Plant Physiol. (1989) 90, 1041-10480032-0889/89/90/1041 /08/$01 .00/0

Received for publication September 27, 1988and in revised form February 20, 1989

Enhancement of Thermal Injury to Photosynthesis in WheatPlants and Thylakoids by High Light Intensity1

Kassim Al-Khatib and Gary M. Paulsen*Department of Agronomy, Kansas State University, Manhattan, Kansas 66506

ABSTRACT

Thermal inhibition and photoinhibition of plants, which mayoccur simultaneously in nature, were investigated to determinewhether the two causal stresses interact and to characterize anyinteractions that occurred. Photosynthetic rates of wheat (Triti-cum aestivum L. cv Len) seedlings declined gradually after tem-perature treatment increased from 22 to 420C or after photosyn-thetically active radiation (PAR) treatment increased from 450 to2000 micromoles per square meter per second and fell rapidlyafter the stresses were simultaneously imposed. Stomatal con-ductance and internal CO2 were affected little, indicating theinteraction occurred in chloroplasts. Thylakoid whole chain elec-tron transport, quantum yield, and saturating PAR intensity weredecreased by high temperature and an additional amount by highPAR treatments. Photosystem reactions involving water oxidationwere inhibited more than other reactions, and chlorophyll fluores-cence transients indicated most inhibition was on the photooxi-dizing side of photosystem II. Injury was influenced little by theorder in which the stresses were imposed and was always mostsevere when they were combined. Release of proteins fromthylakoid membranes was not detected. Lability to the stresseswas lowest in thylakoids from vegetative stage plants and in-creased as plants matured. We concluded that thermal injury isaccentuated by high PAR, the two stresses may act at a commonsite near the water oxidizing complex, and their interaction maybe involved in photosynthetic decline during adverse conditions.

Photosynthesis is highly sensitive to thermal inhibitionwhether the stress is applied to intact tissues (1, 5, 27) orisolated organelles (14, 19, 26). High temperatures inhibitthylakoid activities more than chloroplast envelope or stromalenzyme components (14, 26). Within the thylakoids, PSII isvery sensitive to heat (8, 19, 26), whereas PSI is stable (5).Low to moderate PAR guards plants against thermal injury

(13, 27, 30) by stabilizing the water-splitting complex withprotons (30). High PAR, on the other hand, causes photo-inhibition, which is also localized within thylakoid mem-

branes near PSII reaction centers (4, 22).Injury from stresses such as chilling, freezing, drought, and

salinity that restricts photosynthesis is strongly exacerbatedby photoinhibition (9, 15, 23, 24, 28). Interactions betweenthermal inhibition and photoinhibition, in contrast, have beengiven little attention. The alleviation of thermal inhibition bylow to moderate PAR (13, 27), however, suggests that inter-

'Contribution No. 88-491-J of the Kansas Agricultural Experi-ment Station.

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actions could also well occur at high PAR intensities. More-over, as noted by Powles (22), intense heat combined withfull sunlight typifies much of the earth's arable surface duringsummer.The possibility that high temperature might interact with

high PAR prompted these investigations. Objectives were todetermine whether thermal injury was alleviated or aggravatedby high PAR and to characterize the nature ofany interactionsthat occurred.

MATERIALS AND METHODS

Plant Materials

Wheat (Triticum aestivum L. cv Len) seedlings for evalu-ating temperature/light interactions on whole plants weregrown in 15-cm-diameter containers with a mixture of 2 partsloam: 1 part peat moss moistened with half-strength nutrientsolution (11). Seedlings for thylakoid extracts were grown insimilar containers of vermiculite, which was also moistenedwith half-strength nutrient solution. Both sets of seedlingswere sampled at the two-leaf stage after 2 weeks' growth.Plants for sampling at different developmental stages weregrown in 2-L opaque containers with aerated half-strengthnutrient solution (11), which was replaced weekly. Compositesamples of leaf blades were harvested at seedling, stem elon-gation (jointing), anthesis, and 2- and 4-week postanthesisstages.Growing conditions for all plants were 450 /mol m-2 s-'

PAR (400-700 nm) measured with a LI- 1 88B quantum meterand LI- 190SB sensor (Li-Cor, Inc., Lincoln, NE), 16/8-h day/night periods, 22°C/20°C temperatures, constant 40% RH,and 370 AL L' CO2.

