Plant Physiol. (1982) 70, 1748-17580032-0889/82/70/1748/1 1/$00.50/0

Characterization of the Formation and Distribution ofPhotosynthetic Products by Sedum praealtum Chloroplasts1

Received for publication May 20, 1982 and in revised form August 19, 1982

GEORGE J. PIAZZA, MARSHA G. SMITH2, AND MARTIN GIBBSInstitutefor Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02254

ABSTRACT

Photoassimilation of 'CO2 by intact chloroplasts from the Crassulaceanacid metabolism plant Sedumpraeakum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate(DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction.Only a minor amount of glycolate was produced. A portion of neutralsaccharide synthesis was shown to result from extrachloroplastic contam-ination, and the nature of this contamination was investigated with lightand electron microscopy. The amount of photoassimilated carbon parti-tioned into starch increased at both very low and high concentrations oforthophosphate. High concentrations of exogenous PGA also stimulatedstarch synthesis.DHAP and PGA were the preferred forms of carbon exported to the

medium, although indirect evidence suported hexose monophosphate ex-port. The export of PGA and DHAP to the medium was stimulated byhigh exogenous orthophosphate, but depletion of chloroplastic reductivepentose phosphate intermediates did not occur. As a result only a relativelysmall inhibition in the rate of CO2 assimilation occurred.The rate of photoassimilation was stimulated by exogenous PGA, ribose

5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose6-phosphate. Inhibition occurred with phosphoenolpyruvate and high con-centrations of PGA and ribose 5-phosphate. PGA inhibition did not resultfrom depletion of chloroplastic orthophosphate or from inhibition of ribu-lose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpy-ruvate were shown to interact with the orthophosphate translocator.

There have been only a few reports of CO2 assimilation byisolated chloroplasts from CAM plants. Levi and Gibbs (19)reported assimilation by mechanically isolated chloroplasts fromKalanchoe daigremontiana. Nishida and Sanada (21) isolated chlo-roplasts from several species of CAM plants. In either case,chloroplasts could be obtained only from leaves with below nor-mal starch content. Spalding and Edwards (29) devised a tech-nique for the isolation of enzymically derived protoplasts fromSedum praealtum, which upon disruption yield chloroplasts ofrather high starch content. These authors determined the pH andPi optima for CO2 assimilation, and obtained some evidence forthe presence of a functioning Pi translocator in the chloroplastmembrane.An improved variation of the Spalding and Edwards technique

has been used for chloroplast isolation from S. praealtum, and thepurity of this preparation has been investigated with electron and

'This research was supported by National Science Foundation GrantPMC-8141742.

2Present address: Cancer Research Institute, New England DeaconessHospital, Boston, MA 02215.

light microscopy. One goal of this study was to compare thephotosynthetic products of Sedum with those of more commonlystudied plastids from C3 spinach. Inasmuch as starch accumulationand utilization play a key role in CAM, another aspect of thisstudy was the investigation of factors involved in the partitioningof photosynthate into starch. Finally, the ability of several phos-phorylated compounds to penetrate the chloroplast membranewas detected by their influence on CO2 assimilation. The inter-action of these compounds with the Pi translocator was investi-gated.

MATERIALS AND METHODS

Chemicals and Supplies. Cellulase and Macerozq'me R-10 wereobtained from Yakult Biochemicals Co., Japan. [ C]Sodium bi-carbonate was supplied by Amersham. AG l-X8 anion exchangeresin and Bio-Gel P-2 were from Bio-Rad Laboratories. Dextran(15,000-20,000) was obtained from United States BiochemicalCorporation. Silicone oil was purchased from William F. Nye,Inc., New Bedford, MA. Aluminum planchets were purchasedfrom Coy Laboratory Products, Ann Arbor, MI. Enzymes, Ficoll(type 400), and Percoll were from Sigma. All other chemicals orsolvents were reagent grade. Water was distilled from an all glassapparatus.

Plant Material. Sedum praealtum D. C., supplied by Edwardsand Spalding, was propagated with stem cuttings in 15-cm potscontaining a vermiculite-soil (1:1) mixture. The plants were keptin a greenhouse, but natural light was supplemented with banksof fluorescent lights in the winter months. The plants were wateredwhen dry, approximately every 2 to 3 days. They were fertilizedwith one-quarter strength Hoagland solution once a week. Leafmaterial was not removed from the plants until 2 months after thecuttings became established. Young, but fully expanded leaveswere used for chloroplast isolation. Protoplast yields diminishedsignificantly when the established plants were older than about 8months.

Protoplast Isolation. Protoplast isolation was modeled after theprocedure developed by Spalding and Edwards (29), except sev-eral changes were made in the procedure which increased theyield. Isolation was begun near the end of the solar day. Theleaves were thinly sliced and placed in cold 0.3 M sorbitol. Theslices were washed once, very gently vacuum-infiltrated for a fewseconds, and washed once again with 0.3 M sorbitol. The slices(40g) were then transferred to 65 ml cooled digestion medium ona bed of ice. The digestion medium contained 0.3 M sorbitol, 0.9%(w/v) cellulase, 0.45% Macerozyme, and 4% BSA at pH 5.6. Thenext morning, the digestion mixture was manually swirled, passedthrough a 210-,pm nylon net, and centrifuged for 10 to 15 min at20g in a swinging bucket rotor. The pellets were resuspended in40 ml 30%o (w/v) dextran, 0.3 M sucrose, I mm CaCl2, and 50 mMHepes-NaOH (pH 7.8). This suspension was swirled for about 15s in a small beaker. This process increases protoplast yields. Asolution of 20%o dextran of the same composition as before was

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SEDUM CHLOROPLASTS

layered to a depth of 1.5 cm above the 30%o dextran in a centrifugetube, and a solution of 0.3 M sorbitol 1 mm CaCl2, and 50 mMHepes-NaOH (pH 7.8) was added on top to a depth of 0.5 cm.This gradient was centrifuged at 165g for 5 to 10 min. Duringcentrifugation, the intact protoplasts rose to the top of the 20odextran solution. They were removed with a Pasteur pipette andresuspended in breaking medium (40 ml) containing 0.2 M sorbitol,5 mm EDTA, 5mM NaHCO3, 1 mM MgCl2, 1% (w/v) BSA, and0.2 M Tricine-NaOH (pH 8.2).

Chloroplast Isolation. The protoplasts were ruptured by draw-ing the breaking medium into and expelling it quickly from a 5-ml disposable syringe fitted with a 22-gauge needle. This processwas repeated, and the medium was expelled into a polycarbonatecentrifuge tube containing a 1.5-cm cushion of 8% (w/v) Ficoll,0.3 M sucrose, 5 mM EDTA, 1 mM MgCl2, 1 mm MnCl2, and 50mM Hepes-NaOH (pH 7.6). This gradient was centrifuged for 3 to5 min at 100g. The intact chloroplasts formed a pellet at thebottom of the cushion. Chloroplasts prepared by this method weregenerally about 90%o intact based on ferricyanide-dependent 02evolution before and after osmotic shock (20).When chloroplasts of a higher state of purity were needed, the

following procedure was followed. The chloroplast pellet from theprevious step was resuspended in 2 ml 0.33 M sorbitol, 5 mmEDTA, 1 mm MnCl2, 1% (w/v) BSA, and 50 mm Hepes-NaOH(pH 7.6). This was layered on top of a preformed continuousPercoll gradient and spun at 165g for 15 min. Intact chloroplastsbanded near the bottom. Spread throughout the Percoll werechloroplasts contaminated with various amounts of membrane.Broken chloroplasts remained on the top of the gradient.The Percoll gradient was prepared as follows. Percoll was

dialyzed against 3 x 40 volumes of glass-distilled H20 for 24 h.An 80%o Percoll solution (10 ml) containing the same componentsas the chloroplast resuspension medium, except with no BSA, wasplaced in a 100 x 16 mm polycarbonate tube, and was centrifugedin a Sorvall type SS-34 fixed position rotor at 3 1,000g for 25 min.

