Plant Physiol. (1976) 57, 673-680

Alterations in Chloroplast Thylakoids during an in Vitro Freeze-Thaw Cycle1

Received for publication July 9, 1975 and in revised form December 8, 1975

MELVIN P. GARBER2 AND PETER L. STEPONKUSDepartment of Floriculture and Ornamental Horticulture, Cornell University, Ithaca, New York 14853

ABSTRACT

Plastocyanin and chloroplast coupling factor 1 (CF,) are releasedfrom spinach (Spinacia oleracea L.) thylakoids during a slow freeze-thaw cyde. CF, addition increases the proton uptake of thylakoidspreviously frozen in sucrose concentrations of 15 mm to 100 mM.Addition of CF, and plastocyanin restores the proton uptake of thyla-koids frozen in 100 mm sucrose. Plastocyanin and CF, release is amanifestation, not the cause, of freeze-thaw damage.

Frozen-thawed thylakoids appear to exhibit two levels of response tosucrose as measured by light;dependent proton uptake. Different levelsof protection afforded by sucrose may be due, in part, to quantitativedifferences in CF, release. The results suggest at least three freeze-induced lesions in light-dependent proton uptake by thylakoids: plasto-cyanin release, CF1 release, and disruption of the semi-penneability ofthylakoids.

The mechanism by which a freeze-thaw cycle damages or, inextreme cases, kills portions of or entire plants remains to beelucidated. Current evidence implicates cellular membranes asthe primary site of freeze-thaw injury in plants (4, 5, 11, 19).Chloroplast thylakoids have numerous biochemical functionswhich are easily measured and dependent on membrane integ-rity (19). Recent work establishes the validity of using in vitroexperiments with thylakoids to make inferences concerning invivo phenomena (6), Therefore, thylakoids appear to represent auseful system for studying the effects of a freeze-thaw cycle oncellular membranes.

In vitro freezing of thylakoids leads to partial or completeelimination of biological functions, including light-dependentproton uptake (4, 20), cyclic photophosphorylation (4, 5, 19),Mg2+-ATPase activity (4), osmotic response (4, 19), and theability to store succinate ions (19). Injury to chloroplast thyla-koids during a relatively fast freeze-thaw cycle has been ascribedto altered permeability properties and not the release or inacti-vation of a specific protein(s) (4, 19).

In cortical tissue of poplar, the degradation of phosphatidyl-choline appears closely associated with freezing injury (21). Theprimary site of freeze-thaw injury in cellular membranes and thecharacterization of lethal membrane alterations, with the excep-tion of altered permeability properties (4, 19), remain unre-solved.The objective of this study was to determine the effects of an

in vitro freeze-thaw cycle with rates comparable to those ofnatural conditions on light-dependent proton uptake of spinach

This work is part of the Ph.D. thesis of M. P. G.Present addresss: Department of Horticulture, Iowa State Univer-

sity, Ames, Iowa 5001 1.

thylakoids. Close attention was paid to possible release of mem-brane components.

MATERIALS AND METHODS

Chloroplast Isolation and Measurement of Biochemical Func-tions. The procedure for isolation of chloroplasts from spinach(Spinacia oleracea L.) was similar to Heber (4), except chloro-plasts were osmotically ruptured by resuspending in 10 mMNaCl. Measurement of light-dependent proton uptake by chlo-roplast thylakoids was essentially that of Neumann and Jagen-dorf (18). Thylakoids corresponding to 60 gg of Chl/ml wereresuspended in 5 ml of solution containing 15 mm NaCl, 0.5 mMMgC12, and 40 jaM phenazine methosulfate. Unfrozen controlswere assayed immediately after isolation. The temperature ofthe reaction mix was maintained at 16 C and initial pH was 6 to6.2. Chl determination was according to Arnon (2). Activationof membrane-association CF13 by trypsin and measurement ofCa2+-ATPase activity was after Lien and Racker (9). The deter-mination of Pi content was according to J. Fessenden-Raden(personal communications).

