Moderate heat stress reduces the pH component of the transthylakoid proton motive force in...

Moderate heat stress reduces the pH component of thetransthylakoid proton motive force in light-adapted, intacttobacco leavespce_2018 1538..1547

RU ZHANG1, JEFFREY A. CRUZ2, DAVID M. KRAMER2, MARIA E. MAGALLANES-LUNDBACK3,DEAN DELLAPENNA3 & THOMAS D. SHARKEY1,3

1Department of Botany, University of Wisconsin-Madison, 430 Lincoln Drive, Madison, WI 53706, USA, 2Institute ofBiological Chemistry, 289 Clark Hall, Washington State University, Pullman, WA 99164, USA and 3Department ofBiochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA

ABSTRACT

We measured the DY and DpH components of the transthy-lakoid proton motive force (pmf) in light-adapted, intacttobacco leaves in response to moderate heat. The DY causesan electrochromic shift (ECS) in carotenoid absorbancespectra. The light–dark difference spectrum has a peak at518 nm and the two components of the pmf were separatedby following the ECS for 25 s after turning the light off.The ECS signal was deconvoluted by subtracting the effectsof zeaxanthin formation (peak at 505 nm) and the qE-related absorbance changes (peak at 535 nm) from a signalmeasured at 520 nm. Heat reduced DpH while DYslightly increased. Elevated temperature accelerated ECSdecay kinetics likely reflecting heat-induced increases inproton conductance and ion movement. Energy-dependentquenching (qE) was reduced by heat. However, the reduc-tion of qE was less than expected given the loss of DpH.Zeaxanthin did not increase with heat in light-adaptedleaves but it was higher than would be predicted given thereduced DpH found at high temperature. The results indi-cate that moderate heat stress can have very large effects onthylakoid reactions.

Key-words: chlorophyll a fluorescence; electrochromic shift;ion movement; moderate heat stress; proton conductance;qE; transthylokoid proton motive force; zeaxanthin.

INTRODUCTION

Heat stress reduces photosynthetic capacities of leaves(Berry & Björkman 1980; Sharkey 2005). In the moderatelystressful range of 35 to 42 °C, the reduction of photosynthe-sis is often reversible in light-adapted leaves but less so indark-adapted leaves (Havaux, Greppin & Strasser 1991).Reversible heat stress can reduce both the capacity forcarbon metabolism (Berry & Björkman 1980; Weis 1980;Kobza & Edwards 1987; Feller, Crafts-Brandner & Salvucci

1998; Salvucci et al. 2001; Salvucci & Crafts-Brandner 2004;Sharkey & Schrader 2008) and electron transport (Weis1981; Weis 1982; Schrader et al. 2004). Cyclic electron flowaround PSI (CEF) is frequently reported to be induced bymoderately high temperature (Havaux 1996; Pastenes &Horton 1996; Bukhov et al. 1999; Bukhov, Samson & Car-pentier 2000). Another effect of moderate heat stress is anincreased proton conductance of thylakoid membranesin dark-adapted leaves (Weis 1981; Havaux et al. 1996;Bukhov et al. 1999; Schrader et al. 2004). Our previous workfocusing on thylakoid reactions under moderate heat stressshowed that the proton conductance was also reversiblyincreased by heat in light-adapted intact leaves of Arabi-dopsis (Zhang & Sharkey 2009).

The electrochromic shift (ECS) can be used to probe thetransthylakoid pmf and proton circuits with intact leaves(Avenson et al. 2005; Baker, Harbinson & Kramer 2007).The ECS results from an electric field effect on carotenoidabsorbance bands, and is strongest at 518 nm (Witt 1979).In steady-state light, when influx of protons is balanced byefflux, a steady membrane energization (steady baselineabsorbance signal) is achieved (Sacksteder et al. 2000). Fol-lowing a light–dark transition, proton efflux without influxresults in a rapid dissipation of the pmf. The relaxation ofthe electric potential can be measured as a fast decay inabsorbance of carotenoids at 518 nm.The counterion move-ments that cause the DpH are slower than that of protons(Schröppel-Meier & Kaiser 1988; Thaler, Simonis &Schröppel-Meier 1992; Cruz et al. 2001). After the initialrapid absorbance decline, the signal partially reverses overa number of seconds reflecting slow movement of ions backto their concentrations in steady-state darkness (Cruz et al.2001). This slowly-recovering phase, ECSinverse, is propor-tional to the DpH component of pmf; the non-recoveringphase, ECSsteady-state, is proportional to the DY component ofpmf. Thus, measuring ECS for 25 s after the light is offallows the contribution of the DpH and DY to the pmf to beassessed.

