Plant Physiol. (1992) 99, 21-250032-0889/92/99/0021/05/$01 .00/0

Received for publication August 14, 1991Accepted December 3, 1991

Effect of Cold Treatments on the Binding Stability ofPhotosystem 11 Extrinsic Proteins and an Associated

Increase in Susceptibility to Photoinhibition'

Wei-Qiu Wang, David J. Chapman, and James Barber*AFRC Photosynthesis Research Group, Wolfson Laboratories, Department of Biochemistry, Imperial College of

Science, Technology, and Medicine, London SW7 2AY, United Kingdom

ABSTRACT

When pea plants (Pisum sativum L. cv Feltham First) aresubjected to freezing conditions (-18°C) followed by a thaw to18°C, there is a significant inhibition of water-splitting capacityjudged by the rate of light-induced reduction of 2,6-dichloro-phenol indophenol using isolated thylakoid membrane fragmentsenriched in photosystem 11 (PSII). The freeze-thaw-induced inhi-bition of water-splitting activity has been correlated with the lossof the 17- and 23-kilodalton extrinsic protein of PSII and with aweakening of the binding of the 33-kilodalton protein. There wasno apparent loss of bound manganese. Addition of 10 millimolarCaC12, however, allowed a full recovery of the water-splittingactivity of these modified PSII-enriched particles. The freeze-thaw-induced changes in the organization and functional capacityof PSII was found to increase its susceptibility to photoinhibitionin agreement with the concepts presented in the accompanyingpaper, that oxidative damage can occur within the PSII reactioncenter as a consequence of extending the lifetime of P680+.

In the preceding paper (25), we have presented data ob-tained by various treatments of isolated PSII-enriched mem-branes that indicate that PSII is more vulnerable to photoda-mage when electron donation to P680+ is impeded. As aconsequence, we have suggested that it is the increased lifetimeof the P680+ state that gives rise to deleterious oxidationreactions leading to pigment and protein degradation and tothe loss of functional activity. Such conclusions have beenreached by others (4, 9, 18, 19, 22, 23).

It is well known that in vivo the degree of photoinhibitionis enhanced if other stress conditions are imposed (16). Onesuch stress condition is low temperature (2, 15), and indeedthe combination oflow temperature and light has been shownto induce significant levels of photoinhibition in the field (6,10, 1 1). In this paper, we report the results of experimentsdesigned to investigate the possible mechanism of the en-hancement of photoinhibition in leaves that have been sub-jected to low temperatures. We conclude that such an en-hancement could partly be due to effects on the donor side ofPSII.

'This work was financed by the Agricultural and Food ResearchCouncil.

MATERIALS AND METHODS

Pea plants (Pisum sativum L. cv Feltham First) were grownin trays of vermiculite in a controlled environmental cabinet(Fisons model 600G3/TTL) at 18°C with a 16-h photoperiodand light intensities between 160 and 240 ,mol photon m-2s-

Plants were exposed to various low-temperature treatmentsfor 18 h under dark conditions. Some plants were thentransferred to 18°C, with or without light, for 1 h prior toisolation of thylakoids or PSII-enriched membranes, usingpreviously described procedures (5).Oxygen evolution and the reduction ofDCPIP2 were meas-

ured as described in the preceding paper (25).SDS-PAGE using a linear 7 to 17% gradient of acrylamide

was used to analyze the protein composition of PSII mem-branes. Isolated PSII membranes were pretreated by incuba-tion in 5% TCA on ice for 15 min, and precipitated bycentrifugation. Proteins were solubilized on gels and separatedby electrophoresis at 550 V, 120 mA, and 4°C. The proteinswere visualized by staining with Coomassie brilliant blue.ELISA of the 33- and 23-kD extrinsic polypeptides of PSII

were carried out as described previously (5), as was the pro-cedure for pH washing (5, 25).A Perkin-Elmer 2280 atomic absorption spectrophotometer

with an HGA graphite furnace was used with a Perkin-ElmerHGA 400 Programmer and argon supply to determine theamount of manganese in samples. A standard manganese(II)nitrate solution was used for calibration.