Negative Staining and Freeze-Etch Electron Microscopy. Adrop of thylakoid suspension was placed on a 300 mesh carbon-coated formvar-covered grid, and the excess was removed. Adrop of 2% (w/v) K phosphotungstate, pH 7, was momentarilyplaced on the grid, and the excess was removed. The grid wasimmediately viewed with a Phillips EM 300 electron microscopeoperated at 80 kv.The procedure for freeze-etch electron microscopy was essen-

tially that of Moor and Muhlethaler (17). Freeze-etch work wasdone with a Balzer BA-360 M freeze-etch unit, and replicaswere viewed with a Phillips EM 300 electron microscope oper-ated at 60 kv. The direction of shadowing is indicated in thelower right-hand corner of each figure and electron micrographsare processed to show the shadow in white.

Cooling and Warming Chloroplast Thylakoids. Thylakoidswhich had been washed twice in NaCI were resuspended in 5 mlof varying concentrations of sucrose in plastic centrifuge tubes.The tubes were tightly capped and placed in a 4 C methanolbath, with the suspension completely submerged. The freezerwas programmed to cool at 2.8 C/hr and allowed to remain atthe desired isothermal for 3 hr, prior to warming at 5.6 C/hr.Treatment of Thylakoids with DCCD. Thylakoids were resus-

pended in 5 ml of the proton uptake assay medium, and 5 Ml of0.1 M DCCD were added. This resulted in a ratio of 100 nmolesof DCCD/60 Mug of Chi. At least 3 min elapsed between additionof DCCD and the assay for proton uptake. The procedure wassimilar to that of McCarty and Racker (15).

Reconstitution of Frozen-Thawed Thylakoids with PurifiedCF,. The procedure was essentially that of Lien and Racker (10).

3Abbreviations: CF1: chloroplast coupling factor 1; DCCD: N,N'-dicyclohexylcarbodiimide; STA: silicotungstate.

673

GARBER AND STEPONKUS

After reconstitution, thylakoids were given two 1 0-min washingsin 10 mm NaCI at 20,000g to remove residual Tricine buffer.Controls (no CF, addition) were carried through the entireoperation. The procedure for isolation and purification of CF1was that of Lien and Racker (9).

Reconstitution of Thylakoids with Plastocyanin. The proce-dure for isolation and purification of plastocyanin was essentiallythat of Anderson and McCarty (1). To circumvent the loss ofplastocyanin during a freeze-thaw cycle, plastocyanin was addedto the thylakoid suspension at a final concentration of 100 AMprior to freezing. Following freezing and thawing. thylakoidswere given two 10-min washings in 10 mm NaCl at 20,000g toremove external plastocyanin.

RESULTS AND DISCUSSION

Thylakoids Frozen in Varying Concentrations of Sucrose atVarying Temperatures. Thylakoids subjected to a slow freeze-thaw cycle in the absence of sucrose were unable to form a light-dependent proton gradient, while those frozen in sucrose re-tained this capacity (Fig. 1). The extent to which the capacity forproton uptake was protected depended on the sucrose concen-tration, but two distinct response regions were evident andsuggests that there are at least two freeze-induced lesions inlight-dependent proton uptake. Protection of proton uptake wasafforded by the lowest concentration of sucrose utilized (0.5mM), with increasing amounts of protection to 15 mm. Withfurther increases in sucrose concentration to 40 mm no furtherincrease in proton uptake was observed. Increases in sucroseconcentration above 40 mm resulted in further increases inproton uptake, with a second plateau occurring between 50 and250 mM.Current theories (13) indicate that increasing electrolyte con-

centrations attendant with freezing are of primary concern withrespect to freezing injury, and substances like sucrose can affordprotection on a colligative basis by reducing the effective con-centration of electrolytes (5, 1 1). Since the total mole fraction of

10l o60

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40

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1

1110

10 20 30 40 SO

solute in a partially frozen solution is a direct function of temper-ature, the concentration of sugar required for protection shoulddecrease with warmer temperatures. Accordingly, thylakoidswere frozen in varying concentrations of sucrose to -7, -18,and -29 C. At all temperatures the two response areas tosucrose were evident, and maximum protection was afforded by50 mM sucrose. Initiation of the second sucrose response re-quired higher concentrations of sucrose as the freezing tempera-ture was lowered (Fig. 2) and occurred at 15 mm at -7 C, 25mM at -18 C, and 35 mm at -29 C. As S mm was the lowestconcentration used, it was not possible to ascertain whether asimilar effect was manifested for the first sucrose response.

