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Journal of Plant Physiology 162 (2005) 181—194

KEYWORDChlorophyfluoresceninduction;Heat stresPhotosysteRecovery;

0176-1617/$ - sdoi:10.1016/j.

Abbreviationat 100ms; GPXP700, reactionsecondary elecsubstances; TL�Correspond

Tel: +41-(0)22-E-mail addr

www.elsevier.de/jplph

Biophysical studies of photosystem II-relatedrecovery processes after a heat pulse in barleyseedlings (Hordeum vulgare L.)

Szilvia Z. Totha,b,�, Gert Schanskera, Judit Kissimonb, Laszlo Kovacsc,Gyo+zo+ Garabc, Reto J. Strassera

aLaboratory of Bioenergetics, University of Geneva, Chemin des Embrouchis 10, CH-1254 Jussy, SwitzerlandbFaculty of Horticultural Science, Department of Molecular Plant Biology, Szent Istvan University, PO Box 53, BudapestH-1518, HungarycInstitute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, PO Box 521, SzegedH-6701, Hungary

Received 7 April 2004; accepted 25 June 2004

This paper is dedicated to Gabor Horvath (1944–2000)

Sll-ace

s;m II;

ee front matter & 200jplph.2004.06.010

s: DA515, flash-induce, guaiacol peroxidase;center chlorophylls otron acceptor quinone, thermoluminescenceing author. Laboratory759-9940; fax: +41-(0ess: toth1@etu.unige.

SummaryLeaves of 7-day-old barley seedlings were subjected to heat pulses at 50 1C for 20 or40 s to inhibit partially or fully the oxygen evolution without inducing visiblesymptoms. By means of biophysical techniques, we investigated the time course andmechanism of photosystem II (PSII) recovery. After the heat treatment, the sampleswere characterized by typical heat stress symptoms: loss of oxygen evolution activity,strong decrease of Fv/Fm, induction of the K-step in the fluorescence inductiontransient, emergence of the AT-thermoluminescence-band and a dramatic increase inmembrane permeability.

In the first 4 h in the light following the heat pulse, the AT-band and the K-stepdisappeared in parallel, indicating the loss of this restricted activity of PSII. This phasewas followed by a recovery period, during which PSII-activity was gradually restored inthe light. In darkness, no recovery, except for the membrane permeability, wasobserved. A model is presented that accounts for (i) the damage induced by the heat

4 Elsevier GmbH. All rights reserved.

d electrochromic absorbance transient; APX, ascorbate peroxidase; F100ms, fluorescence intensityLHCII, light-harvesting chlorophyll a/b protein complex of PSII; MDA, malondialdehyde; P680 andf PSII and PSI, respectively; PSI, photosystem I; PSII, photosystem II; QA and QB, primary ands of PSII, respectively; SOD, superoxide dismutase; TBARS, thiobarbituric-acid reactive

of Bioenergetics, University of Geneva, Chemin des Embrouchis 10, CH-1254 Jussy, Switzerland.)22-759-9945.ch (S.Z. Toth).

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pulse on the membrane architecture and on the PSII donor side, (ii) the light-dependent removal of the impaired reaction centers from the disorganizedmembrane, and (iii) the subsequent light-independent restoration of the membranepermeability and the de novo synthesis of the PSII reaction centers in the light.& 2004 Elsevier GmbH. All rights reserved.

Introduction

Several photosynthetic processes are sensitive toheat stress. Rubisco activase, the protein thatfacilitates the release of sugar phosphates fromRubisco ceases to function properly at elevatedtemperatures, inactivating rubisco (Crafts-Brand-ner and Law, 2000). Heat stress also affectsprocesses related to membrane integrity, ionconductivity, and phosphorylation activity. It hasbeen shown that elevated temperatures acceleratethe decay of the flash induced absorbance changeat 518 nm (Weis, 1982; Havaux et al., 1996), whichis thought to be related to ion transport acrossthylakoid membranes (Peters et al., 1984). Gou-naris et al. (1984) demonstrated that a heattreatment induced grana destacking and the for-mation of cylindrical-inverted lipid micelles. Withrespect to the photosynthetic electron transportchain, exposure of plants to elevated temperatureshas two contrasting effects. It was observed thatphotosystem I (PSI) (measured as the reduction rateof P700+) is stimulated by heat, which is related toa reduction of the plastoquinone pool by electronsfrom the stroma (Havaux, 1996; Bukhov et al.,1999). In contrast, PSII and especially the oxygen-evolving complex is very heat sensitive. Heatinduces the dissociation of the manganese-stabiliz-ing 33 kDa protein from the photosystem II (PSII)reaction center complex followed by release of themanganese atoms (Enami et al., 1994; Yamane etal., 1998). Heat-inactivation of PSII may befollowed by dissociation of the light-harvestingChl a/b protein complexes of PSII (LHCII) (Schreiberand Armond, 1978; Takeuchi and Thornber, 1994;Yamane et al., 1997). A further heat-induced effectthat has been described is a shift of the redoxequilibrium between QA and QB, increasing theresidence time of an electron on QA relative to QB

(Ducruet and Lemoine, 1985; Havaux, 1989).For plants, the rate of recovery from heat-

induced damage might be even more importantthan the extent of the damage itself. Gour et al.(1997) reported that in cyanobacteria, the degreeof recovery depends very much on the presence oflight during the repair processes. It was also shown(Heckathorn et al., 1997) that heat-related effectson PSII and particularly on the oxygen evolution are

important limitations to post-stress recovery. Ingeneral, however, little is known about theprocesses involved in post-stress repair.

