Effects of low temperature stress on excitation energypartitioning and photoprotection in Zea mays

Leonid V. SavitchA,D,E, Alexander G. IvanovB,D, Loreta Gudynaite-SavitchB,C,Norman P. A. HunerB and John SimmondsA

AAgriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre (ECORC),Central Experimental Farm, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada.

BDepartment of Biology, University of Western Ontario, London, ON N6A 5B7, Canada.CPresent address: Iogen Corporation, 310 Hunt Club Road East, Ottawa, ON K1V 1C1, Canada.DThese authors contributed equally to the writing of this manuscript.ECorresponding author. Email: savitchl@agr.gc.ca

Abstract. Analysis of the partitioning of absorbed light energy within PSII into fractions utilised by PSII photochemistry(FPSII), thermally dissipated via DpH- and zeaxanthin-dependent energy quenching (FNPQ) and constitutivenon-photochemical energy losses (Ff,D) was performed in control and cold-stressed maize (Zea mays L.) leaves. Theestimated energy partitioning of absorbed light to various pathways indicated that the fraction ofFPSII was twofold lower,whereas the proportion of thermally dissipated energy throughFNPQwas only 30%higher, in cold-stressed plants comparedwith control plants. In contrast,Ff,D, the fraction of absorbed light energy dissipated by additional quenchingmechanism(s),was twofold higher in cold-stressed leaves. Thermoluminescence measurements revealed that the changes in energypartitioning were accompanied by narrowing of the temperature gap (DTM) between S2/3QB

� and S2QA� charge

recombinations in cold-stressed leaves to 8�C compared with 14.4�C in control maize plants. These observationssuggest an increased probability for an alternative non-radiative P680+QA

� radical pair recombination pathway forenergy dissipation within the reaction centre of PSII in cold-stressed maize plants. This additional quenchingmechanism might play an important role in thermal energy dissipation and photoprotection when the capacity for theprimary, photochemical (FPSII) and zeaxanthin-dependent non-photochemical quenching (FNPQ) pathways arethermodynamically restricted in maize leaves exposed to cold temperatures.

Additional keywords: cold stress, non-photochemical quenching, PSII photochemistry, thermoluminescence.

Introduction

Environmental changes in temperature, irradiance and otherfactors (e.g. nutrients, water availability) result in imbalancesbetween the light energy absorbed through photochemistry andenergy utilisation through photosynthetic electron transportcoupled to carbon, nitrogen and sulfur reduction. It has beendemonstrated and reviewed that the potential for such energyimbalance in all photosynthetic organisms is significantlyincreased under conditions of cold temperatures, which lead toincreased PSII excitation pressure (Huner et al. 1996, 1998).The increased excitation pressure estimated by the chlorophyllfluorescence parameter 1 – qL (Kramer et al. 2004), measures therelative reduction state of QA, the first stable quinone electronacceptor of PSII. This, in turn, reflects the redox state of theplastoquinone pool and the intersystem electron transport chain(Gray et al. 1996; Huner et al. 1996, 1998). Such an imbalanceimposed by low temperature might lead to photoinhibitionof photosynthesis, which in turn might result in photodamageof the D1 reaction centre polypeptide of PSII (Krause 1988;Aro et al. 1993).

Low temperatures impose strong thermodynamic restrictionson CO2 assimilation in maize (Zea mays L.; Greer and Hardacre1989; Kingston-Smith et al. 1997; Fryer et al. 1998; Foyer et al.2002) and this might also lead to photoinhibition ofphotosynthesis and photodamage of PSII (Nie et al. 1992;Baker 1994). The inhibitory effects of low temperature on thephotosynthetic performance of maize have been extensivelystudied using chlorophyll fluorescence measurements underboth controlled and field conditions (Havaux 1987; Krall andEdwards 1991; Andrews et al. 1995; Massacci et al.1995;Haldimann et al. 1996; Fracheboud et al. 1999; Koscielniakand Biesaga-Koscielniak 2006). As there is a close, althoughnot always linear, correlation between CO2 fixation and thequantum efficiency of PSII in maize (Foyer et al. 2002), cold-induced limitations on photosynthesis, that is, a decrease in thephotochemical use of absorbed light energy, can induce theproduction of potentially dangerous reactive oxygen species(ROS) (Baker 1994) and would require the use of one or moremechanisms fornon-photochemicaldissipationofexcitation lightenergy to prevent photodamage of PSII (Ortiz-Lopez et al. 1990).

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The major mechanism for the thermal dissipation of excesslight energy in higher plants is considered to be the DpH- andxanthophyll-cycle dependent non-photochemical quenching(NPQ) occurring within the light-harvesting antennacomplexes (LHCII) pigment bed of PSII (Demmig-Adams andAdams 1992; Horton et al. 1996). The role of the DpH- andzeaxanthin-dependent shifts in the oligomerisation state ofLHCIIin developing the rapidly relaxing energy-dependent component(qE) of NPQ is well established, with qE representing the majorprotective mechanisms against photoinhibitory damage of PSII(Walters and Horton 1991; Horton et al. 1996; Niyogi 1999;Müller et al. 2001). Non-photochemical quenching (qN or NPQ)can be kinetically resolved into at least three componentscorresponding to different non-photochemical processes(Horton and Hegen 1988; Walters and Horton 1991). Thepredominant and most rapid component is the DpH- or energy-dependent component, qEorNPQf, and itsmagnitude is regulatedby inter-conversion of pigments in the xanthophyll cycle(Demmig-Adams and Adams 1992; Ruban and Horton 1995).A second component, qT, relaxes within minutes because ofstate transitions. The third component identified as qI or NPQs

and exhibiting the slowest dark relaxation kinetics is related tophotoinhibition of PSII and requires light-dependent chloroplastprotein synthesis to repair damaged PSII reaction centrecomplexes (Horton and Hegen 1988; Walters and Horton1991; Krause and Weis 1991).

The partitioning of the light energy absorbed by PSII based onfluorescence quenching analysis in the C4 species maize hasdemonstrated that under suboptimal temperatures the reductionin photochemical quenching (qP) is accompanied by increasedlevels of non-photochemical quenching (Labate et al. 1990; Kralland Edwards 1991; Fryer et al. 1995; Koscielniak and Biesaga-Koscielniak2006). It hasbeendemonstrated that in addition to thezeaxanthin-dependent quenchingof excitation energy,whichwasdetermined to be the most important quenching mechanism, thepersistent depression of photosynthetic efficiency in cold-acclimated maize might also result from a reduced capacity forrepair and/or replacement of damaged PSII centres, although noquantitative analysis has been presented (Fryer et al. 1995).

