Photosynthesis, Photorespiration and of Chloroplasts From ... · Photosynthesis and CO2 production in light by chloroplast preparations are in many respects similar to that of bean

Plant Physiol. (1969) 44, 946-954

Photosynthesis, Photorespiration and Respiration of ChloroplastsFrom Acetabularia mediterranial

R. G. S. Bidwell2, W. B. Levin, and D. C. ShephardDepartments of Biology and Anatomy, Case Western Reserve University, Cleveland, Ohio 44106

Received January 27, 1969.

A bstract. A chloroplast fraction isolated from Acetabularia mediterrania carries on photo-synthesis at rates essentially equal to those of whole cells. Electron and phase contrastmicroscopy reveals that the chloroplasts are intact and well preserved. Preparations containno identifiable peroxisomes, but some cytoplasmic and mitochondrial contamination is present.Photosynthesis and CO2 production in light by chloroplast preparations are in many respectssimilar to that of bean leaves, although the measured rates are somewhat lower. Respirationand photosynthesis of chloroplast preparations and whole cells of Acetabularia is essentiallysimilar except that oells have a strong dark-type respiration which continues in light and isCO, dependent, the substrate being mainly recent photosynthate. The data suggest thatchloroplasts are the site of photorespiration.

WVe have recently prepared chloroplasts from thegiant algal cell, Acetabularia mediterrania, which are

ajble to carry otut normal photosynthesis for severalhr under laboratory conditions (15). Preliminarygas analysis of photosynthesis indicated that the rateof photosynthesis of this chloroplast preparation was

comparable with that of intact cells on a chlorophyllcontent basis. The products of 14CO2 fixation bychloroplasts were normal and indistinguishable fromthose of cells. Mitochondrial contamination in thepreparation was minimal; the addition of Kreb'scycle substrates and ADP did not stimulate the lowrate of dark CO., evolution. Oxygen evolution was

proportional to bicarbonate concentration and was

not affected by the addition of glycerate-3-P, glycolicacid, ribulose- 1,5-diP, fructose-I `6-diP, ferredoxin,NADP or ADP, when adequiate bicarbonate was

present. Glycerate-3-P supported oxygen evolutiononly when the concentration of bicarbonate was

limiting. All these observations (15) indicate thata normal photosynthetic process occurs in thesechloroplasts, which therefore form an excellent systemfor the study of photosynthesis and related metabolicprocesses.

This paper describes the characteristics of lightand dark CO., exchange in the presence of varyinglevels of oxygen. By this means the photosynthetic,photorespiratory and respiratory behavior of chloro-plast preparations is compared with wvhole Aceta-bnlaria cells and bean leaves.

-'This work was supported by grants to R. G. S.Bidwell and D. C. Shephard from the National ScienceFoundation, and to R. G. S. Bidwell from the NationalResearch Council Canada, which are gratefully ack-nowledged.

2 Present address: Department of Biology, Queen'sUniversity, Kingston, Ontario, Canada.

946

Materials and Methods

Plant Material. A4. miiediterrania cells were main-tained in laboratory culture by methods similar tothose of Keck (11) details of the methods are inpress (14). Cells were grown at 200 ft-c, 12 hrdays, in a synthetic medium shown in table I. Whenwhole cells were tested, they were suspended in thesame culture medium, but with the pH adjusted to7.5 and with the addition of 0.5 mg/ml carbonic

Table I. Mediwniii Used to Grozw, Acetabularia Cells.The values are quantities per liter of final medium.

Salts NaCl 24 gMgSO4*7H2o 12 gCaCl2,2H,6 1 gTris 1 gKCl 750 mgNaNO3 40 mgKH,PO4 1 mg

The phosphate is added last, the pH is adjusted to7.8 with N HCl before autoclaving.

Micronutrients Na EDTA 12 mgZnSO4*7H2O 2 mgNa,MoO4*2H 0 1mgFeCI3-6H O 0.5 mgMnCI,24H10 0.2 mgCoCl.,*6H.,O 2 ugCuSO405H.,O 2 ug

The micronutrient solution is made up separately andadded before autoclaving.

Bicarbonate NaHCO3 100 mgVitamins Thiamin HCI 300 ug

p-Aminobenzoate 20 ugCa-pantothenate 10 ,ugVitamin B12 4 Ag

The vitamin and bicarbonate solutions are made upseparately, sterilized by filtration, and added afterautoclaving.