14CO2 Assimilation. Unless otherwise indicated, assays wereperformed in a medium containing 0.33 M sorbitol, 5 mm EDTA,1 mM MgCl2, 1 mM MnCl2, 0.25 mM Pi, 5 mM NaH14CO3 (2-6,uCi/,umol), 1000 units catalase/ml, 20 to 50 ,ug Chl/tube, and 50mM Hepes-NaOH (pH 7.6). Reaction mixtures of 1.0-ml volumewere incubated in 55 x 17 mm test tubes at 30°C which wereilluminated from both sides by banks of 150-w flood lampsproviding 620 w/m2. Sedum chloroplasts are very dense comparedto other more commonly studied plastids. Before sampling, thereaction solution was rapidly mixed by drawing it up into andexpelling from a disposable tip of a Gilson P- 1000, Pipetman.14CO2 incorporation was determined by spotting 100-,ul aliquotson acidified aluminum planchets containing lens paper at 3- to 4-min intervals. Sample radioactivity was determined with a NuclearChicago gas-flow counter. After an initial lag period of 3 to 5 min,the rates of assimilation were linear for the next 15 to 20 min.

Separation of Stroma and Extrachloroplastic Medium. Siliconeoil centrifugation was used to separate chloroplasts from theirsurrounding medium. The silicone oil was a 4.17:0.85:1 (w/w)mixture of Dow-Coming 550, 200, and 710 silicone oils withviscosities of 125, 2, and 500 centistokes, respectively. For exper-iments where the levels of photosynthetic metabolites were deter-mined by ion exchange chromatography, the following procedurewas used. In the bottom of a 100 x 14 mm glass centrifuge tubewas placed 1 ml 14% HC104, and a 2-ml layer of silicone oil. Thereaction solution (3.5 ml) containing chloroplasts (100-200 ,ugChl) and 5 mm NaH14CO3 (57 ,uCi/,umol) was layered on thesilicone oil in the dark. After illumination for 15 to 20 min, thetubes were centrifuged at 9000g for 1 min after reaching full speed.The chloroplast medium which remained above the silicone oilwas removed and added to 0.5 ml 14% HC104. The oil layer wasremoved with a Pasteur pipette. The aqueous solution remaining

in the centrifuge tube contained the stromal contents, since thechloroplasts break upon contact with the acid. The pellet con-tained starch and other water-insoluble components. The chloro-plast medium and stromal solutions were taken to pH 5 with 5 MK2CO3 and were bubbled with N2 for 5 min, cooled in ice, andcentrifuged. After removal of the supernatants and one 1- to 2-mlwash, each fraction was taken to pH 7.6 with NaOH.For the subsequent measurement of RuBP3 levels, the stroma

and medium were separated by centrifugation in 400-,l plasticMicrofuge tubes containing 100 pl 14% HC104 and 75 A1 siliconeoil. At 5-min intervals, 200 pl of assimilation medium were layeredon top of the silicone oil. The tubes were spun in a BeckmanMicrofuge B for 30 s. The 14% HC104 containing the stromalcontents was neutralized as before.

Starch Determination. Two hundred ,ul of assimilation mediumwere spun in a Microfuge tube. The supernatant, silicone oil, andHC104 fraction were removed and the pellet containing the starchwas washed once with 100 pl of water. To each Microfuge tube,100 A 1.0 N HCI were added, and the tubes were placed in boilingwater for 30 min. The tubes were centrifuged. The aqueoussupernatant was spotted onto a planchet along with a 100-pl wash.Sample radioactivity was determined as described above. Starchformation rates were linear when the rate ofCO2 assimilation waslinear. Paper chromatographic analysis of hydrolyzed starch in-dicated the presence of glucose and a trace amount of maltose.

Metabolite Analysis Using Anion Exchange Chromatography.Neutralized reaction samples were applied to a 94 x 1.7 cmcolumn of anion exchange resin (AG I-X8, 20000 mesh, Clform). The neutral and cationic material was eluted with 130 mlwater, and then a 1500-ml linear gradient (0-0.33 N HCI) wasapplied. Fractions of approximately 8 ml were collected. Eachfraction was mixed with a Pasteur pipette, and an appropriateamount was spotted on a planchet along with 50 pi 0.33 M sorbitol.The first 50 fractions were acidified to remove any traces ofbicarbonate remaining from fixation. Peaks were identified bycomparison of the elution profile with those of previous work (13).Additionally, FBP, G6P, F6P, PGA, and DHAP were identifiedby enzymic assay using a slight modification of Latzko and Gibbs(18). The glycolate peak was analyzed according to Calkins (8).Aspartate was identified by descending paper chromatography.RuBP Determination. RuBP assays were performed with the

neutralized stromal fractions containing 10 mm NaH'4CO3 (38gCi/Imol), 15 mM MgCl2, and 0.04 unit RuBP carboxylase. After12 h, the reaction medium and one 200-,lI wash were transferredto 4.5-ml glass scintillation vials, and 200 ,ul 1 N HCI was added.The acidified assay medium was completely dried in a ventedoven at 90°C. To each vial was added 0.5 ml water and 3.5 mlaqueous counting scintillant. Sample radioactivity was determinedusing a Beckman LS-150 scintillation counter. Countin§ efficiencywas determined by doping each vial with 0.07 ,uCi [U-' C]glucose.RuBP carboxylase was prepared by a published procedure (25)

except that the chloroform step was omitted. To check the purityof this preparation, the assay procedure described above wasrepeated with standard RuBP in the presence of ribulose-P, PEP,or DCPIP, an inhibitor of P-ribulokinase. None of these additionsaffected the results, which shows that the assay is free of PEPcarboxylase and P-ribulokinase.

Fractionation of NF on Bio-Gel P-2. The neutral fraction fromthe stroma was evaporated to dryness in vacuo, redissolved in 0.3ml water, and applied to a 213 x 1.5 cm column of Bio-Gel P-2.

3 Abbreviations: RuBP, ribulose 1,5-bisphosphate; DHAP, dihydroxy-acetone phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphos-phate; G6P, glucose 6-phosphate; HMP, hexose monophosphates; NF,neutral saccharide fraction; PEP, phosphoenolpyruvate; PGA, glycerate 3-phosphate; R5P, ribose 5-phosphate; SBP, sedoheptulose 1,7-bisphosphate;a-GP, a-glycerol phosphate; DCPIP, 2,6-dichlorophenol-indophenol.