Despite these studies, no attempts have been made toquantitatively assess the partitioning of the total absorbed lightenergy by PSII in maize, although several models have beenproposed (Demmig-Adams et al. 1996; Hendrickson et al. 2004;Kramer et al. 2004; Kornyeyev and Hendrickson 2007) andsuccessfully applied to various photosynthetic organismsunder a range of environmental conditions (Hovenden andWarren 1998; Niinemets and Kull 2001; Hikosaka et al. 2004;Hendrickson et al. 2005; Ivanov et al. 2006a; Kornyeyev et al.2006). In the present study, chlorophyll fluorescence andthermoluminescence measurements were used to assess theeffects of low temperature stress on the photochemicalefficiency and energy partitioning within PSII as well ascharge recombination events between the acceptor and donorsides of PSII in maize plants exposed to low temperatures. Wereport that cold-stressed maize plants exhibit a higher proportionof constitutive non-photochemical energy losses (Ff,D) than non-stressed controls, which indicates the operation of additionalelectron sink(s) and/or quenching processes for thephotoprotection of maize when exposed to cold stress.

Materials and methodsPlant material and growth conditions

ZeamaysL. plants (inbred line CK44)were grown in a controlledgrowth environment under a 16 h photoperiod with a 25�C/20�C(day/night) temperature regime and an irradiance of 500PPFD. For cold stress, 3-week-old plants were placed for3 days in a 15�C/15�C (day/night) temperature regime and thesame photoperiod and irradiance as the control plants.Chlorophyll a fluorescence and gas exchange measurementswere carried out on 25�C-grown plants at either 25�C or 15�Cand for 15�C-stressed plants at 15�C. All measurements werecarried out on the middle portion of the fourth fully developedleaves of 3-week-old plants. Gas exchange and fluorescencemeasurements were initiated 6 h after the onset of thephotoperiod and continued for 4 h. Within this period of timethe steady-state rates of photosynthesis when measured at theprevailing growth temperature and irradiancewere constant (datanot shown). Themid portion of the fourth fully expanded leafwassampled in the growth chamber under the prevailing growthcondition 8 h after the onset of the photoperiod and quicklyfrozen in liquid N2 for subsequent biochemical analysis.

SDS–PAGE and immunoblotting

Mesophyll chloroplasts were isolated according to Darie et al.(2005). The purity of the mesophyll chloroplasts and the lack ofcontamination by the bundle sheath chloroplast fraction wereevaluated by the estimation of Rubisco activity using thespectrophotometric method described by Sharkey et al. (1991).

Thylakoidmembranes for SDS–PAGE from control and cold-stressedmaize leaves were isolated as described previously (Krolet al. 1999). Benzamidine and aminocaproic acid were present inthe homogenisation buffer at concentrations of 2mM. Thylakoidpreparations were solubilised in a 60mM Tris–HCl (pH 7.8)buffer containing 1mM EDTA, 12% (w/v) sucrose and 2%(w/v) SDS to achieve a SDS : Chl ratio of 20 : 1. Solubilisedsamples containing equal amounts of protein (20mg lane�1)were separated on a 15% (w/v) linear polyacrylamide gelusing aMini-Protean II apparatus (Bio-Rad,Hercules,CA,USA).

Immunoblotting was carried out by transferring the proteinsfromSDS–PAGE to nitrocellulosemembranes (0.2mmpore size;Bio-Rad). The membranes were probed with antibodies raisedagainst PsbA (D1), PsbS and Lhcb5 (AgriSera AB, Vanas,Sweden). The dilutions used were 1 : 2500 for D1, 1 : 5000 forPsbS, 1 : 750 for NDH-H and 1 : 5000 for Lhcb5.

Pigment analysis

The pigments from control and cold-stressed maize leaves wereextracted, separated and quantified by HPLC as describedpreviously (Ivanov et al. 1995; Gray et al. 1996). The systemwas composedof aBeckmanSystemGold programmable solventmodule 126, a diode array detector module 168 (BeckmanInstruments, San Ramon, CA, USA) and an analytical CSC-Spherisorb ODS-1 reverse phase column (5-mm particle size,25 cm� 0.46 cm internal diameter) with an Upchurch Perisorbguard column (both columns from Chromatographic Specialties,Concord, ON, Canada). The samples were injected using aBeckman 210A sample-injection valve with a 20-pL sampleloop. The pigments were eluted isocratically for 6min with a

38 Functional Plant Biology L. V. Savitch et al.

solvent system of acetonitrile : methanol : 0.1 M Tris–HCl(pH 8.0), (72 : 8 : 3.5, v/v/v), followed by a 2-min lineargradient to 100% methanol : hexane (4 : 1, v/v), whichcontinued isocratically for 4min. The total run time was12min and the flow rate was 2 cm3min�1. A11 solvents wereof HPLC grade. The absorbance was detected at 440 nm using adiode array detector and the areas of baseline separated peakswere integratedbyBeckmanSystemGold software.The retentiontimes and response factors of Chl a, Chl b, lutein and b-carotenewere determined by injection of known amounts of purestandards purchased from Sigma (St Louis, MO, USA). Theretention times of zeaxanthin, antheraxanthin, violaxanthin andneoxanthin were determined using pigments purified by thin-layer chromatography as described by Diaz et al. (1990). Theepoxidation state (EPS) of the xanthophyll-cycle pigments in themaize leaf samples was estimated as in Thayer and Bjorkman(1992) using the equation: EPS= (V+ 0.5A)/(V +A+Z), whereV, A and Z correspond to the concentration of violaxanthin,antheraxanthin and zaexanthin, respectively.

Methylviologen treatment and antioxidant content

Exposure to methylviologen (MV) is known to induce oxidativestress through the generation of active oxygen species andsusceptibility to MV treatment is considered to be an indicatorof the capacity for active oxygen scavenging enzymatic systems(Asada 1996). The susceptibility of 25�C-grown and 15�C-stressed Z. mays plants to different MV concentrations wascarried out according to Savitch et al. (2000). Leaf discsmeasuring 1 cm2 collected from the central part of the fourthfully developed leaves of control and cold-stressed plants wereinfiltrated in thedarkunder amildvacuumfor30minwith2mLof0, 0.25, 0.5 or 1.0mM MV solutions. The MV-dependentinhibition of PSII photochemistry was estimated bymonitoring the changes in Fv/Fm after the exposure of leafsamples to either 4 h of darkness or 25�C and 500 PPFD for4 h. The total levels of antioxidantsweremeasured on the samplesfrom 25�C-grown and 15�C-stressed plants collected at theprevailing growth condition by the method of Lado et al.(2004). The ferric reducing ability of plasma (FRAP) methodused in the present study estimated the total antioxidant capacity,that is, the total level of ascorbate, glutathione and a-tocopherolinvolved in non-enzymatic ROS scavenging systems.