Energy-dependent quenching (qE), the predominantcomponent of non-photochemical quenching (NPQ),reduces the rate of PSII electron transport under excess

Correspondence: T. D. Sharkey. Fax: +1 517 353 9334; e-mail:[email protected]

Plant, Cell and Environment (2009) 32, 1538–1547 doi: 10.1111/j.1365-3040.2009.02018.x

© 2009 Blackwell Publishing Ltd1538

light conditions (Niyogi 2000). The DpH component of pmfacidifies the thylakoid lumen, which activates the conver-sion of violaxanthin to zeaxanthin through the intermediateantheraxanthin by the lumen-localized enzyme violaxan-thin de-epoxidase (VDE) and the protonation of lumen-exposed residues of PsbS, a polypeptide associated with thelight-harvesting complex of PS II (Li et al. 2000, 2002). Itis proposed that zeaxanthin binds to protonated PsbS anddissipates extra energy from excited chlorophylls in theform of heat (Niyogi 2000; Müller, Li & Niyogi 2001; Li et al.2004). The alternative model of qE response is that lightharvesting complexes of PSII (LHCII) is the site of qE andprotonated PsbS (probably together with zeaxanthin) inter-acts with an LHC protein to promote the conformationalchange of LHCII which forms a quencher (Horton et al.1991; Horton, Ruban & Walters 1996; Horton,Wentworth &Ruban 2005). Zeaxanthin has also been proposed to stabi-lize the lipid bilayer of the thyakoid membrane (Havaux &Tardy 1996). Although the formation of qE in response tolight is well studied, its role during heat is not yet as welldescribed. The relationship between qE and the DpH com-ponent of pmf under moderately high temperature is alsounknown.

Here, we use an in vivo technique, taking advantage ofthe ECS to examine the effect of moderate heat stress ontransthylakoid pmf, DpH and DY components of pmf,proton conductance, and ion movements within light-adapted intact tobacco leaves before, during and after heattreatment. Chlorophyll a fluorescence was used to monitorPSII efficiency and qE response. The levels of zeaxanthinwere assessed both by optical methods and by high-performance liquid chromatography (HPLC) to examinehow the change in DpH at high temperature affects thexanthophyll cycle.

MATERIALS AND METHODS

Plant material

Nicotiana tabacum WU38 (tobacco) plants were grown in agreenhouse with the supplemental 1000 W high-pressuresodium lights that provide approximately 200–300 mmolphotons m-2 s-1, 16 h photoperiod, day/night temperaturearound 23 °C. Young, fully expanded intact leaves from6-week-old plants were used.

Heat treatment

Spectroscopic experiments with heat treatments wereperformed using a non-focusing optics flash spectro-photometer instrument (NoFOSpec) as described before(Avenson, Cruz & Kramer 2004; Takizawa, Kanazawa &Kramer 2008). Intact tobacco leaves were clamped into theleaf chamber in the NoFOSpec instrument under flowingambient air (372 ppm CO2, 21% O2, 79% N2), which washumidified by bubbling in distilled water. Actinic light wasprovided by red light-emitting-diodes (LEDs, 650 nm) at

intensities from 150 to 1000 mmol photons m-2 s-1. Heattreatment was imposed by flowing water from either of twothermostated water baths, one set at 23 °C and the other at40 °C, through a metal block that was attached to the actinicand measuring light pipe of the NoFOSpec, just above theleaf. The temperature of the leaf (monitored by thermo-couple attached to the leaf) was switched between 23 and40 °C in 2 min. Spectroscopic measurements were first con-ducted at 23 °C with five light intensities sequentially, 150,257, 347, 500, 612 mmol photons m-2 s-1, each for 30 min, toestablish the relationships among the measured parametersat 23 °C.Then, the leaf was again illuminated with 500 mmolphotons m-2 s-1 and the temperature was switched to 40 °Cfor 30 min, followed by switching back to 23 °C with500 mmol photons m-2 s-1 light for 30 min. This protocol wasused so that the relationships among the parameters couldbe determined and the effect of heat could be comparedwith those relationships. The 500 mmol photons m-2 s-1 lightwas chosen for the heat treatment and recovery becauseit is typically saturating for photosynthesis of C3 plantsand it is consistent with our previous work (Zhang& Sharkey 2009). Each light treatment lasted 30 minbecause we wanted to assay the system under steady-statephotosynthesis.