RESULTS

As Table I shows, when compared with untreated plants,there was an inhibition of electron transport from water toDCPIP, but not DPC to DCPIP, in PSII membranes isolatedfrom leaves of plants that had been treated at -1 8°C for 18 hand then thawed at 1 8°C in low light for 1 h. The effects onelectron transfer caused by freezing in vivo appears to bedirectly analogous to the situation when isolated PSII-en-riched membranes were treated in vitro with high concentra-tions ofsalts, citrate buffer, pH 3.0, or alkaline pH as describedin the preceding paper (25). A further similarity between theinfluence of these chemical treatments and the effect of cold

2Abbreviations: DCPIP, 2,6-dichlorophenol indophenol; DPC, di-phenyl carbazide; P680, primary electron donor of PSII.

21

Plant Physiol. Vol. 99, 1992

Table I. Electron Transfer Rates and Degree of Photoinhibition ofPSII-Enriched Membranes Isolated from Plants that Had, or Had Not,Been Subjected to Freeze-Thaw Treatment

Dark, no pretreatment; light, pretreatment with 2000 gumol photonm-2 s-1 white light at 1 00C for 10 min with 30 /g Chi mL-1. Frozen,-1 8°C in the dark for 18 h and 1 80C, 1 h; +Ca2 , supplemented with10 mM CaCl2 and 20 mm NaCI. Rates of NADPH reduction are givenin ,Amol mg-' Chi h-1 and are the average of at least three measure-ments with deviations from the average being no greater than 5%.

H20-- DCPIP DPC -* DCPIPSample

Dark Light Dark Light

Control 40 27 44 33Control + Ca2 42 34 44 35Frozen 8 1 57 29Frozen + Ca2+ 42 36 57 48

treatment is the ability to recover water oxidation by addingCaCl2 to the photoinhibited PSII membranes (Table I).Although the freezing and thawing treatments only inhib-

ited the water oxidation-supported DCPIP photoreductionand not the DPC-supported rate, it was found that the DPC-supported rate was more susceptible to subsequent photo-inhibition in membranes from frozen compared with un-frozen plants (Table I). Again, this is reminiscent of theobservations with chemically induced inhibition of wateroxidation (25). Also paralleling the results with chemicaltreatments was the reduction in the susceptibility to photo-damage given by restoration of water oxidation activity byadding 10 mm CaC12. Similarly, protection against photoinhi-bition of the DPC to DCPIP activity was achieved by addingDPC prior to the preillumination treatment (data not shown).

Because exposure to -18°C for 18 h is a severe treatment,we also investigated the effect of exposures to milder temper-atures. Incubation of pea plants at air temperatures just belowfreezing for 18 h in the dark did not result in freezing of theleaf tissue and did not lead to a substantial inhibition ofelectron transfer activity in isolated thylakoids (data notshown) or PSII membranes (Table II). The water oxidation-dependent activity in preparations isolated from plants thathad been frozen by treatment at about -8°C for 18 h in thedark was significantly inhibited, although not to quite thesame extent as that in - 18°C samples. As reported in TableII, the DPC to DCPIP rate was not inhibited by these treat-

Table II. Comparison of Treatment at Different Sub-ZeroTemperatures on Electron Transfer Activities

Pea plants were grown at 180C under light of 200 ,umol photonm-2 s-i and then exposed to temperatures of -2, -8, or -18°Cbefore assays were carried out on PSII-enriched membranes. Controlplants were taken directly from growth conditions.

Treatment Chlalb Relative Rate Relative Rate

H20 --. DCPIP DPC --* DCPIP

Control 2.1 100 100-20C 1.9 88 135-80C 2.0 30 141-180C 2.1 15 144

ments; in fact, there was a distinct stimulation of this activity.Table II also shows that the various cold treatments had noinfluence on the Chl a/b ratio of the isolated PSII-enrichedmembranes, indicating that no degradation of the intrinsicPSII complex had occurred.To distinguish between the effects caused by freezing and

freezing with subsequent thawing in the dark or light, frozenpea plants (-1 8°C, 18 h, dark) were treated in three differentways before isolation of PSII membranes: (a) used directly forisolation with a minimum thaw period; (b) transferred to 1 8°Cin the dark for a period of 1 h in which there was completethawing; (c) transferred to 18°C in white light of about 200,umol photon m-2 s-' for 1 h. Electron transfer activity inthese preparations was assayed by DCPIP photoreduction inthe presence or absence of DPC and the rates expressed as