Gas exchange measurements and chlorophyll fluorescenceThe CO2 exchange rates and PSII fluorescence parameters weremeasured on the mid portion of the attached, fully expandedfourth leaves using LI-6400 with an attached leaf chamberfluorometer (LI-COR Biosciences, Lincoln, NE, USA). Thisintegrated system gives complete control of the leafenvironment for simultaneous collection of gas exchange andchlorophyll fluorescence data. The relative humidity of the airstream was maintained at 50%. The light response curves of theCO2 assimilation andfluorescence parametersweremeasured at ap(CO2) of 35 Pa and a p(O2) of 21 kPa. The temperature of thechamber was controlled to maintain a leaf temperature of either25�C or 15�C. Gas exchange measurements were made 6 h afterthe onset of the photoperiod. The gas exchange parameters weremeasured after the steady-state rates of CO2 exchange,

transpiration and light-adapted steady-state Fm0 values were

established (15–20min).Chlorophyll a fluorescence of dark-adapted (35min) control

leaves and cold-stressed maize plants was measured at growthtemperatures of 25�C or 15�C under ambient CO2 conditionsusing a PAM 101 chlorophyll fluorescence measuring system(HeinzWalzGmbH, Effeltrich, Germany) (Schreiber et al. 1986)as previously described (Ivanov et al. 1995). The photochemicalfluorescence quenching (qL) and Fv

0/Fm0 parameters were

calculated when the steady-state Fs level was reached. Thenomenclature of van Kooten and Snel (1990) was used for theparameters of Chl fluorescence. The PSII ‘excitation pressure’ orthe relative reduction state of PSII at the growth temperature andgrowth irradiance was measured as 1 – qL (Kramer et al. 2004).Partitioning of the absorbed light energywas estimated accordingto the model proposed by Hendrickson et al. (2004). Theallocation of photons absorbed by the PSII antennae tophotosynthetic electron transport and PSII photochemistry wasestimated as FPSII = 1� (Fs/Fm

0). The quantum efficiencies ofregulated DpH- and/or xanthophyll-dependent non-photochemical dissipation processes within the PSII antennae(FNPQ) were calculated as: FNPQ = (Fs/Fm

0)� (Fs/Fm).Constitutive non-photochemical energy dissipation andfluorescence (Cailly et al. 1996) was calculated as:Ff,D =Fs/Fm.

As the bundle sheath chloroplasts of C4 plants exhibitminimalfunctional PSII and the measured chlorophyll fluorescence wasexcited by week light that was quickly attenuated as it penetratedthe leaf tissue we assumed that the contribution of bundle sheathchloroplasts to the fluorescence signals was negligible and, ingeneral, the fluorescence was elicited from chloroplasts (inmesophyll cells) near the leaf surface. The linear electrontransport rate (ETR) was calculated as the product ofFPSII�PPFD� 0.5� 0.84. The two final terms arise from theassumptions that half of the absorbed photons are allocated to PSIand the absorbance of the leaf is 0.84 and this did not change withtemperature treatment. In the current study, no statisticallysignificant changes were observed in the Chl a/Chl b ratio andChl(a+b) content. This allowed us to assume that the short-termlow temperature treatment in the present study did not affect oraffected only minimally leaf absorptivity and the pattern ofexcitation distribution in the thylakoids.

P700 redox transients

Flash-induced transients of P700 were used to estimate thefunctional fraction of PSII in maize leaf discs essentially asdescribed by Losciale et al. (2008). The redox state of P700was determined in vivo under ambient O2 and CO2 conditionsusing a PAM-101 modulated fluorometer equipped with a dualwavelength emitter-detector ED-P700DW unit and PAM-102units (Heinz Walz GmbH) (Klughammer and Schreiber 1991) asdescribed in detail by (Ivanov et al. 1998). The far-red lightinduced steady-state oxidation of P700 (P700+) wasmeasured bythe absorbance change around 820 nm (DA820–860) in a custom-designed cuvette. Far-red light (FR; lmax = 715 nm, 10Wm�2,Schott filter RG 715) was provided by an FL-101 light source(Heinz Walz GmbH, Effeltrich, Germany). Single turnover (ST)flashes of white saturating light were provided by an XST-103power/control unit (HeinzWalzGmbH) and superimposed on the

Energy partitioning in cold-stressed Zea mays Functional Plant Biology 39

continuous far-red light for transient reduction of the steady-stateP700+ signal. Themeasurementswere carried out at 25�Cor 15�Cfor control and cold-stressed plants, respectively.

Chlorophyll a fluorescence decay measurements

The re-oxidation kinetics of QA� were measured as the decay of

chlorophyll a fluorescence using a PAM fluoremeter aspreviously described (Ivanov et al. 2001). Saturating singleturnover flashes obtained from an XST 103 xenon dischargelamp connected to a PAM 103 unit (Heinz Walz GmbH) wereused to convert all QA toQA

�. The low-intensitymeasuring beamprovided by a pulsed light emitting diode (660 nm) wasmodulated at 100 kHz. The variable fluorescence decay,reflecting the re-oxidation of QA

�, was detected at a resolutionof 20ms. The signals were recorded using a PDA-100 dataacquisition system (Heinz Walz GmbH) installed in an IBM-compatible personal computer.Toavoidpossiblegating artefacts,the first data point used for the analysis was at 150ms after theactinic flash reached its maximum intensity. Data from at least 10recordings were averaged. The final curve fitting was performedby a non-linear data analysis using aMicrocal Origin Version 6.0software package (Microcal Software, Northampton,MA,USA).

Thermoluminescence measurements

Thermoluminescence (TL) measurements of intact control andcold-stressed maize leaves were carried out on a personal-computer-based TL data acquisition and analysis system aspreviously described (Ivanov et al. 2001). A xenon-dischargeflash lamp (XST103; HeinzWalz GmbH) was used to expose thesamples to a single turnover flash (1.5ms peak width at 50% ofmaximum). Dark-adapted leaves (30min at 20�C or 10�C) werecooled to 2�C before exposure to the flashes. For the S2QA

�

recombination studies, the leaves were vacuum infiltrated withDCMU (20mM) in darkness before the flash illumination. Thenomenclature ofVass andGovindjee (1996) andSane (2004)wasused for characterisation of the TL glow peaks. The experimentswere performed at a heating rate of 0.6�C s�1.