Negative Stain Electron Microscopy. Thylakoids washed in 10mM NaCl and negatively stained with K phosphotungstate have asurface morphology similar to Figure 3. They are tubular inshape and have ±+90 A particles, identified as CF, particles (3.10). present on the outer surface. Thylakoids frozen and thawedin the absence of sucrose (presence of distilled H2O) (Fig. 4), 25mM sucrose (Fig. 5), and 50 mm sucrose (Fig. 6), change from atubular to a vesiculate structure. Vesicle size is random and nodetectable correlation exists between vesicle size and freezingmedium. Thylakoids frozen in the absence of sucrose do notform a proton gradient, unlike thylakoids frozen in 25 and 50mM sucrose, suggesting vesiculation is not the only possiblereason for loss of biological activity.

In addition to vesiculation, there is apparent removal of CF,particles. Thylakoids frozen in the absence of sucrose (Fig. 4)have very few of the CF1 particles that are present on unfrozenthylakoids (Fig. 3). Thylakoids frozen in 25 mm and 50 mMsucrose have numerous CF, particles (Figs. 5 and 6). Vesicula-tion was apparent in all freezing media, whereas CF, release wasmore pronounced with thylakoids frozen in the absence of su-crose.

Freeze-Etch Electron Microscopy. The + 150 A particles onthe outer surface of deep etched thylakoids have been identifiedas CF, particles (3). Thylakoids frozen in the absence of sucrose(Fig. 7) are nearly devoid of the + 150 A particles on the outer

75 1S0 250

SUCROSE mmFIG. 1. Light-dependent proton uptake of thylakoids frozen to -29 C in varying concentrations of sucrose.

-29C

so0 + +

I

;

Plant Physiol. Vol-, 57, 1976674

V4

FREEZE-THAW INJURY IN THYLAKOIDS

wv2

a b,_

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0 ao n o no nso

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sucmo nM

FIG. 2. a, b, and c:. Light-dependent proton uptake of thylakoidsfrozen at various temperatures in varying concentrations of sucrose.

surface (OS) of deep-etched thylakoids. Thylakoids frozen in 25and 50 mm sucrose have numerous -+-150 A- particles on theouter surface (Figs. 8 and 10),although some are nearly devoidof these particles (Fig. 9). The results raise the possibility thatthe freeze-thaw process is an "all or nothing" phenomenon,implying that thylakoids are completely inactivated or not dam-aged at all. This would be in agreement with Uribe and Jagen-dorf(II9)-The -+-165 A particles on the inner fracture face are apparently

removed when CF, particles are removed by freeze-thaw or STAtreatment. They might represent some membrane componentclosely associated with CF, or a portion of the CF,molecule.The inner fracture face of water-frozen thylakoids (Fig. IIlb) islacking the -+-165 A particles present on the inner fracture faceof unfrozen thylakoids (Fig. Illa). The particle diameter fre-quency distributionof water-frozen thylakoidsis essentially thesame asSTA-treated thylakoids (3). The inner surface and outer

fracture face in water-frozen and 25 and 50 mm sucrose-frozenthylakoids resemble that of unfrozen thylakoids.

Trypsin-activated CA2+-dependent ATPase. The Ca2+-ATP-ase activity of unfrozen and frozen-thawed thylakoids (Table I)indicates a decrease due to freeze-thaw treatment. The Ca21-ATPase activity increases as the concentration of sucrose inwhich the thylakoids are frozen is increased. At concentrationsof sucrose that afford maximum protection (50 mM), the Ca2-ATPase is slightly less than unfrozen thylakoids.A decrease in Ca2+-ATPase activity could be an indication

that CF, particles are either released or inactivated. The resultsof Figures 4, 7, and 9 suggest that CF, particles are removed by aslow freeze-thaw cycle. Furthermore, previous work suggeststhat CF, particles are not inactivated by freezing, even in highsalt solutions (4).

Stimulation of Proton Uptake by DCCD and CF,. DCCD canincrease the extent of proton uptake in thylakoids with CF1particles removed; however, only a slight stimulation is observedwith CF1 particles present (14, 15). CF1 particles probably have astructural and a catalytic role in proton uptake, and DCCD isthought to "plug-up the holes" in the membrane created by orduring CF, removal (14).

Frozen-thawed thylakoids show an increased proton gradientwith the addition of DCCD (Table II). Thylakoids frozen in theabsence of sucrose show a proton gradient equivalent to 8% ofthe unfrozen control, an increase over similarly treated thyla-koids without DCCD. After the addition of DCCD, thylakoidsfrozen in 25 or 50 mm sucrose show similar proton gradients.