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Plant Physiol. Vol. 70, 1982

The saccharides were eluted with water. After one void volumehad passed, 2-ml fractions were collected and aliquots werecounted. The P-2 column was calibrated with partially hydrolyzeddextran as follows. In 10 ml 0.3 N H2SO4 was dissolved 0.2 gdextran (15,000-20,000). This was placed in boiling water for 20min, neutralized with BaCO3, and then evaporated to 2 ml invacuo. In this solution, 10 mg glucose was dissolved, and 0.3 mlwas applied to the P-2 column. Fractions were collected as before.The carbohydrate was hydrolyzed and reacted with anthrone togive a complex which absorbs at 625 nm (2).

Extraction of Sedum Leaf Saccharides. Two hundred g ofSedum leaves were completely ground in a blender containing 500ml 95% ethanol. The solution was centrifuged at 10OOg for 10 minto remove debris and evaporated in vacuo. Seventy ml water wereadded, and the suspension was centrifuged at 36,400g for 20 min.The aqueous supernatant was extracted three times with 50 mlethyl acetate and two times with 50 ml petroleum ether (38.4-49.0°C). The remaining aqueous solution was reduced in volumeto 15 ml in vacuo. The pH was adjusted to 7.2 with NaOH, andthe resulting precipitate was removed by centrifugation at 36,400gfor 10 min. Twenty g of AG 1-X8 (Cl form) anion exchanger wasadded, and the slurry was stirred for 15 min. The resin wasremoved by filtration and was washed with about 50 ml water.The solution volume was again reduced, and the solution wasstored overnight at -20°C. The precipitate which formed uponthawing was removed by centrifugation. The solution was thentreated with 20 g Dowex 50W-X8 as before, and the pH wasadjusted to 3.8 with NaOH. The solution volume was reduced to3 ml in vacuo, and 0.3 ml was applied to Bio-Gel P-2.

Paper Chromatography. The identities of the compounds in theneutral fraction as well as aspartate were determined by descend-ing chromatography using Whatman No. 1 chromatography pa-per.The following chromatographic solvents were used. A: 1-pen-

tanol saturated with 5 M formic acid (7); B: phenol-water, preparedby dissolving 0.5 kg phenol in 88 ml water and adding 84 ml ofthis solution to 16 ml water and 0.2 ml 0.5 M EDTA; C: 1-butanol-acetic acid-water (4:1:5); D: acetone-water-chloroform-methanol(8:0.5:1:1); E: butanol-pyridine-water (6:4:3); F: ethylacetate-aceticacid-water (9:2:2).

Except for the following, all sprays used for the visual detectionof compounds on paper were prepared as described previously (7,11). Ninhydrin spray was prepared by adding 1.5 ml 1% aqueousCu(NO3)2. 3H20 to a mixture of 0.4 g ninhydrin, 100 ml ethanol,20 ml acetic acid, and 4 ml collidine. 2,4-Dinitrophenylhydrazinespray (0.4% in 2N HCI) was followed by a spray of 10% aqueousNa2CO3.

RESULTS

Chloroplast Contamination. When viewed under a light micro-scope, Sedum chloroplasts appeared to be free of substantialcontamination (29). However, electron microscopy revealed thepresence of clumps of chloroplasts partially or completely sur-rounded by a membrane (Fig. IA). An intact protoplast is shownin Figure lB. It can be seen that rupturing caused the loss of thevacuolar material. Apparently, most of the cytoplasm (containingPEP carboxylase) had also been lost, since the rate of dark CO2assimilation was less than 1% of that in the light. A reinvestigationof the chloroplasts with light microscopy revealed that the resealedprotoplast membrane could be observed upon the addition ofmethylene blue (Fig. IC). A number of attempts were made toachieve a pure chloroplast preparation, but none was successful.However, the amount of contamination could be substantiallyreduced by centrifugation on a preformed Percoll gradient (Fig.ID). Many experiments were conducted with the more heavilycontaminated chloroplasts, but a number were repeated using thepurified preparations. In the discussion that follows, differences

in results obtained with the two preparations will be explained asnecessary.

Metabolites of CO2 Photoassimilation. Figure 2 shows an elu-tion profile from an AG l-X8 anion exchange column of water-soluble extracts of Sedum and C3 spinach chloroplasts, togetherwith their surrounding medium, after photoassimilation withNaH'4CO3. Identical conditions were used except the temperaturewas 30°C at pH 7.6 with Sedum, and with spinach, 25°C at pH8.1. Spinach chloroplasts produced mostly glycolate, DHAP, andPGA. In contrast, Sedum chloroplasts produced mostly NF,DHAP, and HMP. A major peak does appear in the Sedum profilenear to glycolate. After paper chromatography, the material re-acted with bromophenol blue spray, indicative of its acidic nature.Although not enough material was available to give a positivereaction with ninhydrin, the radioactive compound from thecolumn did co-chromatograph on paper with unlabeled aspartatein solvents A, B, and C.

Metabolite Distribution between the Stroma and Medium. Thedistribution of Sedum photosynthate between the stroma andmedium is shown in Figure 3. Both DHAP and PGA werepredominantly exported out of the chloroplast and HMP alsoappeared in the medium. Aspartate, NF, and HMP were the majorcomponents of the stroma. FBP, SBP, and RuBP eluted at higheracid concentrations. They are not visible in Figure 3 because theirconcentrations are very low.Two experiments were performed to gain some understanding

of the origin of the various metabolites. In the first experiment(Table I), assimilation was conducted in the presence of a-GPdehydrogenase and NADH, which act as a trap for DHAP.Overall, the metabolite levels were lower in the presence oftrapping enzyme, reflecting a lower assimilation rate. The per-centage of each metabolite (based on carbon content) is indicatedin parentheses. In the presence of trapping enzyme, the percentageof DHAP in the medium (in the form of a-GP) increased at theexpense of other metabolites, except NF. However, the percentagesof HMP, SBP, and PGA decreased only 3-5 fold, while thepercentage of FBP decreased 12-fold. It is unlikely that mediumPGA is derived from DHAP, since the concentrations of thenecessary cofactors, MgADP and NAD(P), are very low. Thus,the much reduced level of medium PGA resulted from its muchreduced stromal level, and by inference, this could be true forHMP and SBP, but not for FBP. The possibility that some materialin the medium was derived from broken chloroplasts cannot becompletely discounted, but is unlikely for two reasons. RuBP hasnever been observed in the medium. Also, in the control experi-ment, the umol HMP in the medium is higher than in the stroma,but the pmol medium NF is much smaller than is found in thestroma. If all medium HMP and NF were from breakage, it wouldbe expected that they be found in equal concentration, unlessthere is some protective effect by the resealed protoplast mem-

branes.In the second experiment (Table I), the metabolites from chlo-

roplasts purified on a Percoll gradient were compared with un-

purified chloroplasts. Higher assimilation rates gave a uniformlyhigher concentration of metabolites in these experiments com-

pared to those discussed above, but there were only minor differ-ences in the relative concentrations of metabolites. With thePercoll-purified chloroplasts, the percentage ofNF in the mediumdecreased about 3-fold, and that in the stroma about 2-fold,compared to its control. The percentage of the other metabolitesin the medium remained nearly unchanged. Only the percentageof stromal HMP increased by a substantial amount. These dataindicate that synthesis of some or all NF was associated withchloroplast contamination such as the resealed protoplast shownin Figure lA. Furthermore, NF must have been carried into thestromal fraction by its entrapment in these resealed protoplasts.