Statistical analysis

The data presented are the means of three independent biologicalexperiments, with 3–10 replicates for each experiment (�s.e.).The number of replicates for eachmeasurement is provided in thetable descriptions and figure legends. Statistical analysis of thedata was performed using the ANOVA statistics package inMicrosoft Excel 2002 (Microsoft Corporation, Redmond, WA,USA).

Results

Gas exchange and chlorophyll fluorescence

Exposure ofmaize plants to a cold temperature of 15�C for 3 dayssignificantly decreased the photochemical efficiency of PSII andtheFv/Fm valueswere almost 50% lower (0.41� 0.01) comparedwith the control plants (0.77� 0.01). The light response curves ofCO2 assimilation measured at ambient CO2 (35 Pa) and O2

(21 kPa) also exhibited the expected effects of the measuringtemperature and cold stress in control maize plants grown at25�C. The CO2 assimilation rate was 2.5-fold lower at 15�Ccompared with the photosynthesis rate measured at the growth

temperature of 25�C (P< 0.0001). Plants exposed to cold stress at15�C for 3 days exhibited slightly lower photosynthesis whenmeasured at the same temperature (Fig. 1A). These datacorresponded well with the ETRs and as expected the ETR inthe control plants was strongly reduced at the lower measuringtemperature. However, cold-stressed plants exhibited twofoldlower light-saturated ETRs compared with control plantsmeasured at 15�C (Fig. 1B) (P < 0.0001).

As expected, decreasing themeasuring temperature from25�Cto 15�C increased the excitation pressure (1 – qL) in the leaves ofthe control plants irrespective of the measuring irradiance(Fig. 1C). A similar increase in excitation pressure wasobserved for cold-stressed plants (Fig. 1C, closed symbols).Thus, although cold-stressed plants exhibited twofold lowerlight-saturated ETRs compared with the control plantsmeasured at 15�C (Fig. 1B), the response of excitationpressure to cold stress was similar to that in the control plantsmeasured at 15�C(Fig. 1C).This indicates that excitationpressuremight account, in part, for the lower temperature suppression inphotosynthetic rates (Fig. 1A), but it cannot account for theapparent differences in the light response curves for electrontransport between cold-stressed plants and control plantsmeasured at 15�C.

Interestingly, the relationship between the parameterFv/Fm�Fv0/Fm0, reflecting the level of energy dissipationwithin the PSII antennae (Demmig-Adams et al. 1996), and qN,representing all non-photochemical dissipation processes, clearlydemonstrated that the proportion of DpH- and/or zeaxanthin-dependent energy quenching occurring in the LHCII is muchlower in cold-stressed (3 days) maize leaves than in controlleaves at either 25�C or 15�C (Fig. 1D) (P< 0.0001).

Carotenoid composition and immunoblotting

In contrast to previous reports (Haldimann et al. 1995; Haldimann1998), the quantitative analysis of pigment compositiondemonstrated only a minor (11%) reduction in total chlorophyllcontent and no changes in the Chl a/b ratio in 3-day cold-stressedmaize leaves (Table 1). However, as previously reported (Fryeret al. 1995; Haldimann et al. 1995; Haldimann 1997), exposureto cold stress induced a sharp increase in the amount of thexanthophyll cycle pigments antheraxanthin and zeaxanthin andminimal changes in the amounts of neoxanthin, lutein andb-carotene (Table 1). As a consequence, the total xanthophyllcycle pool size (V+A+Z) and the EPS of the xanthophyllcycle pigments were increased by 64% and 43%, respectively, incold-stressed leaves compared with control plants. However,although the amounts of antheraxanthin and zeaxanthin wereincreased by ninefold and 12.2-fold, respectively, the reductionin violaxanthin was unexpectedly small and only a small portion(22%) of violaxanthin appears to be enzymatically converted toantheraxanthin and zeaxanthin through the xanthophyll cycle incold-stressed leaves (Table 1). Thus, it appears that the increasedamount of xanthophyll cycle pigments in cold-stressed leavesprobably results from de novo synthesis of antheraxanthin andzeaxanthin rather then de-epoxidation of violaxanthin.

Interestingly, the relative abundance of Lhcb5 (Fig. 2) and theother LHCII proteins (data not shown) as well as PsbS protein,which are suggested to be the major binding sites for the

40 Functional Plant Biology L. V. Savitch et al.

xanthophyll cycle pigments (Horton et al. 1996; Niyogi 1999),exhibited minimal changes as a result of exposure to 3 days ofcold stress (Fig. 2). In contrast, the apparent relative abundanceof D1 decreased by at least 50% after cold stress comparedwith the controls (Fig. 2). Furthermore, cold stress appeared toinhibit LHC phosphorylation, but stimulate D1 phosphorylation(Fig. 2).

Estimation of the functional fraction of PSII

Exposure ofmaize plants to cold stress significantly decreases thephotochemical efficiency of PSII measured as Fv/Fm and therelative abundance of the PSII reaction centre protein D1. Inaddition, we applied a recent method for in vivo determination ofthe functional fractionofPSII (Losciale et al. 2008).The chemicalreduction of far-red light induced P700+ signal by a ST flash ofwhite light followed by its re-oxidation to the steady-state level(Fig. 3) characterises the transient electron flow arriving fromPSII and represents a reliable and accurate measure of thefunctional PSII complexes in various C3 and C4 plants(Losciale et al. 2008). The traces presented in Fig. 3 clearlydemonstrate that the ST-flash-induced reduction of the P700+

signal in cold-stressed plants is much shallower than thatobserved in control maize. This indicates that a much smallernumber of electrons are produced in cold-stressed plants,

0

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A (

μmol

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s–1

)

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)

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(B)

0 800 1600

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Light irradiance (μmol m–2 s–1)

qN0.25 0.50 0.75

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Fv/F

m –

Fv′

/Fm

′

(C)

(D)

Fig. 1. Effects of light irradiance and low temperatureon (A) the rates ofCO2

assimilation, (B) linear electron transport, (C) PSII ‘excitation pressure’(1 – qL) and (D) the relationship between the total level of energydissipation in the PSII antennae and the combined DpH-dependent andDpH-independent non-photochemical quenching of chlorophyllfluorescence (qN) in Zea mays plants. Light response curves of CO2 gasexchange and chl a fluorescence parameters weremeasured on control (25�C)leaves grown at either 25�C (open symbols) or 15�C (triangles) and on cold-stressed (15�C for 3 days) leaves at 15�C (solid circles). The data represent theaverage of three independent biological experiments (mean� s.e.; n= 4 foreach experiment).