In this work, we focus our attention on therecovery processes related to the repair of heatdamage to PSII in planta. To this end, full inhibitionof oxygen evolution was induced using heat pulses:immersion of leaves of young barley seedlings for40 s in a water bath of 50 1C. Pilot experimentsshowed that homogeneous starting material ofwhole plants with fully inhibited oxygen evolvingcomplex could most readily be obtained withpulses, rather than with prolonged heat treatmentsat somewhat lower (40–45 1C) temperatures. Thistreatment, while it induces the typical effects ofheat stress, virtually causes no visible symptoms. Asa comparison, we also subjected some plants to ashorter, 20 s, heat pulse that caused partial inhibi-tion of oxygen evolving activity. Several biophysicaltechniques were used to investigate the recoverykinetics of heat-inactivated PSII and also the role oflight in these processes.

Materials and methods

Plant material and growth conditions

Barley (Hordeum vulgare L. cv. Triangel) seeds weresoaked in tap water for 4 h and then sown onperlite. The seedlings were grown in a growthchamber (Conviron S10) under long day conditions(16 h light and 8 h darkness) at 22 1C and 100 mmolphotons m�2 s�1 photosynthetic photon flux den-sity. Seedlings were watered with Hoagland solu-tion and heat treatments were carried out whenthe barley seedlings were 7 days old.

Heat treatment and plant growth conditionsfollowing the treatment

The heat treatment of 7-day-old barley seedlingsconsisted of turning a pot containing 30–40 seed-lings upside down into a water bath of 50 1C for 20 sor 40 s. The primary leaves were entirely sub-merged. After the heat treatment the plantswere returned to the growth chamber. Some

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heat-treated and untreated plants were kept incomplete darkness after the heat treatment, undersimilar temperature conditions as in the growthchamber.

Chlorophyll-a fluorescence measurements

Chlorophyll-a fluorescence emission of intact bar-ley leaves or thylakoids was measured by a portablefluorometer (PEA-Plant Efficiency Analyser, Hansa-tech Instruments, King’s Lynn, Norfolk, UK). Sam-ples were dark-adapted for 10–15min before themeasurements and then illuminated with contin-uous light (2400 mmolm�2 s�1, 650 nm peak wave-length, for 5 s) provided by an array of six light-emitting diodes focused on a circle of 5mmdiameter of the sample surface. For evaluatingthe fluorescence induction transients, the Biolyzerv.3.0.6 software (developed by Ronald Rodriguez inthe Laboratory of Bioenergetics, University ofGeneva) was used.

Isolation of thylakoid membranes

Thylakoids were isolated from leaves of the heat-treated barley seedlings. The leaf tissue washomogenized in 40mM HEPES buffer (pH 7.5),containing 0.4M sucrose, 1% (w/v) BSA, 5mMMgCl2, 2mM EDTA-Na2 and 15mM NaCl. The homo-genate was filtered through two layers of fine nylonmesh and centrifuged at 3000g for 5min. Theplastid pellet was resuspended in the same bufferand was centrifuged at 3000g for 5min. Then themembrane pellet was resuspended in 40mM HEPESbuffer (pH 7.5) containing 0.4M sucrose, 5mMMgCl2 and 15mM NaCl. The chlorophyll content wasadjusted to 200 mgml�1 (using the method of Porraet al., 1989) and the thylakoid suspension wasdiluted to the desired concentration before eachmeasurement. Thylakoids were kept on ice indarkness and used within 3 h after isolation.

Measurement of oxygen evolving activity

The oxygen evolving activity of thylakoids (25 mg chl(a+b) ml�1) was measured with a Clark-type oxygenelectrode (Hansatech, Kings’ Lynn, UK) in atemperature controlled cell at 25 1C under saturat-ing light intensities (1800 mmolm�2 s�1) for 2min.Measurements were carried out using 500 mMphenyl-p-benzoquinone (PPBQ) as an electronacceptor of PSII that binds to the QB-site (Rengeret al., 1988).

Thermoluminescence measurements

Thermoluminescence was measured using a cus-tom-made TL apparatus described by Wiessner andDemeter (1988). For TL measurements, thylakoidsuspension (0.3ml, 50 mg Chl (a+b) ml�1) wasplaced on a copper sample holder. The sampleholder was connected to a ‘‘cold finger’’ immersedin liquid nitrogen. A heater coil, placed under thesample holder, ensured the desired temperature ofthe sample during the measurements. Dark-adapted samples were illuminated at �20 1C by asingle turnover flash and cooled down to �40 1C.Thermoluminescence was measured while heatingthe sample to 96 1C in darkness with a constantheating rate of 20 1Cmin�1. The emitted thermo-luminescence light was measured with a Hamamat-su end-window photomultiplier.