Figure 4 shows graphically the unusual light-dependent increase

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Ao B

FIG. 1. Sedum chloroplasts and protoplasts. A, Electron micrograph of resealed protoplast containing chloroplasts (C) with starch grains (S) (bar= 10 fLm). B, Electron micrograph of an isolated protoplast showing the large central vacuole ( V) surrounded by cytoplasm containing chloroplasts (C)(bar = 10 um). C, Light micrograph of isolated chloroplasts contaminated with resealed protoplasts (arrow). The preparation was stained on amicroscope slide with 0.1 mM methylene blue (magnification x 240). D, Light micrograph of chloroplasts purified on a Percoll gradient (magnificationx 240).

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Plant Physiol. Vol. 70, 1982

I0SPINACH

6PGA

21x

SEDUM

NF

3-

0 .05 .1 .15 .2 M[HCI]

FIG. 2. Elution profiles of extracts of spinach and Sedum chloroplastson anion exchange resin AGI-X8. CO2 assimilation (20 min) was con-ducted as described in "Materials and Methods" with Sedum chloroplasts(85 ,g Chi/ml) except the stroma and medium were not separated. Withspinach chloroplasts (25 ug Chl/ml), the conditions of assimilation wereidentical, except 50 mm Tricine-NaOH (pH 8.1) was used, and the tem-perature was 25°C. Spinach chloroplasts were prepared as previouslydescribed (30). The radioactivity is for a 10- and 100-Ml aliquot of spinachand Sedum column fractions (-8 ml), respectively. The HCI concentrationwas calculated from the total effluent volume, and is the concentration atthe top of the column.

of 'stromal' NF with time, while the concentration of HMP,DHAP, and PGA remained relatively unchanged after the firstfew minutes. Thus, the resealed protoplast membrane must beimpermeable to the NF fraction. The stromal fraction alwayscontained very low levels of DHAP and PGA, the state of purityof the chloroplast preparation notwithstanding; the levels of thesemetabolites increased in the medium with time. This suggests thatthe resealed protoplast membrane is permeable to triose-P, orwhen trapped in the membrane, triose-P is completely convertedto NF.

Neutral Fraction Saccharides. An investigation into the natureofNF was undertaken. Figure 5A shows that an elution profile ofthis material on a P-2 gel filtration column consists mainly of twopeaks. When compared to a calibration elution profile (Fig. 5C),it can be seen readily that the material in the neutral fractionconsisted mostly of material in the mol wt range of mono- anddisaccharides. For comparison with the labeled materiaL a sac-charide fraction from Sedum leaves was prepared, and was elutedfrom the P-2 column (Fig. SB). This fraction consists mostly ofmono- and disaccharides, as well as some material of higher molwt.The nature of saccharides extracted from whole Sedum leaves

was investigated, inasmuch as enough material was available togive reactions with a number of reagents which give specific colorswith certain types of sugars. Paper chromatography with solvent

241

14

4CM

x

0080~

48

16

MEDIUM

6 .05 .1 .15 .2 M[HCI]

FIG. 3. Elution profiles of the medium and stroma of Sedum chloro-plasts on anion exchange resin AGI-X8. CO2 assimilation (20 min) wasconducted with 80 Mug Chl/ml. The radioactivity is for a 0.5-ml aliquot ofthe column fractions (.8 ml).

D separated monosaccharides from disaccharides and also effectedfairly good separation of the monosaccharides from one another.Known standards of glucose, fructose, sedoheptulose, and man-noheptulose co-chromatographed with monosaccharides fromSedum leaf. Treatment with dinitrophenylhydrazine, p-anisidine-diphenylamine, naphthoresorcinol-H,PO4, or orcinol-TCA pro-duced colors consistent with fructose. Dinitrophenylhydrazine, p-anisidine, aniline-phthalic acid, and p-anisidine-diphenylamineindicated the presence of glucose. The presence of sedoheptuloseand mannoheptulose were indicated by the bluish green colorgiven with orcinol-TCA. Chromatography with known standards,using solvents E and F, showed that sucrose and a small amountof maltose were dissaccharides present in Sedum leaves. In addi-tion, sucrose reacted with orcinol-TCA to give a yellow colorindicative of a component fmctose.

Two-dimensional paper chromatography was performed withsolvents D and E, using the labeled NF mixed with knownstandards and with the saccharides from whole Sedum leaves.Four distinct radioactive spots were resolved. Sucrose and maltosewere clearly associated with two of the NF spots. In addition, themaltose and sucrose ofNF were hydrolyzed by a-glucosidase andinvertase, respectively. Known standards of mannoheptulose andsedoheptulose ran close to glucose and fructose, respectively, inthis solvent system. The remaining two NF spots were associatedwith the glucose and fructose standards, but it was not possible torule out completely the presence of some 7-carbon sugars. Chro-matography in solvent B showed that fructose, but not sedohep-tulose, was present in one NF spot. No solvent could be foundthat well separated glucose and mannoheptulose. Nevertheless, inevery case, the remaining NF spot was mainly associated withglucose.The chromatography data showed that NF consisted primarily

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SEDUM CHLOROPLASTS

Table I. Metabolite Levelsfrom Photoassimilation with Sedum ChloroplastsAfter "CO2 assimilation, the stroma and medium were separated and analyzed by anion exchange chroma-

tography. The trapping enzyme consisted of I unit a-GP dehydrogenase with 4 umol NADH. The trappingenzyme experiment and its control contained 23 jig Chl/ml. The concentration of Percoil-purified chloroplastswas 38 pg Chl/ml, and its control was 68,g Chl/ml. Other reaction conditions are as described in "Materials andMethods." Note that the percentage ofeach metabolite (given in parentheses) refers to the metabolites contribution(based on carbon content) to the medium or to the stroma, rather than to total assimilated carbon.

Control Trapping Enzyme Control roplasts

umol carbon/100 mg ChlMediumNF 2.8 (1.7%) 1.8 (4.8%) 44 (4.1%) 8 (1.7%)HMP 38.5 (23.5) 3.3 (8.7) 312 (28.8) 132 (27.5)DHAP or aGP 99.4 (60.8) 30.6 (81.1) 435 (40.1) 201 (42.0)PGA 8.6 (5.3) 0.45 (1.2) 82 (7.6) 38 (7.9)SBP 2.1 (1.3) 0.17 (0.45) 30 (2.8) 16 (3.4)FBP 5.6 (3.4) 0.11 (0.29) 138 (12.7) 65 (13.5)

StromaNF 20.5 (40.8%) 14.6 (45.1%) 24.4 (26.8%) 12.5 (15.8%)ASP 3.1 (6.1) 2.2 (6.7) 3.3 (3.6) 2.9 (3.6)HMP 19.2 (38.3) 13.2 (40.8) 33.1 (36.5) 37.0 (46.9)DHAP 1.0 (1.2) 0.36 (1.1) 2.9 (3.2) 4.0 (5.1)PGA 3.0 (6.0) 0.54 (1.7) 9.7 (10.6) 7.2 (9.2)SBP 0.55 (1.1) 0.10 (0.3) 3.6 (4.0) 2.4 (3.0)FBP 0.46 (0.9) 0.09 (0.3) 5.3 (5.8) 4.6 (5.9)RuBP 0.47 (0.9) 0.06 (0.2) 4.2 (4.6) 5.4 (6.9)