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BIDWELL ET AL.-PHOTOSYNTHESIS AND RESPIRATION OF CHLOROPLAS1S

Scissor mince cells in 2 ml homogenizing (H) medium. Strain through 173mesh bolting silk, muddle debris with glass rod and rinse with 8 ml washing (W)medium

SuspensionDiscards:

Supernatant - 4

Pellet

Lower layer, pellet '- i

- 500 g, 5 min.

- Resuspended in 1 ml W, gently use teflonhomogenizer. Layer suspension on topof a bi-layer consisting of 3 ml W, 2..5%w/v dextran on 2 ml W, 5SO w/v dextran.

50 g, 5 min.Layers 1 and 2 - Add 6 ml W,

500 g, 5 min.

Supernatant -4,%Washed chloroplast pellet

Resuspended in assay (A) medium when needed.

.dium Mannitol E:DTA BSA

H 0.6 M 10 3M 0.1%SoW 0.6 M 10 3M 0.1%oA 0.6 M - -

* *

TES DTT

101M 10-3MS1-3m o-45xl0 3M 10 -M

5x10-3M -

KC1 MgCl2 KH3PO4 pH- 7.8

7. 2

102M 5x10 3M 5r10 4M 7. 5

*EDTA = disodium ethylenediaminetetraacetate, BSA = bovine serum albumin,

TES = n-tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid, DTT =

dithiothreitol, CA = carbonic anhydrase, pH adjusted with KOH.

FIG. 1. Fractionation of Acetabularia cells. All operations done at 4'. The volumes of media refer to Ig. freshcells.

anhydrase. Experiments on bean leaves were donewith detached mature primary leaves of garden beans(Phaseolus vulgaris var. Pencil Pod Black Wax)with their petioles standing in water.

Chloropl.asts. About 1 to 5 grams of cells in theelongation phase of growth, 15 to 25 mm in length,were cut with scissors into small pieces and frac-tionated as shown in Fig. 1. The chloroplast pelletwas held at 4' until needed, and then suspended inapproximately 5 ml of A medium (Fig. 1). Analiquot was always removed and its chlorophyll con-

tent determined (1).Gas Exchange Measurement. CO., uptake and

evoltution were determined simultaneouslv bv meas-

uring the concentrations of 12CO2 and i4co. in a

circulating closed system. Gas was circulated by a

membrane pump (Neptune dynapump) through thephotosynthesis chamber, througlh a 20 ml chamnbersealed to the end of a thin-window Geiger-Muellertube driving a Nuclear Chicago recording rate meter(GM), and through a modified Beckman infra-redgas analyzer (IRGA). The photo-vnthetic chamberwas a flat, shallow dish with a lucite top sealed to a

ground glass flange, holding approximately 10 ml ofculture medium with a fluid-air interface of about30 cm2. The flow rate of gas through the svstemnwas about 3 1/min. Gas left the photosyntheticchamber through a small condenser moun,ted in the

lid which prevented the concentration of the mediumby evaporation, and removed water from the gasstream which might interfere with the operation ofthe IRGA.

The system was illuminated and stabilized in air.then 14CO2 was added and 12CO.. and 14CO., absorp-tion measured. Without carbonic anhydrase 15 to20 min were needed to achieve a steady rate, becauseof the time taken for the isotopes of CO. andbicarbonate to reach equilibrium in the system. Theaddition of 0.05 % carbonic anhydrase to the medium(Fig. 1) reduced the equilibration time to 5 to 8min, and had no obvious effect on either the net rateor the products of photosynthesis (table II). Car-bonic anhvdrase was therefore used in all experi-ments. The uptake of 14CO., measured by the GMgave gross CO, uptake, corrected for 2 % discrimi-nations against 14CO2vwhich has been de:erminedexperimentally (21), and the uptake of '2CO., meas-ured by the IRGA gave net CO., uptake (i.e. CO.taken up less 12CO., respired). The difference be-tween 14CO. uptake and '2CO2 uptake provided a

measure of the 12CO, evolution. This technique hasbeen carefully tested and described (3, 21). It hasbeen successfully applied to leaves, but measurementsof photorespiration in subnmerged giant cells tend tobe low for 2 reasons. The long diffusion path per-

mits considerable refixation of re-pired CO.,, which

Me CA*

0.05%

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PLANT PHYSIOLOGY

Table II. Effect of Carbonic Anhydrase (CA) on theProducts of 30 Min Photosynthesis in 14CO2

by Acetabularia ChloroplastsRadioactivity as cpm measured on chromatograms of

whole chloroplasts (2).

