 0.4 mm Pi begins exporting considerable label to the medium while that without Pi does not. Thus the lag in CO2 fixation is correlated with the rate of carbon exported but appears independent of the CO2 fixed and retained in the chloroplast. With further experimentation it was observed that an inhibitory concentration of Pi (5 mM) did not increase the percentage of the label appearing in the medium (data not shown). This observation is consistent with high Pi concentrations failing to increase the length of the

lag phase (Fig. 3). Data are presented in Table II which support the suggestion that the increased export of labeled compounds in the presence of Pi (Fig. 6) is due to activity of the Pi translocator. Preincubation of the chloroplasts with pyridoxal-5'-P, an inhibitor of the Pi translocator (7), resulted in a 50%o inhibition of the amount of label exported to the medium in the presence of 0.4 mM Pi (versus 20o inhibition with no Pi added) but had no significant effect on amount of label retained in the chloroplast. In CAM chloroplasts direct studies on metabolite transport and pool sizes of metabolites are needed analogous to those which succeeded the isolation and initial response of functional chloroplasts of C3 plants. LITERATURE CITED 1. ARRON GP, MH SPALDING, GE EDWARDS 1979 Isolation and oxidative properties of intact mitochondria from the leaves of Sedum praealtum. A Crassulacean acid metabolism plant. Plant Physiol 64: 182-186 2. BAMBERGER ES, M AVRON 1975 Site of action of inhibitors of carbon dioxide assimilation by whole lettuce chloroplasts. Plant Physiol 56: 481-485 3. BAMBERGER ES, BA EHRLICH, M GIBBS 1975 The glyceraldehyde 3-phosphate and glycerate 3-phosphate shuttle and carbon dioxide assimilation in intact spinach chloroplasts. Plant Physiol 55: 1023-1030 4. BLACK CC 1973 Photosynthetic carbon fixation in relation to net CO2 uptake. Annu Rev Plant Physiol 24: 253-286 5. EDWARDS GE, SC HUBER 1978 Usefulness of isolated cells and protoplasts for photosynthetic studies. In DO Hall, J Coombs, TW Goodwin, eds, Proc 4th Int Congress on Photosynthesis. The Biochemical Society, London, pp 95-106 6. EDWARDS GE, SP ROBINSON, NJC TYLER, DA WALKER 1978 Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat. Influence of orthophosphate, pyrophosphate, and adenylates. Plant Physiol 62: 313-319 7. FLUGGE U, HW HELDT 1977 Specific labelling of a protein involved in phosphate transport of chloroplasts by pyridoxal-5'-phosphate. FEBS Lett 82: 29-33 8. GERWICK BC, GJ WILLIAMS III, EG URIUBE 1977 Effects of temperature on the Hill reaction and photophosphorylation in isolated cactus chloroplasts. Plant Physiol 60: 430432 9. HELDT HW 1976 Metabolite transport in intact spinach chloroplasts. In J Barber,

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ed, The Intact Chloroplast. ASP Biological Medical Press BV, Amsterdam, pp 215-234 LEVIC, M GIBBS 1975 Carbon dioxide fixation in isolated Kalanchoechloroplasts. Plant Physiol 56: 164-166 LILLEY RMcC, MP FITZGERALD, KG RIENTIS, DA WALKER 1975 Criteria of intactness and the photosynthetic activity of spinach chloroplast preparations. New Phytol 75: 1-10 LiLLEY RMcC, DA WALKER 1974 The reduction of 3-phosphoglycerate by reconstituted chloroplasts and by chloroplast extracts. Biochim Biophys Acta 368: 269-278 NISHIDA K, Y SANADA 1977 Carbon dioxide fixation in chloroplasts isolated from CAM plants. Plant Cell Physiol (special issue): 341-346 ROBINSON SP, JT WISKICH 1977 Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts by uptake in exchange for endogenous adenine nucleotides. Plant Physiol 59: 422-427 SPALDING MH, GP ARRON, GE EDWARDS 1980 Malate decarboxylation in

16. 17. 18. 19. 20.

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isolated mitochondria from the CAM plant Sedum praealtum. Arch Biochem Biophys. 199: 448-456 SPALDING MH, MR SCHMITT, SB Ku, GE EDWARDS 1979 Intracellular localization of some key enzymes of Crassulacean acid metabolism in Sedum praealtum. Plant Physiol 63: 738-743 WALKER DA 1976 Photosynthetic induction and its relation to transport phenomena in chloroplasts. In J Barber, ed, The Intact Chloroplast. ASP Biological Medical Press BV, Amsterdam, pp 235-278 WALKER DA, A HEROLD 1977 Can the chloroplast support photosynthesis unaided? Plant Cell Physiol (special issue): 295-310 WERDAN K, HW HELDT, M MILOVANCEV 1975 The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark. Biochim Biophys Acta 396: 276-292 WINTERMANS JFGM, A DE MOTS 1965 Spectrophotometric characteristics of chlorophyll and their pheophytins in ethanol. Biochim Biophys Acta 109: 448453