ECS measurements

The measurements we made are based on the followingphotosynthetic electron and proton transport theory. Whenlight drives photosynthetic electron transport, protons aretranslocated across the thylakoid membrane producing atransthylakoid pmf, the driving force used to make ATP.Because protons are positively charged, the pmf has twocomponents: the pH gradient (DpH) and the electricalpotential (DY). Because of the low electrical capacitance ofthylakoids, a small number of charges moving across themembrane will build up a large DY (Vredenberg & Buly-chev 1976). Meanwhile, the buffering capacity of the lumenis quite large, so that a large number of protons must bemoved into the lumen to alter its pH (Junge et al. 1979). Asa result, when actinic light is initially applied, the pmf isfirst formed almost exclusively as DY (Junge et al. 1979;Cruz et al. 2001). However, after continuous illuminationfor seconds or minutes, slow movements of ions (most likelyMg2+, Cl- and K+) collapse a fraction of the DY, allowinga DpH to build up (Kramer, Cruz & Kanazawa 2003).With the low concentrations of permeable ions (<10 mm)in the chloroplast, a substantial fraction of pmf is main-tained as DY during steady-state photosynthesis (Cruz et al.2001).

The method involved a pulsed measuring beam for ECSmeasurements consisting of green LEDs filtered throughinterference filters that selected for 505, 520 and 535 nmlight. ECS changes are much faster than other processesthat affect green light absorbance, such as zeaxanthin for-mation (505 nm) and qE-related absorbance signals (peakat 535 nm) (Bilger, Björkman, & Thayer 1989; Wise & Ort1989). During the first 500 ms of the dark interval, the

Light-adapted intact tobacco leaves 1539

© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1538–1547

rapid changes in absorbance at 520 nm are almost exclu-sively the result of the ECS. By measuring three wave-lengths (520, 505 and 535 nm), we deconvoluted the signal,allowing the ECS signal to be evaluated over long darkintervals (e.g. 25 s). The deconvolution equation for ECSwas estimated from long light-interval-relaxation-kinetics(described later) using a similar method as in Kramer &Sacksteder (1998). Dark interval relaxation kinetics ofECS were measured during steady-state photosyntheticconditions. Plants were first acclimated for 15 min inactinic illumination provided by red LEDs. The full ampli-tude of initial change in the ECS [DECS, but termed ECSt

in some previous work, reviewed in Baker et al. (2007)]should proportionally reflect the difference in total pmfbetween light and dark. While the fast ECS kinetics in thecontrol could be adequately fitted with the first-orderdecay, the heated samples required a two component fit,with a small slow phase of about 150 ms. When all curveswere fit with the second-order decay, the slow phase con-stituted about 10% of the decay in the control leaves andless than 20% in the heated leaves. The origin of the slowphase is not clear, but may represent heterogeneityin chloroplasts throughout the leaf and perhaps it isincreased by heat stress. Overall, both the amplitude anddecay rate of this slow phase is quite small, and thus con-stitutes only a small fraction of the total proton flux.Consequently, its inclusion had little effect on the inter-pretation of our data. The time constant for the initialdecay, tECS, was estimated by fitting the first 300 ms of thedecay curve with a second-order exponential decay kineticand the time constant of the faster phase was taken astECS. Proton conductance was estimated as the inverse ofthe decay time constant [1/tECS].

The initial dissipation of the pmf is entirely electrical(Baker et al. 2007) and an inverse ECS can develop as aresult of slow movement of ions (Cruz et al. 2001).The DpHcomponent of pmf was estimated by the inverted signal(ECSinverse) and the DY component was estimated by thedifference between steady-state light and steady-state dark(ECSsteady-state). To investigate how fast the counterionsmoved across thylakoid membrane, the relaxation of theECS from its lowest point (total dissipation of the pmf ) tothe final steady state at the end of 25 s of darkness was fittedby first-order exponential decay.