Measurement of flash-inducedelectrochromic absorbance transients(DA515)

Electrochromic absorbance changes, induced bysingle turnover flashes, were measured at 515 nm,the maximum of the electrochromic transients, in aset-up described earlier (Buchel and Garab, 1995).The time constant was set to 100ms; 32 kinetic traceswere collected with a repetition rate of 1 s�1 andaveraged. The transients were recorded at roomtemperature on leaves that were excised from thebarley seedlings just before the measurements.

Determination of antioxidant enzymeactivities and TBARS content

Guaiacol peroxidase activity (GPX, EC 1.11.1.7) wasmeasured by using a modified method of Chanceand Maehly (1955), the ascorbate peroxidase (APX,EC 1.11.1.11) measurement was based on themethod of Nakano and Asada (1981), and for themeasurement of thiobarbituric-acid reactive sub-stances (TBARS) the method of Popham andNovaczky (1991) was used.

Results

Leaves of barley seedlings were given a heat pulseof 50 1C for 20 s or 40 s. Measurements of variousparameters were carried out 30min, 4 h, 24 h and48 h after the heat treatment. These time pointswere chosen in preliminary experiments usingchlorophyll-a fluorescence transients. The mainconsideration was to choose characteristic points

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defining the recovery processes occurring after theheat treatment and to carry out the measurementsuntil considerable (for the 40 s heat pulse) oralmost full recovery (20 s heat pulse) occurred.After the heat-treatment, plants were kept eitherin darkness or in the light (100 mmolm�2 s�1). Theheat pulses did not cause visible symptoms on thebarley seedlings, except for a slight loss of turgorduring the first 10–15min after the treatment.There were no significant differences in thechlorophyll content of the control and heat-treatedplants (data not shown).

Chlorophyll-a fluorescence kinetics

Upon illumination of a dark-adapted leaf, thechlorophyll-a fluorescence emission exhibits a

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Figure 1. Effects of a 40 s, 50 1C heat pulse on the chlorophyseedlings kept in light after the heat pulse; measurements wekept in darkness after the heat pulse (control-dark: plants kcarried out in planta; C: fluorescence curves of panel A,fluorescence). D: chlorophyll-a fluorescence induction curvesleaves 30min after the heat pulse of 50 1C of 40 s and its con

polyphasic rise from F0 to Fm, via two intermediatesteps (FJ and FI) paralleling the closure of thereaction centers (OJIP-transient, Strasser et al.,1995). Figures 1A and B show the fluorescenceinduction curves measured on barley leaves atdifferent intervals after the 40 s heat pulse (the 20 sheat treatment caused similar but less pronouncedeffects; these data are not shown). The followingeffects could be observed:

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F0 slightly increased upon the heat pulses; thevalue of this parameter further increased both inthe light and in darkness (Figs. 1A and B,respectively).

2.

The K-step (Guisse et al., 1995; Srivastava et al.,1997) appeared at 500 ms. It disappeared by the4th hour when plants were kept in the light(Fig. 1A), but it persisted in the dark (Fig. 1B).

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fluorescence induction curves. A: heat-treated barleycarried out in planta; B: heat-treated barley seedlingsin complete darkness for 48 h); measurements werermalized between F0 and Fm (=V; relative variableasured on thylakoids isolated from heat-treated barleyl.

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Recovery from a heat pulse 185

3.

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A second peak appeared in the fluorescencetransients in the hundreds of ms range; itsmaximum determined the Fm (Figs. 1A and B).When plants were kept in the light, the intensityof this peak significantly decreased by the 4thhour after the treatment; in darkness this wasobserved after 24 h. This peak was not presentwhen chlorophyll-a fluorescence was measuredon thylakoids isolated from the heat-treatedleaves (Fig. 1D).

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In Fig. 1C the relative variable fluorescencecurves ½Vt ¼ ðF t � F0Þ=ðFM � F0Þ� of plantskept in light, are plotted. The figure showsthat after 24 h of recovery in light, the formof the induction curve showed again thesteps OJIP, but with a higher J-step. By the48th hour, the relative intensity of the J-stepbecame lower, approaching the level of thecontrol.

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Figure 2. Time course of the changes of Fv/Fm (A) andthe expression 1�F0/F100ms (B) of barley leaves after a50 1C heat pulse of 20 s or 40 s. Plants kept in the light orin the in darkness after the heat pulse.

Keeping barley seedlings in darkness for 48 h didnot change their fluorescence induction curves(control traces in Figs. 1A and B). There were nodifferences between the fluorescence inductioncurves of 7 or 9 days old control plants either(data not shown).