Whole Seedling Treatments

Two-week-old seedlings were treated in chambers main-tained at 22 or 42°C and PAR of 450 or 2000 ,umol m-2 s-'.The lower temperature is near the optimum for growth ofwheat, and the higher temperature is near the maximum inmajor production regions during summer. The higher PARwas from two 500-W EAL Reflector Photolamps (Sylvania-GTE, Salem, MA) after filtering through 10 cm of water andapproximated full sunlight. Treatments were imposed for 0.5to 4 h.

Gas Exchange Measurements

Net CO2 exchange, leaf conductance, and internal CO2concentration of seedling leaf blades were immediately meas-

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AL-KHATIB AND PAULSEN

Table I. Photosynthetic Rate, Stomatal Conductance, and Internal CO2 Concentration of WheatSeedlings after Treatment with Moderate or High Temperatures and Moderate or High PAR Intensities

Plants were subjected to simultaneous temperature and PAR treatments for indicated times, andgas exchange was measured under standard conditions of 220C, 450 Mmol m-2 S-1 PAR, and 360 ,uLL-1 C02.

Duration of Treatment Conditions (OC/Amol m-2 s-' PAR)Treatment 22/450 42/450 22/2000 42/2000

h Photosynthetic rate (pAmoI m-2 s-' + SE)0 6.0 ± 0.5 6.0 ± 0.5 6.0 ± 0.5 6.0 ± 0.50.5 6.0 ± 0.4 5.8 ± 0.7 5.8 ± 0.6 2.0 ± 0.51 6.5±0.3 4.1 ±0.9 3.7±0.1 1.6±0.42 6.4±0.3 3.7±0.4 3.5±0.3 1.4±0.33 6.3±0.4 3.4±0.4 2.4±0.4 1.1 ±0.34 6.2±0.1 2.8±0.5 1.1 ±0.2 0.3±0.1

Stomatal conductance (mol m2 s' + SE)0 0.15 ± 0.03 0.20 ± 0.02 0.15 ± 0.03 0.15 ± 0.030.5 0.14 ± 0.02 0.22 ± 0.05 0.33 ± 0.04 0.38 ± 0.051 0.14 ± 0.04 0.25 ± 0.05 0.26 ± 0.02 0.39 ± 0.052 0.15 ± 0.03 0.28 ± 0.02 0.36 ± 0.01 0.39 ± 0.033 0.16 ± 0.04 0.26 ± 0.03 0.35 ± 0.03 0.21 ± 0.014 0.16 ± 0.03 0.26 ± 0.03 0.35 ± 0.01 0.27 ± 0.01

Internal [CO2J (AL L ' ± SE)0 320 ± 5 314 ± 12 325 ± 9 313 ± 160.5 315 ± 7 329 ± 11 347 ± 11 361 ± 61 315±12 335±11 351 ± 15 357±92 328±9 334±17 352±7 360±93 320 ± 9 350 ± 11 358 ± 8 351 ± 34 325 ± 11 336 ± 11 348 ± 7 359 ± 4

ured after terminating temperature and PAR treatments.Measurements were made with a Li-Cor LI-6200 photosyn-thesis system at 22°C and 450 ,mol m-2 s-' PAR. They wereinitiated when CO2 and RH in the 0.25-L photosyntheticchamber reached 360 uL L` and 40 ± 5%, respectively, andcontinued until CO2 decreased to 340 ,uL L',