Thylakoids frozen and thawed in concentrations of sucrose, 15mM to 100 mm, are capable of increased proton gradient follow-ing CF1 addition (Table III). While the enhancement of theproton' gradient by addition of CF, varies with the sucroseconcentration, the maximum gradient formed is the same (50%of the control) in all cases. This supports the contention that thedifferent levels of sucrose protection between 15 and 100 mmmay be due, in part, to differences in CF1 release. The inability ofCF1 addition to enhance the proton gradient formed by thyla-koids frozen in H20, 2 mm or 5 mm sucrose would indicate thatthere is a second freeze-induced lesion in light-dependent protonuptake, such as altered permeability properties.

Stimulation of Proton Gradient by Plastocyanin. The protongradient of reconstituted thylakoids previously frozen andthawed in sucrose concentrations of 15 to 100 mm was approxi-mately 50% of the unfrozen control (Table III). This suggeststhat CF1 release is not the only manifestation of membranedamage. Thylakoids frozen and thawed in the presence of highconcentration of plastocyanin, in order to maintain the proteinconcentration on the inner surface, show a proton gradientequivalent to approximately 33% of the unfrozen control (TableIV). This suggests that plastocyanin, in addition to CF1, is re-leased from thylakoids during a slow freeze-thaw cycle. As acontrol, thylakoids were frozen in BSA at a protein concentra-tion equivalent to that of plastocyanih. Very little protection wasafforded by BSA, (proton gradient approximately 12% of theunfrozen control) in comparison to plastocyanin, suggesting thatsoluble proteins, per se, are not very effective cryoprotectants.

Addition of DCCD to thylakoids frozen in plastocyanin in-creases the proton gradient to approximately 54% of the un-frozen control suggesting that CF, particles are released (TableIV). When thylakoids are frozen in the presence of 100 aMplastocyanin and100 mm sucrose and following thawing, DCCDor CF1 particles are added, the proton gradient formed is veryclose to the unfrozen control (Table IV).

This is the first work, to our knowledge, to restore the protonuptake activity lost during freezing and thawing of thylakoids.The results strongly suggest three sites of freezing injury in light-dependent proton uptake by thylakoids: the release ofplastocy-

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OSm

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675Plant Physiol. Vol. 57, 1976

GARBER AND STEPONKUS

C-

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FIG. 3. Thylakoids negatively-stained with 2% K phosphotungstate at pH 7 showing the morphology of thylakoids prior to freezing. They aretubular in shape and have numerous ±90 A particles (CF1) protruding from the surface. CF,, chloroplast coupling factor particles. x 100,000.

FIG. 4. Thylakoids frozen and thawed in distilled H20. Thylakoids change from a tubular (see Fig. 3) to a vesiculate structure. Very few of the CF1particles are detectable. x 100,000.

FIG. 5. Thylakoids frozen and thawed in 25 mm sucrose. Thylakoids change from a tubular to a vesiculate structure and CF, particles are present.CF,, chloroplast coupling factor particles. x 100,000.

FIG. 6. Thylakoids frozen and thawed in 50 mm sucrose. Surface morphology is similar to that in Fig. 5. CF,, chloroplast coupling factor particles.x 100,000.

676 Plant Physiol. Vol. 57, 1976

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Plant Physiol. Vol. 57, 1976 FREEZE-THAW INJURY IN THYLAKOIDS

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FIG. 7. Thylakoids frozen and thawed in distilled H20, and then fractured and deep etched. The outer surface is virtually devoid of CF1 particlesand many of the 150 A particles normally on the inner fracture face are absent. OS, outer surface; IFF, inner fracture face; FP, fracture plane. X

100,000.FIG. 8. Thylakoids frozen and thawed in 25 mm sucrose, and then fractured and deep-etched. CF1 particles are present on the outer surface, as are

150 A particles on the inner fracture face. I, ice; IFF, inner fracture face; FP, fracture plane; OS, outer surface. x 100,000.FIG. 9. Thylakoids frozen in 25 mm or 50 mm sucrose. The outer surface was revealed by deep etching and is virtually devoid of CF1 particles. OS,

outer surface. x 100,000.FIG. 10. Thylakoids frozen in 50 mm sucrose and fractured and deep etched. The outer surface has numerous CF, particles present. I, ice; IFF,

inner fracture face; OS, outer surface. x 100,000.