Radioactivity in products+0.05 % CA -CA

cpint Cp?ltInsoluble material at origin 1792 1908Organic phosphates 71 176Sucrose 546 562Glycolic acid 51 26Glyceric acid 40 32Citric acid 29Aspartic acid 39 31Glutamic acid 68 132Glutamine 41 ...

Alanine 150 78Serine and glycine 87 58Leucine 35 24Total 14CO" fixed 2949 3027

thus cannot be detected. In addition, several minare required to equilibrate the 2 isotopes of CO2throughout the gas and liquid phases. Thus beforesteady rates of uptake of the 2 isotopes are obtained,some recent (i.e. labeled) photosvnthate is alreadybeing respired, which depresses the estimated rate ofphotorespiration. For these reasons the rates whichwere determined are too low, but they are propor-

tional to the actual rates.The Equilibration of CO2 and Bicarbonate. A

major problem when measuring CO2 exchange ofsubmerged plants and comparing them with landplants is determining the equivalence of CO2 andbicarbonate concentrations, and maintaining thelatter. Since leaves effectively absorb CO2, whilesubmerged cells and chloroplasts probably absorbbicarbonate (16), the concentration of bicarbonate isextremely important. Bicarbonate concentration isdetermined by the CO2 tension of the gas phase andthe medium pH. Our apparatus measured gaseous

CO.,, not bicarbonate concentration. We thereforeequilibrated a buffered medium with the desired gas

phase CO2 tension. A pH of 7.5 is optimal for thechloroplas's, and the whole-cell medium was adjustedto the same value for ease of comparison. It was

necessary to demonstrate that photosynthesis ofchloroplasts or cells at pH 7.5 was related to theconcentration of gaseous CO2 in the same way as

leaves. regardless of whether CO2 or bicarbonatewas absorbed. It was observed that whenever thegaseous CO2 concentration was changed, the initialrate of CO2 absorption or loss by the system (i.e.cells or chloroplasts in medium) was much greaterthan the resulting steady-state rate of photosynthesis.Therefore, photosynthesis was not limited by the rate

of CO2 diffusion into the medium. Moreover thesame values were obtained relating CO2 uptake toCO.2 concentration whetlher the gaseous CO., con-

centration was raised or lowered to any given valueThis means that the gas phase measurements accu-rately reflected the rate of photosynthesis. and notthe rate of CO, equilibration with the medium.

The rates of photosynthesis of cells and chloro-plasts were greater at pH 7.8 than at pH 7.4, usingthe same air-CO., concentration, though the shapesof the curves were the same. This suggests thatbicarbonate rather than CO, was absorbed by thechloroplasts, since at constant CO2 pressure bicar-bonate concentration increases at higher pH. Fig-ures are available (13) from which it is possible toconstruct the relationship between air-COQ and dis-solved bicarbonate at different pH values. We havenot attempted to determine bicarbonate concentra-tions directly. However, all our media were suffi-ciently buffered that at any given pH the bicarbonateconcentration was linearly proportional to CO2 par-tial pressure, hence the relationship between CO2and photosynthesis is a valid one. Calculations fromthe figures given (13) indicate that at pH 7.5, themedia in equilibrium with air (400 ppm CO.) wouldcontain about 0.3 mm bicarbonate. Normal seawater (pH 8) in equilibrium with air contains about1 mM bicarbonate. In order to achieve this con-centration of bicarbonate at pH 7.5, roughly 1500ppm CO2 is required in the air phase. Thus, achieve-ment of physiological concentration's of bicarbonatein our medium at pH 7.5 required the use of CO2concentrations which were 4 to 5 times higher thanthe physiological CO2 concentrations used for leaves.

Contamination by Microorganisms. The cultureswere not truly axenic, but continuous efforts weremade to monitor and reduce contamination. Cellswere washed carefully with sterile culture fluid priorto use, and sterile technique was observed in handlingthe chloroplasts. Algal contamination was not de-tected either by plating or by hemocytometer exami-nation of media, cells or chloroplasts. A low levelof Pseudomowas infection was detected, but thisamounted to less than one part in 108 on a cell-volumebasis, and was considered insignificant. No singlepreparation of chloroplasts was maintained at assaytemperature in these experiments longer than 3 to4 hr.