0.5

0 4

CID

CZ0.0t F

'4u- 020*

l X0.1

HMP

0 DHAI1 5 9 13 17

TIME (MINUTES)FIG. 4. The time dependence of the amount of newly assimilated

carbon in several metabolites in the stroma of Sedum chloroplasts. "CO2assimilation was conducted, and the stromal fractions were analyzed onanion exchange resin AGI-X8. The assimilation reactions for the sequentialtime points contained 58, 42, 10, and 10 pg Chl/ml, respectively, withincreasing time.

of sucrose, maltose, glucose, and fructose. Inasmuch as it wasshown before that part, if not all, NF was associated with chlo-roplast contamination, the composition of NF from Percoll-puri-fied chloroplast was investigated. The sucrose levels in NF fromthese chloroplasts showed a substantial decrease relative to theother components. This suggests that sucrose synthesis is extra-chloroplastic in origin, but that the other components may be

partially, if not exclusively, synthesized in the chloroplast. Spinachchloroplasts, which lack any contamination, produce a smallamount of NF which consists primarily of maltose and glucose.The possibility that in Sedum the source of maltose and glucose ispartial hydrolysis of starch by the 14% HC104 used in work-up iseliminated by an experiment in which chloroplasts (after "4CO2assimilation) were boiled in ethanol; the amount ofNF was equalto that associated with chloroplasts treated as usual.

Pi Effects on Photoassimilation. Figure 6 shows the effect of Piconcentration on the steady-state rate of CO2 assimilation. Twotraces are shown for Sedum chloroplasts; the upper was takenfrom previous work (29). For comparison, a trace is shown of thePi dependence of C02-supported 02 evolution by pea chloroplaststaken from the work of Walker and Robinson (31). Similar resultshave been obtained with chloroplasts from spinach, wheat, andsunflower (16). The effects of Pi on both Sedum and C3 chloro-plasts are similar in that low concentrations of Pi stimulated andhigh concentrations inhibited assimilation. However, there are anumber of quantitative differences. When no Pi was added, assim-ilation with Sedum continued at a linear rate for at least 20 minwhereas in spinach chloroplasts photosynthesis ceased after a fewminutes (10). When no Pi was present in the medium, Sedumchloroplasts often exported 65 to 75% of assimilated carbon duringsteady-state. This is only somewhat less than the amount ofcarbon(80-90%) which is exported at the optimal Pi concentration. Sedumchloroplasts were also much less sensitive to high Pi concentra-tions, irrespective of the rate of assimilation. The possibility thatthe resealed protoplast membranes provided a protective effectwas eliminated by an experiment in which impure and Percoil-purified chloroplasts were compared; no difference in sensitivitytoward Pi was observed.

Table II shows the metabolite levels in the medium and stromaafter 20 min of assimilation with no Pi present and with 5 mM Pipresent. The rate of assimilation at 5 mm Pi was 1.13 times thatwith no added Pi. In the medium, the percentages of DHAP,PGA, SBP, and FBP increased, at the expense of HMP and NF,in the presence of 5 mm Pi. This suggests that the origin ofmediumHMP and NF was not from DHAP. The overall increase ofcarbon flux into the medium in the presence of high Pi was

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Plant Physiol. Vol. 70, 1982

EFFLUENT VOLUME (ml x 10-1)FIG. 5. Elution profiles on Bio-Gel P-2. A, NF from the stromal

fraction of Sedum chloroplasts. NF was obtained from two CO2 assimila-tion experiments, both containing 81 ug Chl/ml. B, Saccharide fractionfrom Sedum leaves. After elution, 0.4 ml of each column fraction (2 ml)was added to cooled anthrone reagent (0.8 ml), and the blue color was

developed as described (2) and read at 625 nm. C, mol wt calibrationsaccharides. These were prepared as described in "Materials andMethods." The peak at the far right is glucose. Proceeding to the left, eachsuccessive peak is a saccharide containing an additional glucosyl unit.

relatively small, and the stroma was not depleted of reductivepentose phosphate cycle intermediates. In addition, relative con-centrations of metabolites in the stroma were about the same ateither Pi concentration.

Pi did have an effect upon starch synthesis. The relative rate ofstarch synthesis was high when no Pi was added (Table III). At30°C, it decreased to a minimum at 0.25 mm Pi, which is optimalfor assimilation, and then increased at higher Pi concentrations.Experiment 4 was performed at 15°C. The relative rate of starchsynthesis was minimal up to 0.5 mm Pi, and this reflects an upwardshift in the optimal Pi concentration with decreased temperature.Because the overall assimilation rate slows as the Pi concentrationis increased or decreased from the optimum, it is important to notethat the absolute rate of starch synthesis changes very little withchanges in Pi concentration.The rate of starch synthesis was also measured in intact Sedum

protoplasts. At an external Pi concentration of 0.25 mm, thepercentage of photosynthate partitioned into starch varied be-tween 25 and 66%, with a mean of 44%, in six determinations.The amount of photosynthate going into starch is higher than thatseen with isolated chloroplasts. Also, the rate of CO2 assimilation

0 1 2" 3 4 5 25PHOSPHATE CONCENTRATION (mM)

FIG. 6. Effect of exogenous Pi on photosynthesis in Sedum and peachloroplasts. '4CO2 assimilation was measured at 5-min intervals usingstandard reaction conditions. The rates of assimilation for Sedum (lowertrace) were calculated from data taken after the lag period. The uppertrace from more active Sedum chloroplasts was taken from the work ofSpalding and Edwards (29). For comparison, the effect of Pi on peachloroplasts (31) is shown.

Table II. Phosphate Effects on Metabolite Levelsfrom Photoassimilationwith Sedum Chloroplasts

After '4Co2 assimilation, the stroma and medium were separated andanalyzed by anion exchange chromatography. All experiments contained80 jig Chl/ml.

Medium Stroma

0 5mMPi 0 5mMPi

Amol carbon/100 mg ChlNF 1.8 (25.0%) 0.97 (10.3%) 9.4 (37.2%) 10.0 (37.4%)ASP 0.78 (3.1) 1.2 (4.5)HMP 2.0 (27.8) 2.3 (24.4) 13.2 (52.3) 13.6 (50.9)DHAP 2.4 (33.4) 4.3 (45.6) 0.14 (0.55) 0.13 (0.48)PGA 0.62 (8.6) 1.2 (12.7 0.61 (2.4) 0.83 (3.1)SBP 0.03 (0.42) 0.12 (1.3) 0.12 (0.48) 0.18 (0.67)FBP 0.05 (0.70) 0.17 (1.8) 0.13 (0.52) 0.19 (0.71)RuBP 0.26 (1.0) 0.20 (0.75)

with protoplasts was several times higher than with chloroplasts,e.g. 2 to 15 versus 10 to 60 ,tmol CO2 assimilated/mg Chl.h forchloroplasts and protoplasts, respectively.