677

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GARBER AND STEPONKUS

anin, the release of CF, and possible disruption of. the semi-permeability of the thylakoid membrane. Plastocyanin release isprobably due to mechanical breakage of the thylakoids as evi-dence by vesicle formation following freezing (compare Fig. 3 to4, 5, and 6). This loss of plastocyanin could not be prevented byany of the sucrose concentrations used in this study. The release

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FIG. 11. a:. Particle diameter frequency distribution of the innerfracture face of unfrozen thylakoids. There are two size groups, +100A and ± 165 A. x 100,000. b: Particle diameter frequency distributionof the inner fracture face of thylakoids frozen and thawed in the absenceof sucrose (presence of distilled H20). There is one particle size group,±100 A. x 100,000.

of CF, appears to result from physical-chemical alterations dueto increased electrolyte concentrations attendant with freezing,and the sucrose concentration required for maintaining CF, onthe thylakoids was dependent on the freezing temperature. Theloss of semi-permeability of the thylakoid membranes could beprevented by low concentrations of sucrose (15 mM). It is possi--ble, however, that sucrose at these low concentrations is pre-venting the release or damage of other specific membrane com-ponents.The results superficially support the previously stated conten-

tion (13) that the freeze-thaw process subjects membranes tomultiple stresses. However, the multiple stresses in this studywere obtained with a single freeze-thaw rate. It must be empha-sized that the release of plastocyanin and CF, are manifestations,not the cause, of membrane damage resulting from freezing.The results appear compatible with the mechanism of freezing

injury proposed by Lovelock (I 1) and expanded by Mazur (12)which implicates concentrating electrolytes during freezing. Theresults in this paper do not rule out the possibility that sucrosecan protect, at least in part, on a colligative basis.

Freeze-thaw injury has been ascribed not to the concentrationof electrolytes but the reduction in volume which accompaniesthe freezing of water and concentration of electrolytes (16). Thehypothesis predicts that each cell or organelle will have a mini-mum tolerable volume at which the membrane will resist furthershrinkage and become leaky. It is difficult to explain our resultsbased on this hypothesis alone. Plastocyanin is probably releasedwhen the membrane is disrupted and it might be argued thatdisruption occurs at the minimum tolerable volume. However, itis difficult to imagine that simple shrinkage of the membrane canresult in CF, release. In this study, the breakage, per se, ofthylakoid membranes apparently can not account for all lostbiological activity.The sulfhydryl hypothesis attributes membrane damage to

formation of disulfide bonds during freezing, and upon thawing,proteins unfold and are denatured (7). It is unlikely that thishypothesis can account for loss of thylakoid activity for severalreasons. Chloroplast thylakoids are still inactivated when frozenin the presence of excess cysteine and glutathione or under N2atmosphere (5). Also, the authors suggested that oxidation of-SH groups is a secondary event of freezing. The formation ofdisulfide bonds and subsequent inactivation of membranes is anirreversible process (8). This hypothesis might prove viable if thesecondary event of freezing, formation of disulfide bonds, is thecause of lost activity. If this is correct, the activity lost during afreeze-thaw cycle should not be reconstitutable. The data pre-sented in this paper demonstrate that activity can be reconsti-tuted, casting serious doubt on the sulfhydryl hypothesis as aviable explanation of freeze-thaw injury.

Table I. Trypsin-activated Ca2+-dependent A TPase Activity of Frozen-Thawed Thylakoids3~~~~~~~~~~~~~~~~~~~~

Sample1 .moles Pi liberated/mg chl/hr3

+Unfrozen contro1l

Frozen in H20Frozen in 25 mM sucrose

Frozen in 50 mM sucrose

375 -21

126 -17

230 -27

314 ±23

ISolution in which the thylakoids were frozen and thawed.2 Trypsin-activated Ca2+-dependent ATPase activity was determined immediately after chloroplast thylakoid isolation.3Each value represents the average of three experiments.

...

IRaI

in

678 Plant Physiol. Vol. 57, 1976

Plant Physiol. Vol. 57, 1976 FREEZE-THAW INJURY IN THYLAKOIDS

Table II. Effect ofDCCD on Proton Uptake of Frozen-Thawed ThylakoidsThylakoids were frozen to -29 C.