Table 1. Photosynthetic pigment composition and content of control(258C) and cold-stressed (158C for 3 days) maize leaves

Pigmentswere separated andquantifiedbyHPLC.The results are presented asmg pigment per m2. EPS= (V+ 0.5A)/(V+A+Z). The xanthophyll pool size(V+A+Z) was calculated as the sum of violaxanthin, antheraxanthin andzeaxanthin. Mean values� s.e. were calculated from three replicatemeasurements in three independent experiments (n= 9). Asterisks denotesignificant pigment content differences between control and cold-stressedmaize plants at ***P< 0.0001 and **P< 0.005 according to an ANOVA

(single factor) analysis. ns, not significant

Pigments (mgm�2) Control Cold stressed

Neoxanthin 9.5 ± 0.4 8.9 ± 0.5 (ns)Violaxanthin 16.6 ± 1.4 12.9 ± 1.4 (ns)Antheraxanthin 0.7 ± 0.1 6.4 ± 0.6 ***Lutein 42.1 ± 2.5 44.7 ± 3.1 (ns)Zeaxanthin 0.8 ± 0.1 10.0 ± 0.7 ***b-Carotene 35.7 ± 2.7 31.8 ± 3.5 (ns)Chl (a+ b) 544 ± 23 485 ± 13 (ns)Chl a/Chl b 4.19 ± 0.09 4.46 ± 0.24 (ns)V+A+Z 17.8 ± 1.6 29.3 ± 2.2 **EPS 0.96 ± 0.01 0.55 ± 0.02 ***(V+A+Z)/Chl (a+ b) 0.032 0.060 ***

Energy partitioning in cold-stressed Zea mays Functional Plant Biology 41

corresponding to a lower (45.5� 6.2%, n= 10) number offunctional PSII centres than in control plants.

Mehler reaction and antioxidants

The Mehler peroxidase reaction and ascorbate are known to playan important role in the modulation of thylakoid membraneenergisation and non-photochemical quenching of chlorophyllfluorescence in maize mesophyll chloroplasts (Ivanov andEdwards 1997, 2000). If O2 is to be utilised as an alternativeelectron acceptor, onewould expect a concomitant increase in thecapacity of active oxygen scavenging enzymatic systems andantioxidants to counteract the potential for photooxidativedamage. Exposure to MV is known to induce oxidative stressthrough the generation of active oxygen species; thus, thesusceptibility of PSII photochemistry to MV treatment isconsidered to be an indicator of the capacity for active oxygenscavenging enzymatic systems (Asada 1996; Savitch et al. 2000).The results of Fig. 4A support this thesis because maize plantscold stressed for 3 days exhibited the higher tolerance of PSIIphotochemistry toMV-inducedoxidative stress. These results are

in agreement with previous reports showing that the relative fluxof reducing equivalents to O2 via the Mehler reaction is higherwhen maize plants are subjected to low temperatures (Fryer et al.1998). The cold-stress-induced decrease in susceptibility of PSIIphotochemistry to MV treatment correlated with a threefold

25°CMW

kDa

36.8

35.8

29.2

29.0

49.8

35.8

PhThr D1

LHCII

NDH-H

PsbS

Lhcb5

D1

15°C

Fig. 2. Representativewestern blots of SDS–PAGE-separated polypeptidesof thylakoidmembranes isolated from control (25�C) and cold-stressed (15�Cfor 3 days) Zea mays leaves probed with antibodies raised against D1, Lhcb5,PsbS and NDH-H proteins. The phosphorylation pattern of D1 and LHCIIpolypeptides was probed with commercial polyclonal antibody againstphosphothreonine (PhThr).

0 50 100 150 200–0.20

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Fig. 3. Flash-induced transient kinetics of the P700 redox state in control(solid line) and cold-stressed (dotted line) Zea mays leaf discs. The singleturnover (ST) flash was given when a far-red (FR) light induced P700+ signalreached a steady-state level. The data represent the average of threeindependent biological experiments (n= 8–9 for each experiment).

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mol

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)(B)(A)

Fig. 4. Effects of light irradiance and low temperature on the capacity of thereactive oxygen scavenging enzymatic systems and total pool size ofantioxidants (‘antioxidant power’) in leaves of Zea mays plants. Thesusceptibility of plants to (A) methylviologen (MV)-dependent inhibitionof PSII photochemistry and (B) the total pool size of antioxidants wasmeasured on 25�C grown (open circles, open bars) and 15�C stressed(solid circles, filled bars) plants. The data represent the average of threeindependent biological experiments (mean� s.e.; n= 6 for each experiment).The differences between the control and cold-stressedmaize plants in (A)weresignificant at P< 0.05, P< 0.005 and P< 0.05, respectively, at 0, 0.25 and0.5mM concentrations of MV and in (B) the differences were significant atP< 0.0001 according to an ANOVA (single factor) analysis.

42 Functional Plant Biology L. V. Savitch et al.

increase in the pool size of total antioxidants (Fig. 4B), which isconsistent with previous results for cold-treated maize plants(Fryer et al. 1998; Kingston-Smith et al. 1999). As thecontribution of bundle sheath chloroplasts to the fluorescencesignals is negligible, the observed Fv/Fm changes in response toparaquat treatment can be attributed mostly to the effect of ROSon the thylakoid membranes of Z. mays mesophyll chloroplasts.Concomitantly, the increased tolerance to paraquat induced byshort-term low temperature can be attributed to the stimulation ofROS scavenging by either enzymatic or non-enzymatic(ascorbate, glutathione and a-tocopherol) systems in thechloroplasts of the mesophyll cells. The FRAP method used inthe present study estimated the total antioxidant capacity, that is,the total level of ascorbate, glutathione and a-tocopherol,involved in non-enzymatic ROS scavenging systems. As mostof the ascorbate and glutathione has been reported to be located inthe mesophyll chloroplasts of Z. mays at either normal orsuboptimal temperatures (Pastori et al. 2000), we made anassumption that the results of the FRAP assay might be treatedas an indication of antioxidant capacity in the mesophyllchloroplasts. A detailed analysis of the ROS scavengingsystems in Z. mays affected by low temperature has beenpresented previously (Fryer et al. 1998; Kingston-Smith et al.1999; Pastori et al. 2000) and was part of the detailed analysis inthe current study.