Effect of Phospborylated Compounds on Assimilation. Figure7A shows the effect of F6P and G6P upon the rate of CO2assimilation after the lag period. Each trace was obtained from adifferent batch of chloroplasts. There was very little difference inthe magnitude of stimulation by F6P or G6P when compared inthe same experiment. The concentration of HMP which wasrequired for maximum stimulation was variable, but was mostoften around 5 mm. It is possible that F6P is exogenously phos-phorylated to FBP, which is then converted to triose-P throughthe action ofaldolase. It is this triose-P, then, that is the penetratingspecies. However, when ATP and Mg2+ were incubated with F6P,the magnitude of stim-ulation was equal to the sum of that given

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SEDUM CHLOROPLASTS

Table III. Phosphate Effect on the Partitioning of Photosynthate intoStarch

Total CO2 incorporation and carbon assimilation into starch weremeasured at 4- to 5-min intervals using NaH'4CO3 and standard reactionconditions. The rates were calculated from data taken after the lag period.

Photosynthate in Starcha

PiExperiment 1 Experiment 2 Experiment 3 Experiment 4

(30°C) (30°C) (30°C) (150C)mM %

0 32.2 53.1 34.7 21.20.25 13.9 19.6 14.2 11.60.5 16.1 11.61 20.5 15.25 23.8 18.5

25 40.5 52.8 48.0 19.150 20.7a Rate of carbon incorporation into starch

x 100.CO2 assimilation rate

by F6P and ATP-Mg2" alone. This shows that phosphorylation isnot a factor in the observed stimulation, and that HMP penetratesdirectly.

Figure 7B shows the effect of FBP, R5P, and PGA on the CO2assimilation rate. Each trace was obtained with a different batchof chloroplasts. Maximal stimulation of the rate occurred at aconcentration of only 0.5 mm with each phosphorylated com-pound, which is below the value seen with HMP by an order ofmagnitude. FBP stimulated about the same amount as FPB plusaldolase, suggesting that triose-P is probably the transported spe-cies. PGA was inhibitory, and R5P much less effective at highconcentrations, while such effects were much less notable or absentwith FBP, F6P, or G6P.With isolated spinach chloroplasts, Anderson and Gibbs (1)

showed that the concentrations of PGA which are inhibitory toCO2 assimilation are not always inhibitory to 02 evolution. Thus,PGA inhibition is not associated with the light reactions of pho-tosynthesis or with the reductive phase of carbon metabolism.PGA (24) or a secondary metabolite such as FBP (9) may actcompetitively to inhibit the binding of RuBP to carboxylase, orhigh PGA might shift the equilibrium of RuBP carboxylasetoward reactants, in spite of the large AF' of this reaction (3). Ineither event, an increase in the stroma level of RuBP should beobserved with increasing PGA. However, as shown in Table IV,the stromal level of RuBP (measured enzymically) reflected therate ofCO2 assimilation and decreased at inhibitory PGA concen-trations. This rules out any involvement of carboxylase in theobserved inhibition.

Figure 7C shows the effect of PEP upon the rate of CO2assimilation. PEP is a good inhibitor, with maximum inhibitionseen at a concentration of about 3 mm. Inhibition of CO2 assimi-lation in Kalanchoe chloroplasts (19) has been reported, but PEPhas no effect on spinach chloroplasts (6). PEP has been reportedto inhibit RuBP carboxylase (5) and P-glucoisomerase (15).The amount of "'CO2 incorporated into starch by Sedum chlo-

roplasts was measured in the presence of a number of nonlabeledphosphorylated compounds. If these compounds penetrate thechloroplast envelope and are incorporated into starch, then it isexpected that, per unit of time, the amount of CO2 incorporatedinto starch will decrease as a result of competition between carbonfrom CO2 and carbon from these compounds. This expectation isfulfilled with R5P, FBP, and F6P; as their concentrations wereincreased, the relative amount of "'C incorporated into starchdecreased, but the decrease bottomed out at the maximum rate oftotal CO2 assimilation (Table V). Incorporation in the presence ofPGA first decreased to a minimum and then, at higher PGA

20

16

12

8

4

z-Jx

40

30~1

2 20

0J10C.)(1)

0

10l

8

6

4

2

.A

.

.

--foIz---1 5 25

FBP

.5 1 5

0

25

0

0 .25 .5 1 3 5 10 15CONCENTRATION (mM)

FIG. 7. Effect of various phosphorylated compounds on photosynthesisin Sedum chloroplasts: A, F6P and G6P; B, FBP, PGA, and R5P; C, PEP.0'CO2 incorporation was measured at 5-min intervals using standardreaction conditions. The rate of assimilation was calculated from datataken after the lag period.

concentrations, increased. In the presence of PEP, incorporationinto starch was almost unchanged. This demonstrates that theenzymes necessary for conversion of PEP to a form necessary forentry into the reductive pentose-P cycle are not present in thechloroplast, consistent with an enzyme localization study (28).

Interactions of Phosphorylated Compounds and Orthophos-phate. The inhibition of CO2 assimilation seen at high PGA

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Plant Physiol. Vol. 70, 1982

Table IV. Effect ofPGA on Chloroplastic RuBP LevelsTotal "CO2 incorporation was measured at 5-min intervals using stan-

dard reaction conditions. The rates of assimilation were calculated fromdata taken after the lag period. Assimilation with NaH'2CO3 was con-ducted in parallel using standard reactions conditions. At 5-min intervals,200-td aliquots were removed, and the medium and stroma were separated.RuBP levels in the stroma were measured enzymically. The levels givenbelow were from aliquots taken at 18 min of photoassimilation, and arerepresentative of the trends observed at all time points after the lag period.

CO2 Assimilation Rate RuBP LevelPGA

Experiment I Experiment 2 Experiment I Experiment 2

mM ,umol/mg Chl.h nmol/mg Chi0 6.6 5.9 3.1 3.00.1 10.0 11.7 4.4 3.10.5 10.2 6.3 5.8 2.65 4.1 4.1 2.5 1.3

25 3.4 1.1

Table V. Effect of Phosphorylated Compounds on the Partitioning ofPhotosynthate into Starch

Total CO2 incorporation and carbon assimilation into starch weremeasured at 4- to 5-min intervals using NaH'4CO3 and standard reactionconditions. The rates of assimilation were calculated from data taken afterthe lag period. The carbon incorporation into starch refers only to carbonfrom CO2. Each experiment was performed with a different batch ofchloroplasts.