Thylakoid Treatments

Chloroplast thylakoids were extracted from leaf blades witha Polytron (Brinkmann Instruments, Westbury, NY) in amedium containing 330 mm sorbitol, 40 mM Hepes-NaOH(pH 7.0), 30 mm KCl, and 3 mM MgCl2 (30). The homogenatewas centrifuged at 10OOg for 30 s, and the supernatant wasdecanted and centrifuged at 8,000g for 2 min. The pellet fromthe second centrifugation was suspended in 10 mm NaCl,centrifuged at 20,000g for 10 min, washed twice in 10 mMNaCl, and resuspended in the original extraction medium.Chl concentration was 0.6 mg/mL (3).Temperature and light treatments were imposed on 0.2-

mL thylakoid samples in thin glass tubes. Samples were heatedin controlled temperature baths at 22, 32, or 42°C and illu-minated with 0, 1000, or 2000 ,mol m-2 s-' PAR from two500-W EAL lamps after filtering through 10 cm of water.Thylakoids were simultaneously heated and illuminated for10 min, except as noted, when pre- and postilluminationtreatments and time course effects on thermal injury weredetermined.

Thylakoid Measurements

Thylakoid activities and Chl fluorescence were measuredin a DWI oxygen electrode unit (Hansatech Ltd., King'sLynn, England). The unit was modified by replacing theacrylic water jacket with one machined from brass and rebor-ing the two lower apertures for ports for glass bundle arms.Additional ports were bored 90° from the fiber optic ports toaccommodate two Hansatech FDP photodiode detectors pro-tected by 685 and 740-nm center bandpass interference filterswith bandwidths of 12 nm (Ealing Electro-Optics, SouthNatick, MA). Temperature of the unit was maintained at22°C by a Haake A80 refrigerated circulator (Haake-Buchler,Inc., Saddle Brook, NJ).

Illumination was provided by a stabilized 250-W quartzhalogen lamp in a housing (Oriel Corporation, Stamford, CT)equipped with a mirror to reflect the image of the filamentabove itself, collimating lens, 10-cm water filter, filter holdercontaining an Ealing 90° hot mirror, Ilex shutter (MellesGriot, Rochester, NY), and fiber optics focusing assembly.Radiation was transmitted through 4.5-mm-diameter bifur-cated glass bundles at 1600 ,mol m-2 s-' PAR on the samplesfor thylakoid measurements. Fluorescence measurementswere made with a Schott BG-39 shortpass filter (cutoff X, tmax/2 = 586 nm) and neutral density filters (Schott Glass Tech-nologies, Duryea, PA) to give 100 umol m-2 s-' PAR. Oxygenelectrode and fluorescence photodiode signals were processedby an EXP- 16 multiplexer and DAS- 16 converter (MetraByteCorporation, Taunton, MA) interfaced with a Zenith 158computer (Zenith Data Systems Corporation, St. Joseph, MI).

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ENHANCEMENT OF THERMAL INJURY BY HIGH LIGHT INTENSITY

PAR(Lmol irf2r1)

Figure 1. Photosynthetic whole-chain electron transport activity ofwheat thylakoids after treatment at 220C (0), 320C (0), or 420C (U)and 0 (A), 1000 (B), or 2000 (C) jimol m-2 s-1 PAR. Thylakoidpreparations were subjected to simultaneous temperature and PARtreatments for 10 min, and activity was measured at 220C and 0 to1600 jmol m-2 s- PAR as described in "Materials and Methods."Vertical lines through data points are standard errors.

Table II. Photosynthetic Quantum Yields for 02 Uptake by WheatThylakoids after Treatment with Three Temperatures and Three PARIntensities

Thylakoid preparations were subjected to simultaneous tempera-ture and PAR treatments for 10 min, and quantum yield was ascer-tained at 220C by increasing PAR from 0 to 200 umol m-2 s-' in fivesteps.