Energy partitioning

Further analysis of the partitioning of absorbed light energy intofractions utilised by PSII photochemistry (FPSII), thermallydissipated via DpH- and xanthophyll-dependent energyquenching (FNPQ) and the fraction of absorbed light goingneither to FPSII nor FNPQ, that is, the sum of all otherprocesses involved in non-photochemical energy losses and/orfluorescence within PSII reaction centres with QA in the reducedstate (Ff,D), was provided according to the model proposed byHendrickson et al. (2004). The estimated energy partitioning ofabsorbed light to the various pathways indicated that the fractionutilised by photochemistry (FPSII) was significantly lower incold-stressed plants and this effect was independent of lightintensity (Fig. 5A) (P < 0.0001). Lowering the measuringtemperature had a similar effect on the control plants, but theFPSII fraction remained higher than in the cold-stressed leaveswithin the entire light irradiance range (P < 0.05). Concomitantly,although the proportion of thermally dissipated energy throughDpH-regulated non-photochemical quenching (FNPQ) in cold-stressed leaves was higher at lower irradiance compared with thecontrol (P < 0.001), control plants measured at low temperatureexhibited evenhigherFNPQ than either cold-stressed (P < 0.01) orcontrol plants (P< 0.0001) (Fig. 5B). In contrast,Ff,D, that is, thefraction of absorbed light energy dissipated by additionalquenching mechanism(s) was twofold higher in cold-stressedleaves and this effect was independent of the measuring lightirradiance (Fig. 5C) (P < 0.0001).

Fluorescence decay

Thedecay of variable chlorophyllfluorescencemonitoring the re-oxidation of QA

� after a single saturating flash of dark-adaptedcontrol and cold-stressed maize leaves (Fig. 6A) exhibited

complex kinetics that could be resolved into three distinctdecay components (Table 2) similar to those previouslyreported (Cao and Govindjee 1990; Govindjee et al. 1992;Sane et al. 2003). The estimated decay half-times for the firsttwo phases (fast and medium) of QA

� re-oxidation in controlmaize leaves were 347 and 1330ms, accounting for 38.0 and42.4% of the decay, respectively. The slow phase, presumablyassociated with a back reaction of QA

� with the S2-states incentres in which QA is poorly connected to QB and the PQ pool(Etienne et al. 1990), exhibited a half-time ranging from 0.5 to 2 sand accounted for the remaining 19.6% of the decay. In contrast,

Light irradiance (μmol m–2 s–1)

0.2

0.4

0.6

ΦP

SII

(1 –

Fs/F

m′)

0.2

0.4

0.6

ΦN

PQ

Fs/F

m′ –

Fs/F

m

0 800 1600

0.3

0.4

0.5Φ

f,D F

s/F

m

(A)

(B)

(C)

Fig. 5. Light intensity dependence of the energy partitioning in control(25�C) Zea mays leaves measured at 25�C (open circles) and 15�C (triangle)and in cold-stressed (15�C for 3 days) leaves (solid circles). The fractions ofabsorbed irradiance consumed via (A) PSII photochemistry (FPSII), (B) DpH-and xanthophyll-regulated thermal dissipation (FNPQ) and (C) constitutiveenergy quenching processes involved in non-photochemical energy losses(Ff,D) were estimated according to the model proposed by Hendrickson et al.(2004). The data represent the average of three independent biologicalexperiments (mean� s.e.; n= 4 for each experiment).

Energy partitioning in cold-stressed Zea mays Functional Plant Biology 43

the fast andmedium components of theQA� re-oxidation in cold-

stressed leaves were significantly slower than those observed inthe control leaves (Fig. 6B; Table 2). The fast component

accounted for 52.1% of the fluorescence decay, whereas themedium phase (36.1%) was comparable to that recorded in thecontrol leaves.

Thermoluminescence

Recently, non-radiative dissipation of excess light energy withinthe reaction centre of PSII (reaction centre quenching) occurringthrough reversible changes in the redox properties of the twoquinone acceptors of PSII has been proposed as an additionalquenching mechanism supplementing the antenna-based non-photochemical quenching under various environmentalconditions in a wide range of photosynthetic organisms(Ivanov et al. 2003, 2006b; Sane et al. 2002, 2003). The redoxstate of the electron transport components of the PSII acceptorside (QA and QB) in intact leaves of control and cold-stressedmaize plants was studied by TL measurements. Thesemeasurements provided reliable information on the activationenergies associated with the back reactions of electron acceptors(QA and QB) with the electron donors (S2 and S3) and the redoxpotentials of theparticipatingoxidisedand reduceddonorsofPSII(Ducruet 2003; Ducruet et al. 2007). The temperature maxima(TM) of the TL emission peaks after excitation with two singleturnover flashes of white light in combination with DCMU toblock electron flow between QA and QB reflect the chargerecombinations of S2QA

�, S3QA�, S2QB

� and S3QB� redox

pairs (DeVault and Govindjee 1990; Inoue 1996; Sane 2004).Typical TL emission glow curves obtained following

excitation with two successive single turnover flashes in intactleavesof control andcold-stressedmaizeplants in the absenceandpresence of DCMU are presented in Fig. 7. The TL glow curve ofthe control leaves in the absenceofDCMUexhibited a peakwith acharacteristicTMof 42.6�C (Fig. 7A; Table 3). The band at 42.6�Cwas greatly decreased in the DCMU-treated leaves and hencerepresents the S2/S3QB

� charge recombination (Vass andGovindjee 1996; Sane 2004). In the presence of DCMU, theoverall TL emissionwasmuch lower and yielded a newpeakwithaTMof28.3�C(Table3),whichwas assigned to theS2QA

� chargerecombination (Vass and Govindjee 1996; Sane 2004). Theoverall TL emission of cold-stressed maize leaves in theabsence of DCMU was significantly lower and the TM of theS2/S3QB

� charge recombination was down-shifted by 3�C(Fig. 7B; Table 3) compared with the control leaves. Incontrast to the control leaves, the S2QA

� peak in cold-stressedleaves appeared at slightlyhigher temperatures than in the control.Consequently, the temperature gap (DTM) between the S2/S3QB

�

and S2QA� charge recombinations in the cold-stressed leaves

(DTM= 8.0�C) was ~6�C narrower than in the control leaves(DTM= 14.4�C) (Table 3).

Discussion

Several models based on the quenching parameters derived fromchlorophyll fluorescence measurements have been proposed toquantify the partitioning of total absorbed light energy by PSII(Demmig-Adams et al. 1996; Hendrickson et al. 2004; Krameret al. 2004; Kornyeyev and Hendrickson 2007). Althoughdifferent approaches, pure ‘puddle’ (Demmig-Adams et al.1996) or ‘lake’ antenna models of PSII (Hendrickson et al.2004; Kramer et al. 2004), have been used to estimate the fate

Time (ms)

Time (ms)

0 2 4 6 8 10 12 14

0.0

0.2

0.4

0.6

0.8

1.0

Var

iabl

e ch

loro

phyl

l flu

ores

cenc

eF

luor

esce

nce

(r. u

.)