percentages of those obtained with preparations from un-frozen controls (Table III). As can be seen, freezing followedby thawing (whether in light or dark) severely inhibited theactivity assayed without DPC but caused no inhibition of theDPC-supported rate of DCPIP reduction. When the thawperiod was minimized, the water to DCPIP rate was inhibitedcompared with the control but was greater than if a 1-h thawperiod was given. In contrast, when the assay medium wassupplemented with 10 mM CaCl2 plus 20 mm NaCl, wateroxidation was reconstituted in all cases to levels comparablewith the control.To investigate the possibility that there are changes in

protein composition due to freezing, SDS-PAGE was carriedout on salt and pH-treated or not treated PSII-enriched mem-branes. After electrophoresis, the bands corresponding to theextrinsic proteins were readily distinguishable when largevolumes of supematant were analyzed relative to that usedfor membrane pellets. Washing of PSII-enriched membranepreparations isolated from leaves of pea plants grown underoptimal conditions with 1.2 M NaCl released the extrinsic 23-and 17-kD proteins from the membrane (Fig. 1, lane 3).However, after a freezing treatment (-18°C) of pea plants,the 17- and 23-kD protein bands became very faint in thesupernatant produced by a 1.2 M NaCl wash of the PSIImembrane preparations (Fig. 1, lanes 5, 7, 9), suggesting thatprior to isolation there had been a loss of the 17- and 23-kDprotein. In control samples, only a trace of the 33-kD protein

Table Ill. Effect on PSII Electron Transfer Activity of DifferentProtocols for Thawing Pea Plants

PSII membranes were isolated from plants that had been frozenat -180C for 18 h in the dark and then used directly, or thawed for1 h at 180C, either in the dark or light at 200 ,mol photon m2 s- .

Electron transfer activity was assayed by the photoreduction ofDCPIP, with or without DPC added as an artificial electron donor.The assay medium was 20 mm Mes, pH 6.0, and was supplementedwith 10 mm CaCl2 and 20 mm NaCI as indicated. Rates are given aspercentages of those in PSII preparations from unfrozen controls.

Type of Thaw H20 -* DCPIP H20 -+ DCPIP DPC DCPIP+ CaCI2 DC- CI

Minimum 30 100 94In dark <5 88 120In low light <5 80 129

22 WANG ET AL.

COLD EFFECT ON PSII EXTRINSIC PROTEINS AND PHOTOINHIBITION

1 2 3 4 5 6 7 8 9 1011 1.0

_- _ _|>- 433kD

_ __ ^i ^-~~ - ~~~~~~~~1 7 1kD

_ _ _ _

Figure 1. Effects of freezing treatment in the dark and a subsequentthaw period on the SDS-PAGE profiles of polypeptides in PSII-enriched membrane preparations. Before electrophoresis, washingwith 1.2 M NaCI was used to separate the extrinsic polypeptides(lanes 3, 5, 7, and 9) from the membrane (lanes 4, 6, 8, and 10).Unwashed PSII membranes were analyzed in lane 11, a standardpreparation of the 33-kD protein obtained by a wash with 1 M CaCI2in lane 2, and a mixture of protein molecules with molecular massesof 94, 67, 43, 20, and 14.4 kD (top to bottom, respectively) in lane 1.Control samples from nonfrozen plants are given in lanes 3 and 4.Those from plants frozen at -181C in the dark for 18 h without athaw period are in lanes 9 through 11, those given this freezingtreatment followed by a 1-h thaw in the dark at 1 8OC in lanes 5 and6, or followed by a 1-h thaw at the growth light intensity at 1 80C inlanes 7 and 8. To increase the visualization of proteins in the super-natant fractions, a higher (x7.5) proportion of the supematants (lanes3, 5, 7, and 9) was loaded compared with the pellets (lanes 4, 6, 8,and 1 0).

z

0h-c :3

,>0.5-0 a

Z._z' "O_

.