PAR (UmoI m-2 s-1)Temperature

0 1000 2000

°C mol 02 moh-1 quanta + SE

22 0.487 ± 0.050 0.402 ± 0.040 0.338 ± 0.05032 0.325 ± 0.040 0.250 ± 0.030 0.208 ± 0.05042 0.042 ± 0.005 0.026 ± 0.004 0.015 ± 0.000

Whole-chain uncoupled electron transport activities (H20to MV2), including quantum yield and saturating PAR deter-minations, were measured as 02 uptake in 2.0 mL ofmediumcontaining 330 mm sorbitol, 40 mM Hepes-NaOH (pH 7.6),5 mM MgC92, 10 mm NH4CI, 1.0 mm KCN, and 25 ,iM MVwith 30 ,ug/2 mL Chl (12). Quantum yield was determinedby increasing PAR from 0 to 200 ,umol m-2 s-' in five stepswith neutral density filters. Saturating intensity was similarlymeasured by increasing PAR stepwise from 0 to 1600 jumolm-2 S-Im2 s'Activity of PSII + PSI was assayed with CTL as the electron

donor and MV as the acceptor in 60 mM NaCl, 40 mM tricine-NaOH (pH 8.0), 4 mm MgCl2, 0.5 mm CTL, 0.5 mm ascorbate,1.0 mm NaN3, and 25 j,M MV with 200 units/2 mL superoxidedismutase and 15 ,ug/2 mL Chl (20). Rates were ascertainedas 02 uptake in the absence and presence of2.0 jiM gramicidin,and the ratio of the two activities was taken as a measure ofthe effect of temperature and radiation on coupling factor.

PSII + water oxidizing activity was measured with DCPIPas the electron acceptor in 50 mM tricine-NaOH (pH 7.6), 10mM NH4CI, 5 mM MgCl2, 3 mM NaHCO3, and 30 uM DCPIPwith 50 /Ag/2 mL Chl (10). Light-dependent reduction wasfollowed in a Cary 16 spectrophotometer (Varian Associates,Sunnyvale, CA). Activity of PSII was also measured as elec-tron transport from 4 mM DPC to DCPIP as described forDCPIP reduction. PSII uncoupled electron transport wasdetermined as SIMO-dependent 02 evolution in the presenceof 100 mm sucrose, 40 mm Hepes-NaOH (pH 7.6), 2 mMMgCl2, 0.68 mm glycerol, 4 mM DPC, 0.5 mM SIMO, and 3,gM DCMU with 50 ,ug/2 mL Chl (12).

Activity of PSI was measured as 02 uptake with DHQ,DAD, and DCPIP electron donors (10). Durohydroquinonesolution was prepared by adding sodium borohydride tochilled ethanolic duroquinone and acidifying the solutionwith HCI. The final reaction medium contained 100 mMsucrose, 40 mm tricine-NaOH (pH 7.8), 3 mM MgCl2, 0.5 mMDHQ, 0.1 mm MV, and 2 uM DCMU with 25 jig/2 mL Chl.Oxidation of DAD was assayed in 60 mm NaCl, 40 mMtricine-NaOH (pH 8.0), 4 mM MgCl2, 1.0 mM DAD, 1.0 mMNaN3, 0.5 mm ascorbate, 25 jiM DCMU, 25 jAM MV, and 2,gM gramicidine with 200 units superoxide dismutase and 40jg Chl/2 mL. Oxidation of DCPIP was in 50 mM tricine-NaOH (pH 7.6), 10 mm NH4Cl, 5 mM MgCl2, 5 mM ascorbate,1.0 mm NaN3, 30 jiM DCPIP, 25 jM DCMU, and 25 jiM MVwith 200 units superoxide dismustase and 40 j,g/2 mL Chl.