0 5 10 15 20 25

140

160

180

Fo

Fm

Fm

(A)

(B)

Fig. 6. (A) Chlorophyll fluorescence decay kinetics after single turnoverflash illumination in leaves of Zea mays plants grown under control (25�C,solid line) and cold-stressed (15�C for 3 days, dotted line) conditions. (B) Thelines are exponential fits for the experimental data points. Experimentalfluorescence curves were normalised to the corresponding Fm values andrepresent averages fromthe three independentbiological experiments (n= 8–9for each experiment).

Table 2. Kinetic parameters of variable fluorescence yield decay incontrol (258C) and cold-stressed (158C for 3 days) maize leaves

Exponential analysis yielded triphasic kinetics with different half times (t1/2)and amplitudes (A). Mean values� s.e. were calculated from 8 to 13recordings. Asterisks denote significant differences between parameters incontrol and cold-stressed maize plants at ***P < 0.0001 and **P< 0.005

according to an ANOVA (single factor) analysis. ns, not significant

Parameters Control Cold stressed

t1/2fast (ms) 347 ± 29 579± 86 **t1/2int (ms) 1330 ± 110 3660± 590 ***t1/2slow (ms) >800 >800Afast (%) 38.0 ± 4.8 52.1 ± 1.5 **Aint (%) 42.4 ± 3.8 36.1 ± 4.0 (ns)Aslow (%) 19.6 ± 2.4 10.0 ± 3.5 **

44 Functional Plant Biology L. V. Savitch et al.

of absorbed light energy, all models have defined the sum of allenergy fluxes via PSII as unity and have categorised thepartitioning of total absorbed energy by PSII as eitherphotochemical or thermal dissipation processes. In addition, allthreemodels, regardless of their underlying assumptions, are ableto recognise two easily distinguishable thermal dissipationprocesses, the major one originating from the LHCII anddefined as regulated NPQ (FNPQ) and an additional one

defined as constitutive thermal dissipation (FNO, Ff,D)(Hendrickson et al. 2004; Kramer et al. 2004) or ‘excessexcitation energy’ (E) defined as the fraction of absorbed lightneither going to photochemistry nor to regulated NPQ, that is, theexcitation energy trapped and/or dissipated by PSII reactioncentres with QA in a reduced state (Demmig-Adams et al. 1996).

In thepresent study, thepartitioningof the absorbed light to thevarious pathways in control and cold-stressed maize leaves wasestimated according to themethod ofHendrickson et al. (2004). Itis important to note that as agranal chloroplasts of bundle sheathcells of C4 plants lack grana and bundle sheath thylakoids containextremely low amounts (if any) of PSII complexes (Woo et al.1970; Anderson et al. 1971), the analysis of energy partitioningbased onmodulatedfluorescencemeasurement inC4 plants refersonly to the energy fluxes occurring in themesophyll chloroplasts.

In agreement with previous observations (Greer and Hardacre1989; Kingston-Smith et al. 1997; Fryer et al. 1998; Foyer et al.2002), the exposure ofmaize plants to 3 days of cold stress causeda significant decrease in the photochemical efficiency of PSII(Fv/Fm), the fraction of functional PSII complexes (Fig. 3) andCO2 assimilation and ETRs (Fig. 1). As expected, these effects atthe maximum photon irradiance were associated with a twofolddecrease in FPSII, the fraction of light energy utilised throughphotochemistry (Fig. 5A). This was accompanied by a 30%increase in FNPQ (Fig. 5B), the fraction of the regulated DpH-and zeaxanthin-dependent thermal dissipation process(es)originating from the LHCII, which has been identified as thepredominant quenching process explaining the reducedphotosynthetic efficiency in maize exposed to lowtemperatures (Labate et al. 1990; Krall and Edwards 1991;Fryer et al. 1995). However, it is quite difficult to explain thegreater than twofold decrease in CO2 assimilation and ETRs(Fig. 1A,B) by the observed rathermodest 30% increase inFNPQ.

Concomitantly, the proportion of constitutive thermaldissipation (Ff,D), that is, the fraction of absorbed light energyneither utilised through photochemistry (FPSII) nor FNPQ, wasalmost twofold higher and constituted almost 50% of the totalenergy flux in cold-stressed leaves compared with control leaves(Fig. 5C). This demonstrates that the upregulation of constitutiveenergy dissipation (Ff,D) might compensate for the relativelymodest increase in regulated NPQ (FNPQ) in cold-stressed maizeleaves. Thus, the availability of alternative electron sink(s) and/oradditional quenching mechanism(s) might play an important rolein the dissipation of excess light energy when the capacity for theupregulation of the primary, zeaxanthin-regulated non-photochemical quenching (FNPQ) pathway is restricted at lowtemperatures. The following data are consistent with thisinterpretation.

First, the greater functional stability of PSII in cold-stressedmaize leaves in the presence of MV (Fig. 4A) indirectly indicatesthat the Mehler reaction might effectively serve as an additionalelectron sink, contributing to non-photochemical dissipation ofexcess light in maize plants (Ivanov and Edwards 1997, 2000).Furthermore, cold stress in maize also induced higher levels oftotal antioxidants compared with the control plants (Fig. 4B).These observations are consistent with the proposed role of theMehler reaction and antioxidants as an additional electron sinkalleviating photooxidative damage in cold-acclimated plants(Leipner et al. 1997; Savitch et al. 2000).

S2/3QB–

S2QA– (+ DCMU)

ΔTM

ΔTM

(A)

(B)

10 20 30

Temperature (°C)

The

rmol

umin

esce

nce

(rel

ativ

e un

its)

40 50

Fig. 7. Thermoluminescence glow curves of control (solid lines) andDCMU (20mM) treated (dashed lines) leaves of Zea mays plants grownunder (A) control (25�C) conditions and (B) after 3 days cold (15�C) stressafter illuminationwith two single turnoverflashes. The presented glow curvesare averages of 3–5 measurements in three independent biologicalexperiments.