06 7 8 9

pH

Figure 2. The effect of incubation at various pH values on theremoval of the extrinsic 23-kD protein from PSII membranes. PSII-enriched membrane preparations were isolated from control peaplants (@) and those that had been cold treated in the dark for 18 hat -20C (A) and at -180C (U). Washing of membranes at differentpH values and assay of the amount of the 23-kD protein in thesupernatant by ELISA has been described in detail in refs. 5 and 25.

alkaline pH treatment was used and the extrinsic 23- and 33-kD proteins detected immunologically in supernatants by anELISA method. Results for the 23- and 33-kD proteins aregiven in Figures 2 and 3, respectively, for washes of PSIImembrane preparations isolated from pea plants that hadbeen grown at 18C and exposed to around -2°C and -1 8°Cin the dark for 18 h and transferred back to 18C for 1 h inlow light. PSII membrane preparations from control plantswere characterized by removal of the 23-kD protein by alka-line pH. At pH 7.5, some of the 23-kD protein was removed

100was observed in the supernatant after a 1.2 M NaCl wash (Fig.1, lane 3), and after the treatment of plants by freezing in thedark at -1 8°C for 18 h, a similar result was observed (Fig. 1,lane 9). However, when frozen plants were thawed at 18C inthe dark, a distinct band corresponding to the 33-kD proteinappeared in the supernatant fraction after a 1.2 M NaCl wash(Fig. 1, lane 5), indicating the release of some of the 33-kDprotein from the PSII membrane. When frozen plants wereexposed to weak light during thawing, the band at 33 kDbecame stronger (Fig. 1, lane 7), indicating more 33-kDprotein had been removed from the membranes. A furtherinteresting result was that after a 1.2 M NaCl wash of PSIImembrane preparations from frozen pea plants, four clearlyvisible polypeptide bands and three faint bands between 15and 20 kD were seen in supernatants from plants frozen eitherwith or without thawing treatments (Fig. 1, lanes 5, 7, 9). Thenature of these proteins is unknown but none ofthem reactedto polyclonal antibodies of the 23- or 33-kD proteins.

Further evidence that freezing at -18C induced an altera-tion in the association of the extrinsic PSII proteins with thesurface of the membrane was given by experiments in which

z

i-Ea: E

a-zx0 EZD0o 0

A: .-

50

01:6 7 8 9

pH

Figure 3. The effect of different pH values on the removal of theextrinsic 33-kD protein from PSII membranes. PSII-enriched mem-brane preparations were isolated from control pea plants (0) andthose that had been cold treated in the dark for 18 h at -21C (A)and at -1 81C (U). Washing of membranes at different pH values andassay of the amount of the 33-kD protein in the supernatant by ELISAwas as described in refs. 5 and 25.

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Plant Physiol. Vol. 99, 1992

from the membrane, about one-third as much as at pH 9.5.However, after -18C freezing treatment in vivo, only a traceof the 23-kD protein could be detected in supernatants afterall incubations, even at high pH values (up to pH 9.5). Thisis consistent with most of the 23-kD protein being no longerbound to the membrane after -18C freezing. After treatmentof pea plants at around -2°C, the detected 23-kD proteinwashed off the membrane by alkaline pH was less thancontrols, but significantly higher than from the -1 8°C-treatedplants. This indicates only a partial loss of the 23-kD proteinwith this temperature treatment, with the majority of theprotein remaining bound to the membrane. In control sam-ples, the 33-kD protein (Fig. 3) could only be removed fromthe membrane by treatment at about pH 9.0 and higher asshown previously (5). In contrast, after -180C freezing, the33-kD protein was washed off at much lower pH values. AtpH 7.5 to 8.0, 30 to 40% of the bound 33-kD protein waswashed off, and at pH 8.5 more than 80% of the 33-kDprotein was released from the membrane. This suggests thatthe 33-kD protein is more sensitive to removal after thefreezing treatment. The exposure of pea plants to about -2°Cappeared to induce only a slight change in the removal of the33-kD protein by alkaline pH.To investigate the possibility of changes in manganese

binding to the membrane caused by freezing treatment, man-ganese levels were determined with an atomic absorptionspectrometer in PSII membrane preparations isolated fromcontrol pea plants and plants that had been exposed to -2and -18C in the dark for 18 h and followed by 18C thawin low light for 1 h. No difference was observed in totalmanganese between PSII-enriched membrane preparationsisolated from control plants and those that had been frozen,the level being 3.5 ± 0.3 and 3.3 ± 0.8 Mn atoms to 250 Chlmolecules (±SD) in samples taken from unfrozen and frozenplants, respectively. This indicates that manganese bound toPSII is not released from the membrane after freezing andthawing treatment ofpea plants, or during the isolation of thePSII-enriched membranes.