Figure 2A shows the Fv/Fm values of barleyleaves. This parameter is widely used to determinethe maximum quantum yield of the primaryphotochemistry of PSII (jPo) (Kitajima and Butler,1975). In the case of the 40 s heat pulse, the Fv/Fmdecreased from 0.80 to 0.61, and with the shorter,20 s heat pulse it decreased to 0.70. In the case ofthe 20 s heat pulses, Fv/Fm reached its minimum bythe 4th hour, whereas with the 40 s heat pulses theminimum was to be found after 24 h. In the absenceof light Fv/Fm kept decreasing for both the 20 s andthe 40 s heat treatments. These changes weremainly determined by the emergence and disap-pearance of a second peak in the fluorescencetransient (Figs. 1A and B). To disregard it, the Fv/Fmexpression was modified by replacing the Fm-valuewith the fluorescence intensity at 100ms (F100ms)turning it into 1� F0=F100 ms: The correspondingvalues are shown in Fig. 2B. In the case of thecontrol, the value of the Fv/Fm and the 1�F0=F100 ms did not differ significantly, because Fm

is reached between 100 and 200ms. The maindifference between the time course of Fv/Fm andthe 1� F0=F100 ms is that the latter did not decreaseafter the heat pulse. When the plants were kept inlight, 1� F0=F100 ms showed a slight (40 s heatpulse) or significant (20 s heat pulse) recovery bythe 24th hour. The same parameter did not changewhen the plants were kept in the dark after theheat treatment.

Oxygen evolution

The oxygen evolving activity, as determined bymeasuring the rate of electron transport from waterto phenyl-p-benzoquinone (PPBQ), was severelyaffected (Fig. 3). In the case of the shorter (20 s)heat treatment, it decreased by 62%, while after the40 s treatment the oxygen evolution was fullyinhibited. The activity recovered by the 48th hourin plants kept in light but no recovery occurred whenthe plants were kept in darkness. Similar recoverykinetics can be observed for oxygen evolution and the1� F0=F100 ms parameter (Fig. 2B). The Fv/Fm (Fig.2A) showed completely different kinetics.

Thermoluminescence

The stabilization and recombination of thecharge separation in PSII can be monitored by

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thermoluminescence (TL) (Horvath, 1986; Vass andInoue, 1992).

For TL measurements, thylakoids were isolatedfrom leaves that had been exposed to a heat pulse(50 1C, 20 s or 40 s) and kept in the light or in thedark for different periods of time, up to 48 h. The

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Figure 3. Recovery kinetics of the oxygen evolvingactivity of thylakoids isolated from heat-treated (50 1C,20 s or 40 s) leaves of barley seedlings. Plants were keptin light or in darkness after the heat pulse. The oxygenevolving activity of the control was 325 mmol O2 mg�1 Chlh�1716.7%.

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Figure 4. Thermoluminescence of thylakoid membranes iso50 1C). A: seedlings kept in the light after the heat pulse, andafter the heat pulse, and the control (Control-dark, kepilluminated at �20 1C by one single turnover flash and coolduring heating of the sample to 96 1C in darkness with a con

use of thylakoids instead of leaves was necessary,as upon freezing and heating of leaves, a large TL-band of unknown origin appeared at 50 1C, whichmade the investigation of the B-band quasi im-possible (data not shown).

The results after the 50 1C, 40 s heat pulse areshown in Fig. 4. In the control, two TL bands wereobserved after excitation of the thylakoids with asingle turnover flash at �20 1C: (i) the B-band,formed at around 35 1C, which is generated by arecombination between QB

� and the S2 state of thewater-oxidizing complex, and (ii) a band, appearingat 75 1C, which is tagged as high temperature TL(HTL) band.

The B band was eliminated by the heat treat-ment. When plants were kept in the light, it startedto re-appear after 24 h following the heat treat-ment, and by the 48th hour the intensity of the B-band approached the control level (Fig. 4A). Indarkness, no recovery was observed (Fig. 4B). Theintensity of the HTL band did not change signifi-cantly after the heat treatment in either case.Thirty minutes after the heat pulse, a band wasobserved at �18 1C, which is probably the AT-band(Koike et al., 1986). Four hours after the heatpulse, this band had disappeared when plantswere kept in the light, while it persisted in thedark (Fig. 4B).

In Fig. 5 the intensity of the AT-band is plottedversus the K-step of the fluorescence transient. Alinear correlation was found for the 40 s and also for

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lated from heat-treated leaves of barley seedlings (40 s,the control (Control-light), B: seedlings kept in darkness

t in darkness for 48 h). Dark-adapted thylakoids wereed down to �40 1C. Thermoluminescence was measuredstant heating rate of 20 1Cmin�1.

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the 20 s heat pulses. The difference in slopes maybe due to the partial (20 s heat pulse) or complete(40 s) inhibition of oxygen evolution.