All ECS signals were calibrated and normalized to therapid (microseconds) rise in 520 nm absorbance upon asaturating, single-turnover xenon flash, measured on dark-adapted leaves before experiments. This normalizationaccounted for changes in leaf thickness and chloroplastdensity between leaves (Takizawa et al. 2007). The xenonflash was provided by an EG&G FX1488 H2-quenched fastdecay flash lamp with a 6 uF discharge capacitor charged at1500 V, giving a pulse power of 13 J and a duration ofapproximately 5 ms at half height. The flash was filteredthrough 1 mm thick Schott BG18 filter. Similar amplitudeswere obtained when the discharge power was decreased by50%, indicating that the flash was near saturation, withminimal multiple hits.

Long light-interval-relaxation-kinetics

Long light-interval-relaxation-kinetics (LIRK, 5 min lightinterval) was measured in dark-adapted, intact tobaccoleaves with the NoFOSpec to estimate the deconvolutionformula. Tobacco leaves dark-adapted overnight wereclamped into the leaf chamber of the NoFOSpec. A darkbaseline was measured for 5 min and then the leaf wasilluminated with 1000 mmol photons m-2 s-1 red actinic lightfor 5 min. Finally, the relaxation during a second dark periodwas measured for 5 min. Upon switching on or off the actiniclight, the change in green light absorption was first domi-nated by the ECS signal. The qE-related signal relaxedquickly between 16 and 20 s after the light was switched off.Zeaxanthin relaxed more slowly, and did not fully recover in5 min. The deconvolution formula was estimated by gettingthe relative extinction coefficients for each component(Zeaxanthin, ECS, qE) at three wavelengths (505, 520 and535 nm) as described (Kramer & Sacksteder 1998).

The ECS signal was deconvoluted with the formula

ECS = A A A1 81 0 17 1 62520 505 535. . . .× − × − ×Δ Δ Δ

The zeaxanthin contribution was deconvoluted by theformula

Zeaxanthin A A A= × − × + ×1 12 0 6 0 8505 520 535. . . .Δ Δ Δ

Long LIRK traces with different actinic light intensities(347, 665 and 1000 mmol photons m-2 s-1) were recordedin dark-adapted leaves at both 23 and 40 °C to test theheat effects on absorbance changes caused by zeaxanthinformation.

For optical measurement of zeaxanthin in light-adaptedleaves, three wavelengths (505, 520 and 535 nm) wererecorded simultaneously before heat at 23 °C, during30 min heat at 40 °C and after returning to 23 °C. Thezeaxanthin signal was deconvoluted by the same formuladescribed previously. Because there is no dark-baseline,the deconvoluted 505 signal from light-adapted leavesis a relative measure but reflects changes of zeaxanthinaccumulation.

Chlorophyll fluorescence quenching analysis

The pulsed measuring beam for fluorescence measurementswas a 535 nm light-emitting diode, with fluorescence beingdetected at >720 nm from the side of the leaf illuminated bythe actinic and measuring lights. The qE and the quantumefficiency of PSII (FPSII) was calculated from saturationpulse-induced chlorophyll a fluorescence yields by theequation:

qE F F F F= ′′ ′ − = − ′ ′m m mPSII1 1, ,Φ

where Fm′ is the maximum fluorescence yield caused by asaturating flash pulse in a light-adapted leaf with 15 minactinic illumination, Fm″ is its recovery after 10 min darkperiod at the same temperature and F ′ is the steady statefluorescence from the light-adapted leaf (Baker et al. 2007).