Electron Microscopy of Chloroplast Preparations.A typical chloroplast pellet was fixed in Karnovsky'sfixative (10) for 2 hr, post-osmicated for 1 hr, de-hydrated in acetone and embedded in epon. Thinsections were cut, stained with uranyl acetate andlead citrate and observed at 60 KV in a Phillips 200electron microscope. Fig. 2A shows a low-powerscan of the preparation. The bulk of the pelletconsisted of intact chloroplasts, some with cytoplasmcontaining mitochondria adhering to them. Occa-sional droplets of cytoplasm containing mitochondriaand rarely a dictyosome were seen. A droplet ofcytoplasm adhering to a chloroplast is shown inFig. 2B; the contaminating organelles are unques-tionably mitochondria. The chloroplasts in Fig. 2Ahave distended or swollen vesicles formed from the

948S



BIDWELL ET AL.-PHOTOSYNTHESIS AND RESPIRATION OF CHLOROPLASTS

....B

D, C

FIG. 2. Electroin micrographs of chloroplast preparations. A) A typical field shoving normal levels of cYto-plasmic contamination. The balloon-like vesicles attached to the cliloroplasts are foimed( from their membranes dur-ing fixation. B) A contaminated chloroplast with associated cytoplasm, cytoplasmic vesicles and mitoclhondria. C)Ani intact chloroplast. Scale mark = 1 ,

JiB'? s. iInsert

THE PUBLIC REATi t EARI: 4IJV I\'TJTUTR

949



BIDWELL ET AL.-PHOTOSYNTHESIS AND RESPIRATION OF CHLOROPLASTS

outer membrane system, and such vesicles may beseen free in the pellet. These are artifacts of thefixation. With the phase microscope, they can beseen to form and detach from normal, unswollenchloroplasts (as Fig. 2C) during fixation. Theyare probably the result of rapid entry of the glutar-aldehyde in the fixative followed by osmotic swelling.If anything, they tend to confirm the integritv ofthe chloroplast outer membrane. Nothing resemblingperoxisomes (6,17) could be found in these prepara-tions. A few cytoplasmic vesicles were observed(Fig. 2B), but these did not resemble peroxisomes.Since the treatment of these chloroplasts was ex-tremely gentle compared to the vigorous treatmentused to isolate peroxisomes (17,18), it is unlikelythat they were destroyed or damaged by our treat-ment. We conclude that if peroxisomes exist inAcetabularia cells, they do not sediment at the lowcentrifugal forces used for the chloroplasts in ourprocedure.

Results

Chloroplast Photosynthesis. The rate of net CO2uptake was determined at 2500 ft-c (fluorescent) atvarious CO. concentrations in the circulating gasphase. The experiment was conducted in air, 02and N9, and the results, expressed as CO, uptakeper mg chlorophyll, are shown in Fig. 3. The N.and air curves are composites of data collected fromdifferent preparations on different days, since theirbehavior is precisely repeatable. It may be seenthat at this light intensity photosynthesis was satu-rated in air or No at 1500 to 2000 ppm COO, the

40ACETABULARIACHLOROPLASTS N2

AIR

0~~~~~~~~~~~~

E

ZDIN,

0~~~~~~~~~~0

0 ~ ~ ~ ~

0 500 1000 1500 2000 2500CO CONCENTRATION, ppm2

FIG. 3. Rates of photosynthesis of chloroplasts iso-lated from Acetabularia mediterrania in air, N, or 02,pH 7.5. 0-O, * * Air (different preparations),A A, A-A, N2 (different preparations),0.,.

.L40

0

E' 3DE

Y 20A04

0U,In

1000 24CO2 CONCENTRATION, ppm

FIG. 4. Rates of photosynthesis of Acetabularia medi-terrania cells in air or N2, pH 7.5. 0-0, 0 0Air (different runs), A-\ A N2.

level of atmospheric COO required to give a bicar-bonate concentration which approximates that ofnormal sea water in equilibrium with air. The ratewas somewhat higher in N2 than in air, and thecompensation point in N2, 12 to 15 ppm CO2, wasmuch lower than that in air, 80 to 90 ppm CO2.The curve for 02 was much lower, and the compen-sation point was very much higher, about 500 ppmCOO. Photosynthesis did not appear to be saturatedin O2 at the CO2 concentrations used.