DISCUSSION

The above results clearly show that when pea leaves aresubjected to a freezing period followed by a thaw, the bindingof the extrinsic proteins associated with the oxidizing side ofPSII is weakened. In particular, the 17- and 23-kD proteinsare almost totally lost as a result of this treatment when PSII-enriched membranes are isolated. Although the 33-kD extrin-sic protein remains attached to the isolated PSII-enrichedmembranes after the freeze-thaw treatment, there is clearlysome loosening of the association with the membrane surfacebecause compared with the wild type, this protein is moreeasily removed. The weakening of the binding of the 33-kDprotein with the donor side of PSII does not, however, leadto any loss of manganese. Nevertheless, PSII-enriched mem-branes isolated from leaves subjected to freezing showed asignificant reduction in electron flow from H20 to DCPIP,although the DPC to DCPIP rate was not inhibited comparedwith the control. A similar effect on electron transfer activitywas observed by Terashima et al. (21) with thylakoids fromchilled Cucumis sativus L. leaves. In our experiments, the

H20 to DCPIP rate was reconstituted to the level of thecontrol by inclusion of 10 mM CaCl2 together with 20 mMNaCl in the assay medium. Thus, with CaCl2 present theapparent "loosening" of the association of the 33-kD proteinwith the membrane surface did not affect the capacity toevolve oxygen below that of the preparations isolated fromuntreated plants.As in the previous paper (25), we have found that the extent

of photoinhibition of electron flow activity of isolated PSII-enriched membranes is greatest when the activity ofthe water-splitting system is reduced or not operative. If the PSII mem-branes isolated from cold-treated plants are supplementedwith CaCl2 during the illumination period, there is protectionagainst the enhanced photoinhibition, presumably due tostimulation of water oxidation activity. Because the DPC toDCPIP rates were also inhibited by high-light treatment, weconclude, as in the previous paper (25), that the initial site ofphotodamage occurs within the PSII reaction center, probablyon the oxidizing side.The results and discussions presented above indicate that

some physical changes occur on the oxidizing side of PSII dueto freeze-thaw treatment. It could be that these changes arecaused by alterations in the electrolyte concentration in thelumen of the thylakoids due to ice formation (7) or to modi-fications in the ionization properties of water and pH (24).We have detected these possible effects as an apparent de-crease in the binding capacity of the 17-, 23-, and 33-kDextrinsic proteins of PSII. The modifications in binding affin-ities could be important in increasing the susceptibility ofplants to photoinhibition as a result of lowering leaf temper-ature (2, 15). What is now certain, however, is that the threeextrinsic proteins are not absolutely required for water split-ting. The 17- and 23-kD protein have not been found in theoxygenic photosynthetic cyanobacteria (1), and recent studieswith Synechocystis 6803 have shown that the deletion of thegene encoding the 33-kD protein gave a mutant still able toevolve 02 (3, 14). However, in the case of the latter, theabsence of the 33-kD protein imposed a clear phenotypicproperty, namely a high susceptibility to photoinhibition ascompared with the wild type (14). Such a finding is consistentwith the view that the 33-kD protein, although not absolutelynecessary for water oxidation, does function to optimizeelectron donation to P680+ and, therefore, helps to protectagainst photoinhibition. The results presented in this paperalso support this general concept and further indicate that, inthe case of higher plants, the 23- and 1 7-kD proteins are alsoneeded to protect against photoinhibitory damage. The effectof low temperature treatments on the binding of the 17- and23-kD proteins has also been observed by Shen et al. (17).They observed the loss of these two proteins from PSII mem-branes isolated from leaves of chill-sensitive cucumber treatedat OC. Furthermore, several groups of workers have reportedthat chilling stress impairs 02 evolution (12, 20), although notall link this inhibition with damage to PSII (13). However,our work and that of others (8, 12, 20) supports the view thatPSII is a site vulnerable to chilling or freezing stress.

ACKNOWLEDGMENT

We thank John De Felice for skillful technical assistance.

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COLD EFFECT ON PSII EXTRINSIC PROTEINS AND PHOTOINHIBITION

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