1540 R. Zhang et al.


Extraction and HPLC analysis of pigments

Tobacco leaf samples treated as previously described wereharvested (7–10 mg) directly into liquid nitrogen. Theywere either light-adapted leaves (illuminated by 500 mmolphotons m-2 s-1 light, harvested before heat, at the end of30 min heat or after heat) or dark-adapted leaves (illumi-nated for 5 min by one of the three light intensities, 347,665 and 1000 mmol photons m-2 s-1, with or without heat,harvested after light was off for 5 min). Pigments wereextracted in methanol: chloroform as described (Tian et al.2003). Reversed-phase HPLC separation and quantifica-tion of carotenoids and chlorophylls was performed asdescribed (Tian & DellaPenna 2001) using a gradientsystem consisting of solvent A, acetonitrile-water-triethylamine (900:99:1, v/v/v), and solvent B, ethyl acetate.50 mL samples were injected onto a C18 SpherisorbODS-2, 250 ¥ 4.6 mm column (Column Engineering,Ontario, CA) equilibrated at 1 mL min-1 flow rate withbuffer A and pigments separated with the following gra-dient: 0–13 min, 0 to 66.7% B; 13–13.2 min, 66.7% to 100%B. One hundred percent B buffer was maintained foran additional 2 min at which time the column wasre-equilibrated with 100% buffer A. Carotenoids weredetected by absorbance using a Shimadzu SPD-M10A VPDiode Array Detector and quantified at 440 nm relative toa 5 point standard curve with purified standards. Racemictocol (Matreya Pleasant Gap, PA, USA) was used as aninternal standard to correct for sample recovery duringextraction and was detected by fluorescence at 290 nmexcitation and 325 nm emission.

RESULTS

Heat effects on contributions of DY and DpH tolight-induced pmf

Heat reduced the extent of the DECS (pmf) because of a lossin the ECSinverse (DpH) but the ECSsteady-state (DY) was notgreatly affected (Fig. 1a). To explore the relationshipsamong these parameters, ECS measurements were madeover a range of light intensities and the averages of threeleaves from different plants are shown in Fig. 1b, c and d.The DECS (pmf) increased with increasing light but at hightemperature it was much reduced compared to the level inleaves at the same light intensity at 23 °C (Fig. 1b). Afterrecovery at 23 °C for 30 min the pmf returned to thepre-stress levels. The DpH increased with increasinglight (Fig. 1c) and this accounted for essentially all of theincreased pmf with light. High temperature caused a verylarge decline in DpH, to less than at any light intensity testedat 23 °C, but again, the effect was reversible. The electricalcomponent (DY) was maximal at 257 mmol photons m-2 s-1

light and declined slightly at higher light intensities(Fig. 1d). Heat caused a small increase in DY.

Heat effects on proton conductance andion movement

In light-adapted leaves the decay of the pmf will resultalmost entirely from proton movement through the ATPsynthase. Therefore, the inverse of the time constant of thepmf decay (tECS) in these light-adapted leaves was taken as

Figure 1. Estimating the components oftransthylakoid proton motive force (pmf)by electrochromic shift (ECS). DECS,relative DpH (ECSinverse) and DY(ECSsteady-state) were measured at 520 nmdeconvoluted by 505 and 535 nm signals.DECS is the full amplitude of the ECS,reflecting the pmf in light. DpH and DYare called ‘relative’ because they weremeasured by absorbance change. Oneunit is equivalent to a change of 0.001OD. Controls (ctrl) were measured in anintact tobacco leaf at 23 °C with five lightintensities (150, 257, 347, 500* and612 mmol photons m-2 s-1) sequentially,followed by heat treatment (heat) at40 °C for 30 min with 500 mmol photonsm-2 s-1 light, finally the leaf was returnedto 23 °C for recovery (rec) for 30 minwith 500 mmol photons m-2 s-1 light. Theasterisk after 500 and the star in thegraph denote the control which had thesame light intensity as heat and recovery.(n = 3, mean � SE)

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a measure of proton conductance (1/tECS) through the ATPsynthase (Kanazawa & Kramer 2002), with smaller tECS

representing bigger proton conductance (Fig. 2a). On theother hand, the dissipation of the ECSinverse likely reflectsnet counterion fluxes (Cruz et al. 2001) and the effectiverate of these ion movements should be reflected in the timeconstant for the recovery of ECSinverse (tion) (Fig. 2b). ThetECS increased with light intensity but was reduced at hightemperature, indicating a significant increase in proton con-ductance with temperature (Fig. 2c). The tion was relativelyinsensitive to light intensity, but even more sensitive to heatthan was tECS, indicating heat-induced increase in ionpermeability or ion content (Fig. 2d).