Concentration of Phos- Photosynthate in Starchaphorylated Compounds R5P FBP F6P PGA PEP

mM

0 10.3 18.2 9.0 14.3 17.80.01 9.5 14.4 9.1 10.00.05 7.7 10.2 7.3 7.60.25 4.4 11.9 4.2 4.6 16.90.5 5.0 12.5 3.4 5.5 17.71 5.4 13.5 2.8 7.3 19.05 5.0 12.5 2.6 9.8 23.8

25 4.9 13.7 2.1 27.9 22.4a Rate of carbon incorporation into starch x 100~

CO2 assimilation rate

concentrations with PEP, or to a lesser degree with R5P, couldresult from the interaction between these compounds and the Pitranslocator. In one scenario, these compounds, upon entering thechloroplast, exchange with internal Pi, resulting in depletion ofchloroplastic Pi. In another, these compounds competitively in-hibit Pi entry into the chloroplast. In either event, externallyadded Pi would be expected to overcome these inhibitions. Tocheck this, Pi was varied at a low (0.5 mM) and a high (25 mM)concentration of PGA, F6P, R5P, and PEP. At 0.5 mm PGA, thePi curve was normal, except that low Pi stimulation was somewhatlarger than usual (Fig. 8A). At 25 mM PGA, the rates of assimi-lation were severely inhibited, but increasing Pi overcame thisinhibition to a degree. The inability of Pi to restore completelythe rates of assimilation demonstrates that Pi depletion of thechloroplast is not the only factor involved in the inhibition byhigh PGA. With low and high concentrations of F6P, there wasno difference in the effect of Pi, except that the small amount ofstimulation by low Pi at the lower F6P concentration is absent atthe higher F6P concentration (Fig. 8B). Perhaps this stimulationis not seen because F6P can serve as a Pi source. Reference backto Fig. 7A shows that the stimulation provided by 25 mm F6P andG6P was less than expected. Partial hydrolysis of these phosphateesters to yield a higher than optimal Pi level would explain this,

and the formation of some components ofNF by this route seemslikely. The rates of assimilation were higher at 25 mm R5P thanwith 0.5 mm R5P (Fig. 8C); this appears to contradict the datashown in Figure 7B. However, the optimum R5P concentrationwas higher than usual, and the rates lower than optimal at 25 mMR5P; this type of variability was sometimes seen with differentchloroplast preparations. The effect of Pi, at high 5RP, is not anexact duplicate of its effect at low R5P concentration. At moderatePi concentrations, 25 mm R5P provided a measure of stimulation,but the magnitude of this effect is very small. Figure 8D showsthat high concentrations of Pi stimulate PEP-inhibited (25 mM)chloroplasts. No concentration of Pi could, however, restore therates of assimilation to their optimum levels. In total, these resultsshow that PGA and PEP interact with the Pi translocator; F6Pdoes not, and R5P does only very weakly.

DISCUSSION

In CAM, the carbohydrate source for night PEP synthesis hasbeen ascribed to starch, glucans, and soluble sugars and variouscombinations of these three (4). Although starch is clearly presentin the chloroplast, the compartmentation of glucans or solublesugars is unknown. In this paper, it has been demonstrated thatSedum chloroplasts synthesize glucose and maltose, but the exactpercentage of the photosynthate which partitions into these sac-charides could not be determined due to interference by chloro-plast contamination.

Several pieces of evidence suggest that Sedum chloroplastsexport HMP. One is that, in the presence of DHAP-trappingenzyme, the decrease of medium HMP is no more than wasexpected from its decreased stromal level. The second is that theamount ofHMP in the medium does not depend upon external Pilevels, while export of DHAP and PGA to the medium clearlydoes (Table II). The third, and perhaps most persuasive evidence,is that externally added HMP can stimulate CO2 assimilation atconcentrations as low as 50 ttM, and yet the stromal level ofHMPranged from 0.6 to 2 ams in several experiments (assuming thestromal volume to be 25 pl/mg Chl). It is conceivable that exportedHMP is used for synthesis of glucans or is dephosphorylated tofree sugars. In Sedum, gel filtration analysis of whole leaf extractsdemonstrates that large amounts of intermediate weight glucansare not present.Most of our knowledge of the chloroplast and its function has

come from chloroplasts obtained from short-lived C3 plants. Theseplants flourish in an environment of moderate moisture andsunlight. CAM plants must survive during hot, dry periods-periods of maximum sunlight intensity (22). The almost completeabsence of glycolate (0.2% of total assimilated carbon) as a pho-tosynthetic product is thus viewed with great interest. As shown inFigure 2, spinach chloroplasts produce much larger amounts ofglycolate (13-29% of total assimilated carbon in four experiments).The lack of glycolate production by Sedum chloroplasts in no wayconflicts with reports of photorespiration in CAM (22), becausethis undoubtedly arises from the oxygenase activity of RuBPcarboxylase; under the experimental conditions used here, thesource of glycolate for either Sedum or spinach cannot be fromoxygenase. This is so because 5 mM bicarbonate was added to theassimilation medium, and CO2, the form of carbon used bycarboxylase, is saturating (26). It is only the oxidative pathway(27) of glycolate synthesis which is operational. Possibly becausethe Sedum chloroplast has an efficient oxidant removal system,the flux of carbon to glycolate is minimal. This, in turn, may helpto explain the insensitivity of CAM toward photoinhibition (23),which relates to its survivability during periods of maximumsunlight and little moisture.

It has been discovered recently that isolated Sedum vacuolescontain a large pool of Pi, which moves in and out with time,opposite to the diurnal movement of malate (4). Thus, at predawn,

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SEDUM CHLOROPLASTS

20

ze

16

SW 121--

3-i

0u

-Jo0

:2

1.7

.

1.3S1

O.9 2

8

4

0D25.25 .5 1 10 25 0 25.5 1 5 10 15 25

PHOSPHATE CONCENTRATION (mM)FIG. 8. Effect of Pi on photosynthesis in Sedum chloroplasts in the presence of a 0.5 (0) and 25 mM (-) concentration of several phosphorylated

compounds: A, PGA; B, F6P; C, R5P; D, PEP. "CO2 incorporation was measured at 4-min intervals using standard reaction conditions. The rate ofassimilation was calculated from data taken after the lag period.

it was estimated that the level of Pi in the cytoplasm can beincreased by 2 mm. Although the data are lacking at present,presumably the Pi concentration of the cytoplasm is high in theearly morning hours and decreases throughout the day. Thisprovides a rationale for the insensitivity of Sedum chloroplasts tohigh external Pi levels (Fig. 6).

It was demonstrated that the Pi translocator of Sedum chloro-plasts is different than the more widely studied C3 Pi translocatorin that it responds to PEP. Currently accepted enzyme localizationstudies show that PEP transport across the chloroplast membraneis necessary in the processing of pyruvate (from malate) to a formable to enter the reductive pentose-P cycle. Evidence was obtainedfor the interaction of PGA with the Pi translocator, and theproduct distribution studies (Fig. 3) showed that triose-P is a

major form ofexported carbon. No evidence was obtained for anyinteraction between HMP and the Pi-translocator. It is necessaryto postulate either a distinct translocator, or the movement ofHMP without concurrent Pi exchange. Charge considerationsdemand that either HMP pass across the membrane along withprotons, or in exchange for some other anion, such as bicarbonate.