The photosynthetic behavior of whole cells andbean leaves as affected bv CO2 in air or N2 werecompared with that of chloroplasts, and the resultsare presented in Figs. 4 and 5 respectively. Theshape of curves for the bean leaf (Fig. 5) wassimilar to those for chloroplasts, the differences beingthat bean leaves achieved a 3-fold higher rate on aunit chlorophyll basis. The differences in the CO2concentrations of saturation and compensation be-tween leaves and cells or chloroplasts reflect thehigh atmospheric CO2 reqtuired to achieve naturalbicarbonate concentration in the medium. Thecurves for Acetabularia cells (Fig. 4) were anoma-lous, showing a peculiar sigmoid shape with ap-parently reduced photosynthetic efficiency at lowerCO., concentrations. The maximum rate for cellswas someNvhat higher than that for chloroplasts athigh CO2 concentration, but lower at low CO2 con-centrations. The CO2 compensation points wereessentially the same.

A light intensity curve for photosynthesis ofchloroplasts was made at about one-third CO2 satu-ration, all measurements being made at about 500

951



PLANT PHYSIOLOGY

° 80

E

'E 60 ,

E

A..wa 40 .

CN00 20

200 400CO2 CONCENTRATION, ppm

FIG. 5. Rates of photosynthesis of a bean leaf inair, N2 orG2. 0-O Air, A-/A N2, 011EJ 02.

ppm CO,. The results are shown in Fig. 6. It maybe seen that photosynthesis reached light saturationfor this CO., concentration at 700 ft-c, and the lightcompensation point was about 35 to 40 ft-c. Thiscuirve is normal in all respects (7) except that thereis a suggestion of loss of activity at light intensitieswell above saturation. It was noted that chloroplastpreparations were damaged and suffered a progres-sive irreversible loss of activity if brightly illumi-nated in the absence of CO2, Isolated chloroplaststherefore appear less well protected from excessiveillumination than whole cells.

Cliloroplast Respiration. The rate of dark CO2production of chloroplasts was compared with therates for whole cells and bean leaves (table III).The ra:es for chloroplasts and cells were similar, andmuch lower than the comparable rate for bean leaves.

o 10

EaesE

(P

S

vi

FIG. 6. Rates of photosynthesis of Acetabutlaria miedi-terrania chloroplasts at 500 ppm CO,, pH 7.5, at variouslight intensities.

CO., production in light was measured by the isotopemethod in air at high and low CO2 concentrationsand in N., and 02, with the results shown in tableIII. The rate was highest at low CO, or in 02.;in air it was approximatelv twice the rate of darkrespiration, and fell to 0 in N_. Light CO., produc-tion by cells was muclh lower in air, btut tlle cells didshow some CO., production in N,. Beain leaves hada much higher rate of plhotorespiration, which char-acteristically increased greatly (Yemmii and Bidwell,private communication), and was stimulated by oxy-gen (5), at loNw CO, concentrationi. The muclhlower rates of light CO.,-production in Aceta(biulariachloroplasts or cells may have been largely due tothe slower rates of exchange between cell, mediumand atmospheric CO.), which would permit recyclingof 12CO0.

Table III. Rates of Dark awd Light CO., Rclease

CO., Gas .Acetabularia: BeanCcncIn. phase chloroplasts cells leaves

ppm; Al CO., per myg chlorophyll per w1tinDARK300-400 Air 1.80 1.62 10LIGHT300-400 Air 2.65 1.70 6.2

N2 0 0.80 002 4.28 ... 6.0

40- 80 Air 3.95 0.35 2402 35

Discussion

Th.e behavior of the chloroplast preparation isinteresting for its similarities to as well as its (if-ferences from the behavior of whole Acetabuflariacells and bean leaves. The similarity of the CO.,curves and the light curve of chloroplasts and beanleaves suggests that the overall processes involvedare quite similar. The magnitudes are different, butthe samiie is true for different species of higher plants.The behavior of whole Acetabuilaria cells was un-expected in the light of similarities between theirchloroplasts and bean leaves. The anomalous sig-moidal CO.2 curve for photosynthesis has not beenfound in other marine algae (Tregunna. 4, 16 andprivate comnimnication). It suggests that Aceta-bularia cells have a strong CO.2 dependent respirationin light which is CO., saturated at about 700 to 800ppm CO., (pH 7.5), and falls to a low level at lowCO_. This respiration pattern is apparentlv un-altered in N., (i.c. at low O.,-none of the N., ex-periments were rigorously 02 free, and the O. con-centration was approximately 0.5 %). It is possiblethat this represents cytoplasmic or mitochondrialrespiration of recent plhotosvnthetic products whiclhleave the chloroplasts. This would explain the in-sensitivity to low O.. and the dependence on CO_.Becautise of the difficulties of measuring photorespira-