Heat effects on qE and PSII quantum efficiency

The large effect of heat on the DpH prompted us to examinethe consequences for qE, which is induced by low lumenpH. At 23 °C, both qE and pmf (DECS) increased with lightand the relationship between them was a more-or-less

smooth curve (Fig. 3a). Heating to 40 °C decreased pmf buthad relatively small effects on qE so that the data point forthe heat-stressed condition deviated from the relationshipfound at 23 °C. The relationship of qE as a function of DpH(ECSinverse) at 23 °C was qualitatively similar to that as afunction of pmf.Again, with heat stress the decrease in DpHwas more than the decrease in qE causing the data at hightemperature fall far from the relationship seen at 23 °C(Fig. 3b). FPSII decreased with light intensities at 23 °C(Fig. 4), as reviewed in Baker, Harbinson, Kramer (2007).Heat stress increased FPSII as compared with the controlwith the same light intensity; however, it was completelyrecovered after returning to 23 °C, confirming the finding inlight-adapted, intact Arabidopsis (Zhang & Sharkey 2009).

Heat effects on zeaxanthin accumulation

Another factor affecting qE is the amount of zeaxanthin.We used optical methods to examine whether there werecompensatory increases in zeaxanthin during heating as a

Figure 2. Heat effects on protonconductance and ion movement. tECS isthe time constant of ECS decay duringinitial dark interval, describing how fastprotons can go through the ATP synthase.The inverted ECS signal shown in Fig. 1was plotted up-side-down and the decaytime constant (tion) indicates how fast theions move across thylakoid membrane tocollapse the inverted electrical potentialdue to DpH. Light intensities, 150, 257,347, 500* and 612 mmol photons m-2 s-1,were used for control (ctrl) at 23 °C,500 mmol photons m-2 s-1 light for bothheat treatment (heat) at 40 °C for 30 minand recovery (rec) after returning to23 °C for 30 min. The star in the graphdenotes the control which had the samelight intensity as heat and recovery.(n = 3, mean � SE)

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as well as DpH (ECSinverse) were the sameas described in Fig. 1. The star in thegraph denotes the control which had thesame light intensity as heat and recovery.(n = 3, mean � SE).

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possible explanation for the higher than expected qE. Dark-adapted intact tobacco leaves, which should have only smallamounts of zeaxanthin, were illuminated for 5 min at threedifferent light intensities with or without heat treatment(Fig. 5a).To separate out the ECS and qE-related effects onthe 505 nm signal, the data were deconvoluted using the 520and 535 nm signals. The deconvoluted zeaxanthin signal

reflects the amount of zeaxanthin formed during 5 minlight. Data showed that heat substantially increased theamount of zeaxanthin at all light intensities when theexperiment was started with dark-adapted leaves (Fig. 5a),as has been reported (Havaux & Tardy 1996).The result wasconfirmed by HPLC measurement of zeaxanthin concen-tration in leaves under the same treatments (Fig. 5b). Thezeaxanthin concentration estimated by the deconvoluted505 signal was proportional to that measured by HPLC.There was neither change in chlorophyll concentration(anova, P = 0.8) nor change in total xanthophyll pool(anova, P = 0.4) in all treatments (data not shown). Theamount of antheraxanthin was small with little changecaused by treatments (Fig. 5c) and zeaxanthin plus anther-axanthin showed essentially the same changes as zeaxan-thin only. The amount of violaxanthin showed the oppositetrend as zeaxanthin (Fig. 5d), confirming the complemen-tary loss of violaxanthin.

In light-adapted leaves zeaxanthin levels were followedas temperature was increased from 23 to 40 °C (Fig. 6).Zeaxanthin measured optically (Fig. 6a) and by HPLC(Fig. 6b) showed a small decline at high temperature.Within the noise level, there was little change in zeaxan-thin concentration measured by HPLC going from 23 °Cto 40 °C (P = 0.1). However, the amount of zeaxanthin at40 °C was higher than would be predicted based on thedecreased DpH at high temperature (Fig. 6b). The changein HPLC-measured zeaxanthin concentration follow-ing the temperature switch from 40 to 23 °C was notsignificant (Fig. 6b, P = 0.12) and the recovery level ofthe deconvoluted 505 signal varied from leaf to leaf(Fig. 6a).