Starch accumulation increased in response to low Pi, high Pi,and high PGA. Inasmuch as PEP probably exchanges for externalPi, but does not have a large influence on starch synthesis, it islikely that PGA has a directly stimulatory effect, possibly throughADP-glucose pyrophosphorylase (12). The enhancement by high

Pi concentrations of starch synthesis is unusual and not seen withC3 plastids (12).With Sedum chloroplasts, the stimulation of CO2 assimilation

by R5P was found to be the same regardless of its addition at thebeginning of an assay or after the chloroplasts had attained alinear rate ofCO2 assimilation (30). This suggested that the steady-state concentration of RuBP is below saturation for carboxylase.The direct RuBP measurements presented here support this con-clusion. By and large, Sedum chloroplasts exhibit low rates ofCO2assimilation compared to C3 chloroplasts, and the stimulatoryeffects produced by various phosphorylated compounds (Fig. 7)were derived from increases in the stromal levels of reductive

pentose-P cycle intermediates and not from any specific stimula-tory effect on enzyme activity or light reactions (although someeffect cannot be ruled out).One other issue raised here was the inhibition of assimilation

by high levels of PGA. It was shown that the inhibition did notarise from inhibition of RuBP carboxylase. Part of the inhibitionresulted from efflux of Pi from the chloroplast, but the majorityof the effect could not be from this source. Inhibition of CO2assimilation in a membrane-free reconstituted spinach chloroplastpreparation has been reported (17). In this case, the inhibitioncould not result from depletion of Pi. It is possible that PGAconversion to triose-P, and subsequent efflux oftriose-P, producedan unfavorable chloroplastic ATP/ADP ratio. This, in turn, in-hibited P-ribulokinase (14). It is difficult to conceive, how thisrelates to the inhibition seen with higher levels of R5P. If higherconcentrations of PGA and R5P inhibit in a common manner,then some other source for this inhibition must be sought.

Acknowledgment-We gratefully acknowledge Nancy O'Donoghue for the elec-tron micrographs.

LITERATURE CITED

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2. ASHWELL G 1957 Colorimetric analysis of sugars. Methods Enzymol 3: 73-1053. BASSHAM JA, GH KRAUSE 1969 Free energy changes and metabolic regulation

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4. BLAcK CC, NW CARNAi, WH KENYON 1982 Compartmentation and the regu-lation of CAM. In IP Ting, M Gibbs, eds, Crassulacean Acid Metabolism.American Society of Plant Physiologists, Rockville, MD, pp 51-68

5. BUCHANAN BB, P SCHURMANN 1973 Ribulose 1,5-diphosphate carboxylase: Aregulatory enzyme in the photosynthetic assimilation of carbon dioxide. CuffTop Cell Regul 7: 1-20

6. BucKE C, DA WALKER, CW BALDRY 1966 Some effects of sugars and sugarphosphates on carbon dioxide fixation by isolated chloroplasts. Biochem J 101:636-641

7. BUSH ML, R MoNTroMERy, WL PORTER 1952 Identification oforganic acids onpaper chromatograms. Anal Chem 24: 489-491

8. CALUNS VP 1943 Microdetermination of glycolic and oxalic acids. Anal Chem

~~~C..... . . E

O 1 '' 10 '' 25

PEP \0

-we_

4

B

]- - S-

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_ _.rI _

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1758 PIAZZA ET AL.

15: 762-7639. CHU DK, JA BASSHAM 1975 Regulation of ribulose- 1,5-diphosphate carboxylase

by substrates and other metabolites. Plant Physiol 55: 720-72610. COCKBURN W, CW BALDRY, DA WALKER 1967 Some effects of inorganic

phosphate on 02 evolution by isolated chloroplasts. Biochim Biophys Acta 143:641-624

11. DAWSON RMC, DC ELLIOT, WH ELLIOT, KM JONES 1969 Data for BiochemicalResearch, Ed 2. Oxford University Press, New York

12. HELDT HW, CJ CHON, D MARONDE, A HEROLD, ZS STANKOVIC, DA WALKER,A KRAMINER, MR KIRK, U HEBER 1977 Role of orthophosphate and otherfactors in the regulation ofstarch formation in leaves and isolated chloroplasts.Plant Physiol 59: 1146-1155

13. HELDT HW, AR PORTIS, R McC LILLEY, A, MOSBACH, CJ CHON 1980 Assay ofnucleotides and other phosphate-containing compounds in isolated chloroplastsby ion exchange chromatography. Anal Biochem 101: 278-287

14. JOHNSON EJ 1966 Occurrence of the adenosine monophosphate inhibition ofcarbon dioxide fixation in photosynthetic and chemosynthetic autotrophs. ArchBiochem Biophys 114: 178-183

15. KAHANA SE, OH LOWRY, DW SCHULZ, JV PASSONNEAU, EJ CRAWFORD 1960The kinetics of phosphoglucoisomerase. J Biol Chem 235: 2178-2184

16. KAISER W, W URBACH 1977 The effects of dihydroxyacetone phosphate and 3-phosphoglycerate on 02 evolution and on the levels of ATP, ADP, and Pi inisolated intact chloroplasts. Biochim Biophys Acta 459: 337-346

17. Kow YW, M GIBBS 1982 Characterization of a photosynthesizing reconstitutedspinach chloroplast preparation. Plant Physiol 69: 179-186

18. LATZKO, E, M GIBBS 1969 Level of photosynthetic intermediates in isolatedspinach chloroplasts. Plant Physiol 44: 396-402

19. LEVI C,M GIBBS 1975 Carbon dioxide fixation in isolated Kalanchoechloroplasts.Plant Physiol 56: 164-166

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21. NISHIDA K, Y SANADA 1977 Carbon dioxide fixation in chloroplasts isolatedfrom CAM plants. In S Miyachi, S Katoh, Y Fujita, K Shibuta, eds, Photo-synthetic Organelles, Structure and Function. Special issue of Plant CellPhysiol No 3, pp 341-346

22. OSMOND CB 1976 CO2 assimilation and dissimilation in the light and dark inCAM plants. In RH Burris, CC Black, eds, CO2 Metabolism and PlantProductivity. University Park Press, Baltimore, pp 217-233

23. OSMOND CB, K WINTER, SB POWLES 1980 Adaptive significance of carbondioxide cycling during photosynthesis in water-stressed plants. In NC Turner,PJ Kramer, eds, Adaptation of Plants to Water and High Temperature Stress.John Wiley and Sons, New York, pp 139-152

24. PAULSEN JM, MD LANE 1966 Spinach ribulose diphosphate carboxylase. I.Purification and properties of the enzyme. Biochemistry 5: 2350-2357

25. RACKER E 1962 Ribulose diphosphate carboxylase from spinach leaves. MethodsEnzymol 5: 266-270

26. ROBINSON SP, DA WALKER 1981 Photosynthetic carbon reduction cycle. In MDHatch, NK Boardman, eds, Biochemistry of Plants, Vol 8, Photosynthesis.Academic Press, New York, pp 193-236

27. SHAIN Y, M GIBBs 1971 Formation of glycolate by a reconstituted spinachchloroplast preparation. Plant Physiol 48: 325-330

28. SPALDING MH, MR SCHMITT, SB Ku, GE EDWARDS 1979 Intracellular locali-zation of some key enzymes of Crassulacean acid metabolism in Sedumpraealtum. Plant Physiol 63: 738-743

29. SPALDING MH, GE EDWARDS 1980 Photosynthesis in isolated chloroplasts of theCrassulacean acid metabolism plant Sedum praealtum. Plant Physiol 65: 1044-1048

30. TAKABE T, M NISHIMURA, T AKAZAWA 1979 Isolation of intact chloroplasts fromspinach leaf by centrifugation in gradients of the modified silica "Percoll."Agric Biol Chem 43: 2137-2142

31. WALKER DA, SP ROBINSON 1978 Regulation of photosynthetic carbon assimi-lation. In HW Siegelman, G Hind, eds, Photosynthetic Carbon Assimilation.Plenum Press, New York, pp 43-59

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