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Figure 5. Relationship between the AT thermolumines-cence band and the K-step of the chlorophyll-a fluores-cence transient. The relative intensity of the AT-bandwas determined from the maximum intensity of thethermoluminescence between �20 and 0 1C, divided bythe value for the control. The intensity of the K-band wascalculated as the ratio between the relative variablefluorescence intensity at 500 ms (maximum intensity ofthe K-step) and at 2ms ½V500 ms=V2 ms ¼ ðF500 ms �

F50 msÞ=ðF2 ms � F500 msÞ�:

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Figure 6. Flash-induced electrochromic absorbance transient50 1C. Plants were kept in the light or in darkness after the healight), B: leaves from barley seedlings kept in darkness after tin darkness for 48 h). There was no strict dark-adaptation be(10min in dim light). Curves are averages of two to threehalftimes of the corresponding decay kinetics (t1/2) are indicaflashes.

Flash-induced electrochromic absorbancetransients (DA515)

Absorbance changes around 515 nm reflect electro-genic changes in the membrane (Junge, 1977; Witt,1979). The amplitude of the rapid phase is ameasure for the number of active PSII and PSIreaction centers, which are thought to contributeequally to the signal amplitude. The slower ms-risephase is attributed to the electrogenic activity ofthe cytochrome b6/f complex (Horvath et al., 1979;Farineau et al., 1980). The decay kinetics of thisabsorbance change is proportional to the iontransport across the thylakoid membranes, whichis normally linked to ATP synthesis, but the sameparameter might also serve as a sensitive indicatorof the intactness of thylakoid membranes (Peterset al., 1984).

The heat treatment of 50 1C for 40 s decreasedthe amplitude of the initial phase of DA515 to abouthalf of the control, and drastically (30-fold)accelerated the decay (Fig. 6). The 20 s heat pulseinduced similar effects (data not shown).

During recovery, the initial amplitude was re-stored when the plants were kept in the light, andrecovery of the slow phase could also be observed.When the plants were kept in the dark, there wasno recovery of the initial amplitude. The decaykinetics showed recovery also in darkness. In thecontrol (48 h dark-adaptation) of heat-treated

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Time (ms)

s (DA515) of barley leaves after a 20 s or 40 s heat pulse att treatment. A: heat-treated and control leaves (Control-he heat pulse and dark control (Control-dark, plants keptfore the measurements in the case of plants kept in lightindependent experiments of five leaves each. Apparentted. Arrows indicate the time of firing the single turnover

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plants kept in darkness, the decay of the electro-chromic signal slowed down.

The activity of several antioxidant enzymesand the amount of thiobarbituric-acidreactive substances

It was found that the employed heat pulses inducedno significant alterations in the activities ofascorbate peroxidase (APX: EC 1.11.1.11) andguaiacol peroxidase (GPX: EC 1.11.1.7) (Table 1).Further, NBT-stained native gels showed no obser-vable changes in the pattern or the intensity of theisoenzymes of superoxide dismutase (SOD: EC1.15.1.1, data not shown). The extent of mem-brane lipid peroxidation was measured by theamount of TBARS. There was no significant increasein the TBARS content, independently of the timeafter the heat treatment or the presence orabsence of light (Table 1). In addition, spin trappingwith DMPO (5,5-dimethyl-1-pyrrolin N-oxide) wasused, but we could not detect any free radicals inthe samples (data not shown).

Discussion

The aim of this work was to study the recoverykinetics of PSII activity after heat stress in planta.In order to facilitate these investigations, weinduced complete or partial inhibition of theoxygen evolving activity with heat pulses, immer-

Table 1. Ascorbate peroxidase (APX, EC 1.11.1.11), guaiacof thiobarbituric-acid reactive substances (TBARS) after a 50

Control 20 s, 50 1C

Light Dark Light

APX (mmol AA g FW�1min�1)30min 5.1970.09 5.0970.08 4.6370.24 h 4.8270.16 4.8570.14 5.3170.124 h 4.9970.50 5.1370.65 5.3970.448 h 4.7070.69 4.6970.01 4.7170.7

GPX (mmol H2O2 g FW�1min�1)30min 19.1271.62 19.9370.85 19.4670.74 h 20.0471.33 21.1070.84 20.3371.624 h 20.5770.88 24.5371.46 20.5970.248 h 20.0471.96 21.0970.74 20.3371.7

TBARS (nmol MDA g�1 FW�1)30min 28.1274.10 29.4470.78 28.2570.74 h 31.1471.21 28.2270.38 26.3872.324 h 26.5970.12 26.9271.84 27.9573.148 h 26.7571.95 29.6970.74 33.1872.7

Plants were kept in the light or in the in darkness after the heat pulse

sing leaves of barley seedlings into a water bath of50 1C for 40 s or 20 s. This kind of heat-treatmenthas the advantage that it induces very homoge-neous effects and avoids several secondary effects,such as desiccation, metabolic changes, etc.Similar heat treatments were applied in the worksof Takeuchi and Thornber (1994) and Sazanov et al.(1998), for example. We also note that a heat-treatment at 45 1C for 30min in a temperature-controlled chamber had similar effects on thechlorophyll-a fluorescence transients and the re-covery occurred on the same time scale as after the50 1C, 40 s heat pulse. However, the effects wereless homogeneous and the different recoveryphases were also more difficult to identify (datanot shown).