Table 3. Characteristic thermoluminescence peak emissiontemperatures (TM) of the S2/S3QB

� and S2QA� charge recombinations

of control (258C) and cold-stressed (158C for 3 days) maize leavesThe samples were cooled to 28C and illuminated with two singleturnover flashes of white light. The thermoluminescence glow curves wereobtained immediately after illumination. Mean values� s.e. were calculatedfrom 5–8 measurements in two or three independent experiments. Themeasurements were carried out in the presence and absence of 20mM

DCMU. DTM=TM(S2/3QB�)� TM(S2QA

�)

Sample S2/S3QB

TM (�C)S2QA

TM (�C)DTM(�C)

Control (25�C) 42.7 ± 0.3 28.3 ± 0.1 14.4 ± 0.2Cold stressed (15�C for 3 days) 39.5 ± 0.2 31.5 ± 1.4 8.0 ± 0.7

Energy partitioning in cold-stressed Zea mays Functional Plant Biology 45

Second, it has been proposed that the dissipation of excesslight energy within the PSII reaction centre via shifts in the redoxpotentials of the electron acceptors QA and QB might serve as anadditional quenching mechanism in cold-acclimated plants(Ivanov et al. 2003). Recently, TL measurements haverevealed that narrowing the temperature gap (DTM) betweenthe S2/3QB

� and S2QA� charge recombinations increases the

probability for an alternative non-radiative P680+QA� radical

pair recombination pathway for energy dissipation within thereaction centre of PSII (Sane et al. 2002, 2003; Ivanov et al. 2003,2006b). Indeed, PSII reaction centre quenching of excess lightwas suggested to play a substantial role in supplementing theantenna-basedNPQ in cold-acclimated plants (Ivanov et al. 2003,2006b) when the enzymatic conversion of violaxanthin tozeaxanthin within the xanthophyll cycle is thermodynamicallyrestricted by low temperatures. The existence of such anadditional quenching mechanism is also consistent withprevious observations that significant levels of NPQ can occurindependent of zeaxanthin (Demmig-Adams et al. 1999; Finazziet al. 2004) and cannot be accounted for by antenna quenching(Kramer et al. 2004).

The downshift in the TM of the S2/3QB� charge recombination

in our study is in agreement with earlier observations for cold-stressed maize leaves (Janda et al. 2000, 2004). The shift in theS2/3QB

� characteristic peak to lower temperatures has beenattributed to the build-up of a proton gradient and to areduction in the PQ pool (Miranda and Ducruet 1995). Indeed,it has been reported that maize mesophyll chloroplasts containlarge pools of electrons that can be donated via theNDH complexto the intersystem PQ pool from stromal or cytosolic reductants(Asada et al. 1993). The large stromal electron pool available inmaize (Asada et al. 1993) and the observed higher abundance ofNDH complex (Fig. 2) in the chloroplasts of cold-stressed maizesuggests a higher reduction state of the PQ pool in leaves exposedto low temperatures compared with dark-adapted controls wherethe PQ pool is in a predominantly oxidised state. The increasedexcitation pressure (1 – qL) values (Fig. 1) and the retardedfluorescence decay kinetics used as a measure of the dark re-oxidation of the primary electron acceptor QA (Mohanty et al.1991; Govindjee et al. 1992) (Fig. 6), indicated that QA is in amore reduced state in cold-stressed leaves. As QA is in quasi-equilibrium with QB and the PQ pool, our results strongly implythat the PQ pool is indeed in a predominantly reduced state indark-adapted, cold-stressed maize and this might explain thedownshift in the S2/3QB

� charge recombination. Narrowing ofthe temperature gap (DTM) between the S2/3QB

� and S2QA�

charge recombinations in cold-stressed maize (Fig. 7; Table 3) isaccompanied by an increased proportion of constitutive non-photochemical quenching (Ff,D) (Fig. 5C) and suggests thatdissipation of excess light energy within the remainingfunctional reaction centres of PSII might well serve as anadditional quenching mechanism and supplement the regulatedNPQ. This is in agreement with an earlier study also suggestingthe possible involvement of reaction-centre type non-photochemical fluorescence quenching in maize mesophyllchloroplasts under ascorbate-deficient conditions (Ivanov andEdwards 2000). Ascorbate is required for both the Mehlerperoxidase reaction and violaxanthin de-epoxidase convertingviolaxanthin to zeaxanthin within the xanthophyll cycle

(Neubauer and Yamamoto 1994). Considering that bothprocesses are upregulated in cold-stressed leaves(Table 1; Fig. 4), limited availability of ascorbate could beexpected in maize plants exposed to low temperatures. Indeed,a decreased amount of ascorbate and other antioxidants(glutathione and a-tocopherol) was reported in maize plantsexposed to chilling stress under high light (Leipner et al.1997). Moreover, a zeaxanthin-independent process forthermal energy dissipation was suggested in maize plantsexposed to low temperatures and high irradiance (Leipneret al. 2000).

In addition, theglowcurves and the shift in theS2/3QB�peak to

a lower temperature resemble the TL pattern in photoinactivatedPSII centres (Ohad et al. 1990; Ono et al. 1995; Walters andJohnson 1997). In addition, our data demonstrate a lowerabundance of the D1 reaction centre protein of PSII (Fig. 2)and a 45.5% lower number of functional PSII centres (Fig. 3) inplants exposed to a low temperature for 3 days compared withcontrol plants, indicating a low temperature photoinactivationand degradation of PSII in cold-stressed leaves. One might arguethat the higher constitutive energy dissipation (Ff,D) and thedecreased temperature gap (DTM) between S2/3QB

� and S2QA incold-stressed plants could be attributed to the higher proportion ofinactive and/or damagedPSII centres comparedwith the controls.Indeed, ourdatademonstrated a lower (45.5%) functional fractionof PSII in cold-stressed plants (Fig. 3), although inactive PSIIcentres have previously been demonstrated to constitute ~20% incold-grown maize (Fryer et al. 1995) compared with controlplants where the inactive centres have been reported to be 12% ofthe entire PSII population (Chylla and Whitmarsh 1989). In anearlier study, the damaged or inactive PSII reaction centres wereproposed to function as efficient energy traps (Giersch andKrause1991). More recently, these damaged PSII complexes have beensuggested to function as strong quenchers of excess lightexcitation, effectively dissipating the excess energy via non-radiative charge recombination between QA

� and P680+

(Matsubara and Chow 2004; Sun et al. 2006). We suggest thatsuch amechanismmight also provide effective energydissipationand photoprotection of the remaining functional PSII units. Theexistence of two distinct PSII populations, which mightcontribute to the constitutive energy dissipation (Ff,D) viaenhanced radical pair recombinations requires further detailedstudies to quantify precisely the effects of cold stress on thecapacity for alternative non-photochemical processeswithin bothnon-active and active PSII reaction centres in maize leaves.

Acknowledgements

This work was supported by the OCPA/AAFC Matching InvestmentInitiative, the Canadian Crops Genomics Initiative and NSERC Canada.

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Manuscript received 21 March 2008, accepted 4 October 2008

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