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Figure 4. Heat effects on PSII quantum efficiency (FPSII).FPSII was measured by chlorophyll a fluorescence. Thetreatment sets (ctrl, heat, rec) and light intensities were the sameas described in Fig. 1. The star in the graph denotes the controlwhich had the same light intensity as heat and recovery. (n = 3,mean � SE).

Figure 5. Heat effects on zeaxanthinaccumulation in dark-adapted leaves.Three light intensities (347, 665 and1000 mmol photons m-2 s-1) were givenseparately for 5 min in dark-adaptedintact tobacco leaves at either 23 °C (ctrl)or 40 °C (heat). (a) ZeaxanthinDA505,zeaxanthin concentration measured by505 nm light and deconvoluted by 520and 535 nm light signals. (b) (c) (d)Zeaxanthin, antheraxanthin, violaxanthinconcentrations measured by HPLC.(n = 3 ~ 5, mean � SE).

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DISCUSSION

Heating tobacco leaves from 23 to 40 °C, a moderate heattreatment that does not induce permanent damage to thephotosystems, caused a substantial decline in the pH com-ponent of the pmf but a slight increase in the electricalcomponent (Fig. 1). Bukhov et al. (1999) found a largedecline in nine amino acridine fluorescence quenching inisolated spinach chloroplasts as a result of heat pretreat-ment (presumably in darkness) showing a loss of pH gradi-ent in that system that was not reversible. Our results showa similar, but reversible loss of pH gradient in light-adaptedintact leaves. We also found evidence for this effect inlight-adapted intact Arabidopsis thaliana (R. Zhang andT.D. Sharkey, unpublished, one wavelength was used inthese experiments so a proper deconvolution cannot bepresented).

The ATP synthase in light-adapted leaves will be acti-vated and the decay rate of the ECS will reflect pri-marily proton conductance through the ATP synthase(Avenson et al. 2005). The heat-induced increase in proton

conductance in light-adapted leaves (Fig. 2) may reflect aheat-induced activity of ATP synthase, possibly as a resultof heat-increased photorespiration, which increases ATPusage, or heat-induced structural changes in the ATP syn-thase itself. Thylakoid membranes of dark-adapted leaves,where the ATP synthase is deactivated, were found to beleaky at high temperature (Weis 1981; Havaux et al. 1996;Bukhov et al. 1999; Schrader et al. 2004).

In light-adapted leaves the relaxation of the DpH(ECSinverse) was much faster at high temperature thanbefore heat stress, indicating heat-induced ion movement,which may be related to thylakoid leakiness (Fig. 2). If thesemovements also occur in steady state they would collapsethe ion disequilibrium that supports the DpH, and the DpHwould be reduced. This was observed. This increased ionmovement may be one of the earliest effects of heat alteringthylakoid reactions in light-adapted leaves. Light canprotect the photosynthetic apparatus against heat by build-ing up a transthylakoid pmf (Weis 1982), but this protectionis not complete.The heat-reduced DpH could affect not onlyphotosynthetic electron transport, carbon metabolism butalso other reactions that are dependent on lumen pH, suchas qE and zeaxanthin accumulation.

Moderate heat stress reduced both DpH and qE (Fig. 3).However, the extent of qE as a function of DpH was sub-stantially altered at 40 °C, indicating a change in the qEresponse to changes in DpH. The qE response is controlledby the protonation of PsbS, the conformational change ofLHCII and zeaxanthin accumulation. A change in any ofthese factors could lead to an increase in qE response asa function of DpH. A charge-transfer mechanism hasbeen suggested to mediate qE, in which energy is trans-ferred from coupled chlorophyll and zeaxanthin in LHCII(Avenson et al. 2008). The PsbS peptide (Li et al. 2004)was found to interact with CP29, one of the three minorantenna complexes of PSII involving the charge-transfer(Teardo et al. 2007). It is possible that the heat-reducedDpH was sensed by PsbS and then transduced from PsbSto CP29, which alters the conformational change ofLHCII, resulting in altered qE and DpH relationshipunder high temperature.