In order to investigate the effect of light on therecovery processes some barley plants were put incomplete darkness following the heat treatment,while others were returned to the same conditionsas before the heat treatment. Keeping non-heat-treated plants in darkness for 48 h caused onlyslight changes in most of the investigated para-meters; only the decay time of the DA515-signalincreased in darkness, most probably due to theinactivation of the ATP synthase (Weis, 1981).

Primary heat-stress effects on PSII and onmembrane permeability

The first measurements were carried out 30minafter the heat pulse. At this stage, several typical

ol peroxidase activity (GPX, EC 1.11.1.7) and the amount1C heat pulse of 20 s or 40 s

40 s, 50 1C

Dark Light Dark

3 4.4170.35 5.0870.14 4.4170.400 4.8670.69 5.2370.38 4.8670.375 5.2170.68 5.4770.57 5.2170.589 4.6970.39 5.1370.53 4.6770.21

1 19.5870.27 21.3571.35 17.3570.234 20.9171.84 20.1672.45 22.0071.229 22.5171.14 21.7770.58 21.1170.406 24.2270.17 20.1670.98 23.2571.01

8 27.6470.29 25.7372.56 28.8073.303 22.2471.59 25.4070.46 23.7872.814 23.0371.47 24.0772.22 25.8973.655 26.5970.51 28.6571.45 22.6870.70

as indicated. Data are averages of 4–6 independent experiments.

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and well-characterized effects of heat stress wereobserved:

1.

The F0 value slightly increased (Figs. 1A and B).It may be attributed to a combination of severalprocesses: (i) dissociation of LHCII from thereaction center (super)complexes (Schreiberand Armond, 1978; Yamane et al., 1997), (ii)heat-induced monomerization of LHCII trimers(Takeuchi and Thornber, 1994; Garab et al.,2002), and (iii) dark reduction of QA which isrelated to a heat-induced shift of the redoxequilibrium between QA and QB (Ducruet andLemoine, 1985; Havaux, 1989) and a heat-induced facilitation of the back flow of electronsfrom the plastoquinone pool (Sazanov et al.,1998; Yamane et al., 2000).

2.

The decrease of Fm is related to the heat-inactivation of the oxygen evolving complex(Yamashita and Butler, 1968; Schreiber andNeubauer, 1987). The inactive donor side of PSIIhas a very limited capacity for donating elec-trons. Unless forward electron transfer beyondQA is blocked by DCMU (Strasser, 1997), forexample, or by a reduced PQ-pool (see below),most induced charge separations will lead to afast recombination with the electron holeformed on the donor side. Under recombiningconditions, reaction centers will behave as heatsinks (Strasser and Tsimilli-Michael, 2001). Alsothe loss of oxygen evolving activity and the B-band (recombination of the S2QB

� charge pair),the appearance of the K-step and the appear-ance of the AT-band all indicate the destructionof the manganese cluster. The K-step is sug-gested to be due to an imbalance between thedonation rate of PSII and the re-oxidation rate ofQA� (Strasser, 1997). TyrZ can donate one elec-

tron to P680+, but a second charge separationafter forward electron transfer from QA to QB

would lead either to an accumulation of P680+ ora fast charge recombination (Strasser, 1997). Ineither case, as observed, the fluorescenceintensity would decrease after the first chargeseparation. The AT-band is typical of samplesthat lack a functional Mn cluster (Koike et al.,1986). According to Ono and Inoue (1991) theAT-band is due to charge recombination be-tween the reduced primary quinone acceptor QA

�

and a redox-active histidine residue on thedonor side of PSII, which may be involved inthe binding of the Mn-cluster.

3.

The heat treatment significantly decreased theamplitude of the initial phase of DA515 (Fig. 6),which could be attributed to the heat-inactiva-tion of PSII reaction centers. The dramatic,

about 30-fold, acceleration of the overall decaykinetics is evidently an indicator of a damage inthe membrane permeability, which thus lead toa rapid loss of the electric field component, andmost probably also of the DpH, of the energizedstate.

These changes reflect the primary effects of theheat pulses on PSII. It must also be pointed out thatin the first 30min following the heat pulse nosignificant differences were observed betweenplants kept in light or in darkness.

Measurements related to oxidative stress

Heat shock has been shown to cause oxidativestress that induces genes and promotes the synth-esis of enzymes involved in oxidative stress defense(Morgen et al., 1986; Li et al., 1999). However,under our experimental conditions no oxidativestress was observed. There was no increase in theactivities of the antioxidant enzymes studied (APX,GPX, SOD isoenzymes) and also no increasedcontent of TBARS (Table 1). The HTL-band isthought arise from an interaction between themolecular oxygen and lipid peroxides (Stallaert etal., 1995; Janda et al., 2000). Its intensity alsoremained unchanged (Fig. 4).