(952

z



953BIDWELL ET AL.-PHOTOSYNTHESIS AND RESPIRATION OF CHLOROPLASTS

tion in submerged giant cells, the measured rates ofphotorespiration of cells shown in table II are prob-ably too low. However it can be seen that the ratewhich could be measured was 5 fold greater at highCO., than low CO9 concentration. Furthermore, itwas observed that the CO. evolved into CO.. freeair in light after a period of photosynthesis in 14CO0had a specific activity after equilibration between70 and 80 % of the 14CO, previously fixed. Thisindicates that the main substrate of light respirationis in fact recent photosynthate.

The fact that the chloroplast preparations evolvedCO., in darkness at about the same rate as wholecells suggests contamination by respiring mitochon-dria or cytoplasm. Electron microscopic examinationrevealed a low level of mitochondrial contaminationin the chloroplast preparation, but the preparationdoes not respond to the addition of normal substratesof respiration (15). Recently Walker et al. (19)have prepared chloroplasts which absorb O2 in thedark, bLt they do not release CO9, suggesting thatthey do not have a complete system of respiration oroxidation. Spinach chloroplasts prepared by Wal-ker's method (20) may differ also in their perme-

ability. Although they are capable of high rates ofphotosynthesis, they respond to the addition of severalCalvin cycle intermediates, while Acetabularia chloro-plasts respond only to glycerate-3-P, and then onlywhen bicarbonate is limiting (15). These differ-ences may result from the greater sensitivity ofspinach chloroplasts to the isolation medium, or tothe greater ease of separation of Acetabularia chloro-plasts from the cell sap or vacuolar fluid. It seems

tinnecessary to postulate special attributes of Aceta-bularia chloroplasts, which appear to be normal intheir capacity for in vitro photosynthesis and in theirsensitivity to destructive agents or rough handling(15). It is thus possible that dark respiration is a

normal attribute of intact chloroplast metabolism.The evollution of CO. in light by chloroplast

preparations is similar to photorespiration of beanleaves in that it was completely suppressed bv nitro-gen, and stimulated by lowering the CO2 concentra-tion. The light respiration of cells behaved likedark respiration, since it was only partially sup-

pressed by a nitrogen atmosphere. The stimulationof chloroplast CO2 output by oxvgen at 400 ppm

CO2 is essentially analogous with the effect of O2on leaves at low CO2 (5,8,9, and table III), due tothe limiting bicarbonate concentration in the chloro-plast medium. It has recently been suggested (6,12)that some of the reactions associated with photo-respiration are located in peroxisomes. Electronmicroscopy of the chloroplast fractions from Aceta-bilaria revealed no identifiable peroxisomes. Iso-lated crystalloids similar to those found in peroxi-somes (6, 17) were seen occasionallv, but theseappeared to be vacuolar. It has been establishedthat peroxisomes do not oxidize glyoxylate to CO2(17), and further that peroxisomes are present in

plants lacking photorespiration (18). It thus seems

unlikely that peroxisomes are necessarily involved inphotorespiration. The present data indicate thatphotorespiration is an attribute of chloroplasts. Itseems unlikely that the photorespiration of the chloro-plast preparation was due to mitochondrial contami-nation. In so far as it could be measured, theprocess was influenced by nitrogen, oxygen and CO2in the same way as the photorespiration, not the darkrespiration, of leaves (5, 9, table III).

Acknowledgment

The collaboration of Dr. J. A. Grasso, Departmentof Anatomy, Case Western Reserve University, in theelectron microscopy of the chloroplast fraction is grate-fully acknowledged.

Literature Cited

1. ARNON, D. I. 1949. Copper enzymes in isolatedchloroplasts. Polyphenol oxidase in Beta vulgaris.Plant Physiol. 24: 1-15.

2. BIDWELL, R. G. S. 1962. Direct paper chroma-tography of soluble compounds in small samplesof tissue adhering to the paper. Can. J. Biochem.Physiol. 40: 757-61.