Sample

Unfrozen control

Proton Gradient (% control)3

100

Water-frozen1

Water-frozen + DCCD

25 mM sucrose-frozen

25 mM sucrose-frozen + DCCD

50 mM sucrose-frozen

50 mM sucrose-frozen + DCCD

26

52

34

54

1Thylakoids frozen and thawed in distilled water.

2Thylakoids frozen and thawed in distilled water and then DCCD (100 nmoles/60

pg chlorophyll) added prior to measurement of proton uptake.

3Values represent the average of four experiments. Proton gradient of the un-

frozen control was equivalent to 0.9pjequivalents/mg Chl. The pH of the re-

action mix was 6.0-6.2 at 16 C.

Table III. Effect of CF1 on Proton Uptake of Thylakoids Frozen and Thawed in Varying Concentrations of SucroseThylakoids were frozen to -29 C.

Sample Proton Gradient Sample Proton Gradient

% control' % control'

Unfrozen control2 100 25 mm sucrose-frozen 26Reconstitution control 26

Water-frozen 0 25 mm sucrose-frozen + CF1 48Reconstitution control3 0Water-frozen + CF14 0

35 mm sucrose-frozen 302 mm sucrose-frozen 10 Reconstitution control 29Reconstitution control 10 35 mm sucrose-frozen + CF, 512 mM sucrose-frozen + CF, 11

50 mst sucrose-frozen 405 mM sucrose-frozen 20 Reconstitution control 40Reconstitution control 19 50 mstsucrose-frozen + CF1 505 mm sucrose-frozen + CF, 20

15 mM sucrose-frozen 33 100 mM sucrose-frozen 42Reconstitution control 33 Reconstitution control 4115 mM sucrose-frozen + CF, 50 100 mm sucrose-frozen + CF, 52

Values represent the average of six experiments. Proton gradient of CF1 was not added.the unfrozen control was 0.95 ueq/mg Chl. The pH of the reaction mix 4 Thylakoids were frozen and thawed in respective solutions, and thewas 6.0 to 6.2 at 16 C. described method for CF, reconstitution was carried out. Approximately

2 Thylakoids frozen and thawed in distilled water. 24 hr elapsed between assay of unfrozen and frozen-thawed reconsti-3Thylakoids treated in same manner as reconstituted samples except tuted thylakoids.

679

GARBER AND STEPONKUS Plant Physiol. Vol. 57, 1976

Table IV. Restoration of Proton Uptake Lost during Slow Freeze-Thaw Cycle

Thylakoids were frozen at -29 C.

Sample Proton Gradient (% control)3

Unfrozen control1

Frozen in H 20Frozen in 25 mM sucrose

Frozen in 100 mM sucrose

Frozen in 100luM plastocyanin

+ DCCD2

+ CF1

Frozen in 100 aM plastocyanin and 100 mM sucrose

+ DCCD

+ CF1

100

0

24

31

33

54

32

56

95

97

1The proton gradient of unfrozen thylakoids was determined immediately after

isolation while approximately 24 hrs elapsed before assay of frozen-thawed

reconstituted thylaoids.

2DCCD (100 nmoles/60 yg chlorophyll) was added to thylakoids after the freeze-

thaw cycle but before measurement of proton uptake.

3Values represent the average of four experiments. Proton gradient of the un-

frozen control was 0.9 yequivalents/mg Chl. The pH of the reaction mix was

6.0-6.2 at 16 C.

Acknowledgments-We thank A. T. Jagendorf and M. V. Parthasarathy, Department ofGenetics, Development, and Physiology, and R. E. McCarty and E. Racker, Section of Biochem-istry and Cell Biology, for their valuable advice and helpful suggestions during this study. Aspecial thanks to M. V. Parthasarathy for the generous use of his electron microscopy facilities.Purified CF, and CF, antibody were a gift of E. Racker.

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17. MOOR, H. AND K. MUHLETHALER. 1963. Fine structure in frozen-etched yeast cells. J. CellBiol. 17: 609-628.

18. NEUMANN, J. AND A. T. JAGENDORF. 1964. Light-induced pH changes related to phospho-rylation by chloroplasts. Arch. Biochem. Biophys. 107: 109-119.

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factors in ATP synthesis by spinach chloroplasts. Arch. Biochem. Biophys. 128: 351-359.20. WILLIAMS, R. J. AND H. T. MERYMAN. 1970. Freezing injury and resistance in spinach

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680