The strong acceleration of the decay kinetics ofthe electrochromic absorbance changes (Fig. 6)shows that the membranes became leaky to ionsdue to the heat pulse. Havaux et al. (1996) havealready reported that the increased permeability ofthylakoid membranes does not necessarily involveperoxidative damage of membrane lipids. Thereare several other processes that can lead toincrease in the membrane permeability in heat-treated samples: conformational changes of mem-brane proteins, opening of ion channels, changes inlipid–lipid interactions, redistribution of specificlipids within thylakoid membranes (Santarius,1980), and the formation of non-bilayer phases(Gounaris et al., 1984; Kota et al., 2002).

Fv/Fm and the heat-stimulated dark-reduction of the plastoquinone pool

Changes in the widely used parameter Fv/Fm (Fig.2A) were not in agreement with changes in theoxygen evolving activity, the intensity of the B-band and the amplitude of the DA515 signal. Forexample, in the case of the 40 s heat pulse, Fv/Fmcontinued to decrease until the 24th hour whenthere was already a significant recovery of all otherparameters investigated. Discrepancies had already

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S.Z. Toth et al.190

been observed between the oxygen evolution andthe Fv/Fm in the 50–56 1C temperature range(Yamane et al., 1998).

On the basis of our data, it is possible to proposethat this deviation is caused by a phenomenon thatis unrelated to PSII primary photochemistry. Theshape of the chlorophyll-a fluorescence transientdrastically changed after the heat pulse (Fig. 1). Atshort times after the heat treatment the fluores-cence induction curves showed a second peakbetween 200 ms and 1 s that had a strong effecton the apparent Fm and thus on the value of Fv/Fm(Fig. 1A and B). In plants kept in the light this peakalmost completely disappeared after 4 h, whereasin plants kept in the dark a considerable reductionwas observed only after 24 h (Figs. 1A and B). Thisphase did not occur in thylakoids isolated fromheat-treated leaves (Fig. 1D). These observationscan be explained by considering the heat-inducedstimulation of dark reduction of the plastoquinonepool by a stroma factor, stimulating the re-reduction rate of P700+ (Havaux, 1996; Bukhovet al., 1999). Given the very limited electrondonation capacity of PSII reaction centers withdamaged Mn-cluster, an accumulation of QA

� canonly occur in the presence of a reduced plastoqui-none pool. Initially, red light excitation of PSI willoxidize the plastoquinone pool, but this processbecomes transiently blocked by inactive FNR oncethe redox components on the acceptor side of PSIare reduced (Schansker et al., 2003). In non-heattreated leaves this occurs after 100–200ms (=Fm-level) and this coincides quite well with the onsetof the secondary rise phase of the fluorescence,which is observed in heat-treated leaves. Once theacceptor side of PSI is blocked, the plastoquinonepool will become reduced by electrons coming fromthe stroma and QA

� may slowly accumulate also inthe heat-damaged PSII reaction centers. That thisreally occurs in heat-treated leaves can be shownby simultaneous chlorophyll-a fluorescence and820 nm transmission measurements (Toth andSchansker, unpublished data). After the activationof FNR, which takes a few seconds (Carrillo andVallejos, 1987), the fluorescence level decreasesagain (Figs. 2A and B). In leaves recovering in thelight after a heat pulse, this phenomenon disap-peared after 4 h because, as it will be pointed outbelow, the heat damaged reaction centers areremoved. When plants are kept in darkness, thisdoes not occur and the phenomenon disappearswhen the stimulation of the reduction of theplastoquinone pool by electrons from the stromastops. The heat-induced increase of O2 uptake(measured using DCPIPH2 and MV as electron donorand acceptor) may be a related phenomenon and it

was observed to last as long as 1 to 2 days (Mohantyet al., 1987). In thylakoids this secondary risingphase in the fluorescence was not observedbecause during the isolation process the stroma iseither lost or becomes strongly diluted.

Taking into account the above-mentioned obser-vations a new expression was introduced: (1�F0)/F100ms, which avoided the difficulties with thesecondary rise of the chlorophyll-a fluorescencetransient.

The K-step and the AT-band

A linear correlation was found between the K-stepof the chlorophyll-a fluorescence rise and the AT-band of the TL glow curve (Fig. 5B). The datapresented in this paper indicate that either ofthese two parameters forms a good indicator forthe presence of heat-damaged oxygen-evolvingcomplexes.

Recovery of PSII

Heat induces the dissociation of the manganesestabilizing 33 kDa protein from the PSII reactioncenter complex followed by release of the manga-nese atoms (Enami et al., 1994; Yamane et al.,1998). Experiments on the re-assembly of themanganese cluster (Cheniae and Martin, 1971),e.g. in flashed leaves (Strasser and Sironval, 1972)indicate that these processes need light and can becompleted within a few minutes. The time for therecovery was considerably longer in the case of ourtreatment (48 h). This indicates that the injuredphotosystem II units were not repaired by only a re-assembly of the oxygen-evolving complex but thatthe damage was more serious. It is known that theheat stress may also impair other parts of thereaction center, e.g. the D1 and/or the D2 proteins(De Las Rivas and Barber, 1997).