3. BIDWVELL, R. G. S., W. B. LEVIN, AND I. A. TAMAS.1969. The effects of auxin an photosynthesis andrespiration. In: Biochemistry and Physiology ofPlant Growth Substances. F. Wightman, ed.Ottawa, Canada.

4. BROWN,N, D. L. AND E. B. TREGUNNA. 1967. Inhi-bition of respiration during photosynthesis by somealgae. Can. J. Botany 45: 1135-43

O. FORRESTER, M. L., G. KROTKOV, AND C. D. NELSON.1966. Effect of oxygen on photosynthesis, photo-respiration and respiration in detached leaves. I.Soybean. Plant Physiol. 41: 422-27.

6. FREDERICK, S. E. AND E. H. NEWCOMB. 1969.Microbody-like organelles in leaf cells. Science163: 1353-55.

7. GAASTRA, P. 1963. Climatic control of photosyn-thesis and respiration. In: Environmental Controlof Plant Growth. L. T. Evans, ed. AcademicPress, New York.

8. HESKETH, J. 1967. Enhancement of photosyn-thetic CO, assimilation in the absence of oxygen,as dependent upon species and temperature. Planta76: 371-74.

9. HEW, C. S. AND G. KROTKOV. 1968. Effect of oxy-gen on the rate of CO, evolution in light and dark-ness by photosynthesizing and non-photosynthesiz-ing leaves. Plant Physiol. 43: 464-66.

10. KARNOVSKY, M. 1965. A formaldehyde-glutaralde-hyde fix of high osmolarity for use in electronmicroscopy. J. Cell. Biol. 27: 137A.

11. KECK, K. 1964. Culturing and experimental ma-nipulation of Acetabularia. In: Methods in CellPhysiology. Vol. I. D. Prescott, ed. AcademicPress, New York.

12. KISAKI, T. AND N. E. TOLBERT. 1969. Glycolateand glyoxalate metabolism by isolated peroxi-somes or chloroplasts. Plant Physiol. 44: 242-50.

13. SEVERINGHAUS, J. W. 1965. Blood gas concen-trations. In: Handbook of Physiology, Section www.plantphysiol.orgon January 4, 2020 - Published by Downloaded from

Copyright © 1969 American Society of Plant Biologists. All rights reserved.


PLANT PHYSIOLOGY

B, Vol. II. W. 0. Fenn and H. Rahn, eds. Ameri-can Physiological Society, Washington, D. C.

14. SHEPHARD, D. C. 1969. Axenic culture of Ace-tabularia in synthetic media. In: Methods in CellPhysiology. Vol. IV. D. Prescott, ed. AcademicPress, New York.

15. SHEPHARD, D. C., W. B. LEVIN, AND R. G. S. BID-WELL. 1968. Normal photosynthesis by isolatedchloroplasts. Biochem. Biophys. Res. Commun.32: 413-20.

16. THOMAS, E. A. AND E. B. TREGUNNA. 1967. Bi-carbonate ion assimilation in photosynthesis bySargassumii mutticum. Can. J. Botany 46: 411-15.

17. TOLBERT, N. E., A. OESER, T. KISAKI, H. R. HAGE-MAN, AND R. K. YAMAZAKI. 1968. Peroxisomesfrom spinach leaves containing enzymes related toglycolate metabolism. J. Biol. Chem. 243: 5179-84.

18. TOLBERT, N. E., A. OESER, R. K. YAMASAKI, R. H.HAGEMAN, AND T. KISAKI. 1969. A survey ofplants for leaf peroxisomes. Plant Physiol. 44:135-47.

19. WALKER, D. A., C. W. BALDRY, AND W. COCK-BURN. 1968. Photosynthesis by isolated chloro-plasts, simultaneous measurement of carbon as-

similation and oxygen evolution. Plant Physiol.43: 1419-22.

20. WALKER, D. A., W. COCKBURN, AND C. W. BALDRY.1967. Photosynthetic oxygen evolution by iso-lated chloroplasts in the presence of carbon cycleintermediates. Nature 216: 597-99.

21. YEMM, E. W. AND R. G. S. BIDWELL. 1969.Carbon dioxide exchanges in leaves. I. Dis-crimination between 12CO2 and 14CO, in photo-synthesis. Plant Physiol. In press.

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Photosynthesis, Photorespiration and of Chloroplasts From ... · Photosynthesis and CO2 production in light by chloroplast preparations are in many respects similar to that of bean