On the other hand, zeaxanthin may also contribute to theincreased sensitivity of qE to DpH caused by heat, which,in turn, reflects the competition between violaxanthin de-epoxidation, via the lumen pH-dependent violaxanthinde-epoxidase (VDE) (Pfündel & Dilley 1993; Yamamoto& Bassi 1996), and zeaxanthin epoxidation via zeaxanthinepoxidase (ZE) (Takizawa et al. 2007). We found that theextent of zeaxanthin accumulation upon short-term (5 min)light exposure was higher at 40 than at 23 °C in pre-darkadapted leaves (Fig. 5), as reported earlier by Havaux &Tardy (1996). Zeaxanthin did not increase in heat-treatedlight-adapted leaves, however, its amount was far more thanthat predicted by the dramatically decreased DpH underhigh temperature (Fig. 6), indicating that zeaxanthin accu-mulation became more sensitive to DpH with heat treat-ment. It is possible that increased temperature acceleratedthe activity of VDE more than ZE, leading to relatively

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Figure 6. Heat effects on zeaxanthin accumulation inlight-adapted leaves. (a) ZeaxanthinDA505, relative, deconvoluted505 signal from light-adapted leaves, because there is nodark-baseline, it is a relative measure but reflects changes ofzeaxanthin accumulation. (b) ZeaxanthinHPLC, zeaxanthinconcentrations measured by HPLC. Tobacco leaves illuminatedby 500 mmol photons m-2 s-1 light were harvested at 23 °C (ctrl),at the end of 30 min heat at 40 °C (heat), and after returning to23 °C for 30 min (rec). The measurements of DpH (ECSinverse)were same as at that described in Fig. 1. (n = 3, mean � SE)



more accumulation of zeaxanthin even with a reduced DpH.In dark-adapted leaves, 5 min light is not long enough tofully activate photosynthesis. As a result, DpH induces highqE to protect PSII during the introduction phase. However,in light-adapted leaves, photosynthesis as well as otherphysiological processes are fully activated, all of which willconsume pmf and/or DpH in addition to the collapse of DpHby heat-induced ion movements.With a much reduced DpHat high temperature in light-adapted leaves, it is possiblethat the activity of VDE does not exceed ZE as much as itdoes in dark-adapted leaves, resulting in no heat-induced-zeaxanthin in steady light. Despite this, relatively morezeaxanthin is produced at a given DpH with heat treatmentin light-adapted leaves.

The heat-induced sensitivity of zeaxanthin accumulationto DpH could be seen as an adaptive response since zeax-anthin has some other protective functions independent ofqE. First of all, Havaux and others suggested that zeaxan-thin can stabilize thylakoid membranes by reducing theirfluidity (Sarry et al. 1994; Havaux et al. 1996; Havaux 1998).Zeaxanthin can perform like transmembrane ‘rivets’ byanchoring in the membrane bilayer to stabilize membraneand decrease the permeability to some harmful small mol-ecules produced under high temperature (Sarry et al. 1994).The heat-induced thylakoid membrane permeability indark-adapted leaves can be prevented by treatments thatcaused massive conversion of zeaxanthin (Havaux et al.1996) Second, zeaxanthin can act as an antioxidant andprotect lipid peroxidation (Havaux, Dall’Osto & Bassi2007). When incorporated into liposomal membranes,zeaxanthin can act as chain-breaking antioxidant inperoxy radical-mediated peroxidation and deactivatesinglet oxygen as well as scavenging free radicals (Lim et al.1992; Sarry et al. 1994). Recently, it was reported that theantioxidant activity of zeaxanthin and its capacity to protectthylakoid membrane lipids is comparable to that of vitaminE but evidently higher than that of all other xanthophylls(Havaux et al. 2007).

In conclusion, we propose that moderate heat stressreduces the DpH component of transthylakoid pmf in light-adapted intact tobacco leaves, possibly as a result of alteredchloroplast ion balance or increased permeabilities. Despitethe loss of DpH at elevated temperatures, the qE responseis maintained, in part because zeaxanthin accumulationis accelerated at a given pH. This increased zeaxanthinresponse may represent an adaptation to temperaturestress, protecting the photosynthetic apparatus from photo-damage via the qE response and the membranes fromoxidative damage.

ACKNOWLEDGMENTS

The project was supported by the National Research Initia-tive of the USDA Cooperative State Research, Educationand Extension Service, grant number 2004-35100-14860(T.D.S.) and the US Department of Energy (DE-FG02-08ER15955, D.M.K.).

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Received 25 March 2009; received in revised form 10 June 2009;accepted for publication 15 June 2009



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