The appearance of the K-step and the AT-band istypical of PSII centers with damaged oxygen-evolving complexes. Both phenomena had disap-peared by the 4th hour after the heat pulse whenplants were kept in light. During this period oftime, other parameters indicating the activity ofoperative centers (oxygen evolution activity, in-tensity of the B-band, amplitude of DA515) had notyet started to recover. After this phase, the oxygenevolving activity, the B-band and the amplitude ofthe DA515 signal started to recover. Figs. 1Aand C allow us to visualize some aspects of therecovery process: after 24 h of recovery in the lightthe fluorescence amplitude is still very small, but

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VJ K step

O2

evol. B

bandAT

bandt0 + - ++ ++ - t30min - ++ - - ++ t4h - - - - - t24h ++ - + + - t48h ++ - ++ ++ -

Degradation of damaged PSII unitsin the light

OEC

RC Damaged PSII units

RC

OEC

RC

OECHeat pulse

De novo synthesisof PSII in the light

RC

OEC

RC

OEC

t0

t30 min

t4 ht24 h

t48 h

Figure 7. Model for the recovery of photosystem IIactivity in barley leaves after a heat pulse. At t0 intactPSII units are presented. The heat pulse abruptlydamages the PSII units and also causes an increase inthe membrane permeability. PSII units loose their oxygenevolution and perform only a restricted electron trans-port (appearance of the K-step and AT-band; t30min). Fourhours later even this restricted activity is abolishedimplying that the degradation of the impaired PSII unitsoccurs in the light during this period of time. Followingthis phase, de novo synthesis of PSII units in the lightgives a gradual rise to the observed PSII activities (t24hand t48h), and the ‘‘normal’’ membrane permeability isalso recovered. In the table the presence (++, +) or theabsence (�) of certain signals or activities characterizingphotosystem II are summarized.

Recovery from a heat pulse 191

the form of the induction curve had largelyrecovered.

Recovery of PSII activity did not occur in theabsence of light. Several parameters, such asoxygen evolving activity, the amplitude of DA515,and also the whole form of the TL glow curve,stayed approximately unchanged after the heatpulse. On the other hand, the decay kinetics of theDA515 signal recovered also in darkness, indicatingthat the restoration of ‘normal’ membrane perme-ability did not require light.

To summarize the observed phenomena, a modelis presented in Fig. 7 that describes the recovery ofphotosystem II activity after a heat pulse. Therepair cycle that has been described for thephotoinhibitory damage to the D1-protein was usedas a reference for this model (e.g. Aro et al., 1993;Andersson and Aro, 2001). The heat treatmentcaused a dramatic increase in the membranepermeability, abruptly damaged PSII units, andcaused a loss of oxygen evolving activity. Never-theless, PSII units were still able to performrestricted electron transport (presence of the K-step and AT-band). During the first four hours, theK-step and the AT-band had disappeared withoutrecovery of PSII activity, suggesting that in the lightdegradation of the damaged PSII units occurs. Thisis a strongly light-dependent process; when plantswere kept in darkness it did not occur during theexperiment (48 h). Following this, ‘de novo synth-esis’ of PSII units caused a gradual rise of theobserved PSII activities. The idea of creatingnew reaction centers is supported by the longrecovery time and also by the phenomenonthat after 24 h of recovery the relative intensityof the J-step (VJ) is high and later on it approachesto the level of the control; this is a typical behaviorof young, newly synthesized reaction centers(Srivastava et al., 1999). The restoration of thenormal membrane permeability may be a prere-quisite for the creation of new reaction centers:Muhlbauer and Eichaker (1998) and Zhang et al.(2000) showed that a proton gradient is needed forthe translation and the elongation of the D1protein. Indeed, in our experiments a completerestoration in the membrane permeability occurredbefore recovery of PSII activity (4–24 h and 24–48 h,respectively).

In this study we concentrated on the measure-ment of biophysical phenomena. By detecting someof the cofactors in photosystem II by means ofthermoluminescence and chlorophyll-a fluores-cence as a function of time, we get a glimpse ofthe protein environment too, though biochemicalstudies are necessary to confirm our model. Thedata do indicate that there is a similarity in the way

cells handle damaged photosystem II reactioncenters after both photoinhibition and heat stress.

Note added in proof

At the meeting ‘‘Photosynthesis and Post-GenomicEra’’ (25–28 August 2004, Trois-Rivieres, Canada),prof. Y. Yamamoto (Okayama University, Japan)presented data on the cleavage of the D1-proteinafter a heat treatment. These not yet publishedbiochemical data show that the initial plantresponses to light and heat stresses are very

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similar; it provides additional support for the modelwe present.

Acknowledgements

S. Z. T. is grateful to Dr. Tibor Janda for hisencouragement to write the manuscript and alsofor the critical reading. This work was supported bythe Hungarian Research Foundation (T 026075 and T34188).

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