Chi Fluorescence Transients

Chl fluorescence was measured in the DW1 electrode unitthat was modified to exclude extraneous light, Thylakoidscontaining 2 Ag/mL Chl were dark-adapted 5 min in a me-dium of 50 mM Hepes-NaOH (pH 7.6), 15 mm DCMU, 10mM KC1, and 5 mM MgCl2 before measurements wereinitiated.

2 Abbreviations: MV, methyl viologen; CTL, catechol; DAD, dia-minodurene (2,3,5,6-tetramethyl-p-phenylenediamine; DCPIP, 2,6-dichlorophenolindophenol; DHQ, durohydroquinone; DPC, diphen-ylcarbazide; SIMO, silicomolybdate.

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AL-KHATIB AND PAULSEN

Table Ill. Photosynthetic Whole-Chain and PSII + PSI Electron Transport Activities of WheatThylakoids after Sequential or Simultaneous Treatment with two Temperatures and Three PARIntensities

Thylakoid preparations were subjected to sequential or simultaneous temperature and PAR treat-ments for 10 min, and activities were measured at 220C and 1600 lmol m-2 s-' PAR as described in"Materials and Methods." Percentage values are relative to rates after treatment at 220C and 0 umolm-2s-1 PAR.

Simultaneous220C Sequential Sequential PAR SimultaneousTemperature PAR and Temperature before Temperature

andMOm-2 before Temperature and PAR0Mmolm2~1PAR°C pmoI m-2 S-1 Amol 02 mg-' Chl h' % activity ± SE

Whole-chain activity22 0 245 100 100 100

1000 74±7 77±5 77±72000 60±5 58±5 63±5

42 0 18±2 19±1 18±21000 17 ± 2 16 2 15 32000 16±1 15±2 12±2

PSII + PSI activity22 0 321 100 100 100

1000 86±3 88±4 88±32000 73±6 70±4 71 ±4

42 0 68±4 69±3 68±41000 66±6 62±4 60±32000 61 ±6 56±3 47±3

Release of Proteins from Thylakoid Membranes

Loss of membrane proteins from thylakoid preparationsexposed to 22 or 42°C and 0, 1000, or 2000 ,umol m-2 s-'PAR for 10 min as described above was measured by themethod of Volger and Santarius (29). Treated preparationswere centrifuged at 100,000g for 2 h, and proteins in thepellet and supematant were separated by linear gradient SDS-PAGE (21). Mol wt standards of 14 to 66 kD were references.

Experimental Designs and Data Analyses

Temperature and radiation treatments were arranged incompletely randomized designs. Whole plant treatments weresimultaneously replicated four times and thylakoid treatmentswere sequentially replicated six times. Standard errors oftreatment means were calculated by conventional formulae.Saturating PAR intensities for photosynthetic whole-chainelectron transport were estimated as threshold levels at whichactivities became constant according to standard errors.Quantum yields for whole-chain electron transport were cal-culated by least squares of steady state activities at five stepsfrom 0 to 200 umol m-2 s-' PAR.

RESULTS

Photosynthetic rates of seedlings were stable after moderatetreatment conditions (22°C, 450 ,umol m-2 s-' PAR) and onlydecreased gradually after either the temperature or PAR wasincreased (Table I). When both temperature and PAR werehigh, however, photosynthetic rates fell within 0.5 h, indicat-ing a marked interaction between the two factors. Loss ofphotosynthetic activity from high temperature, high PAR, ortheir combined effects was not attributable to stomatal clo-

sure: conductance remained high and even increased slightlyafter plants were treated (Table I). Internal leaf CO2 concen-tration also rose slightly, particularly after exposure of plantsto high light under both temperature regimes. The data veri-fied the detrimental individual effects of high temperatureand high PAR on photosynthesis, and they demonstrated amarked interaction between the two stresses that was nonsto-matal in origin.High temperature greatly reduced maximum electron trans-

port activity of thylakoids from seedlings, and high PAR wasonly slightly less injurious (Fig. 1). Each stress was mostdeleterious when the other factor was at the optimum level,but the two stresses interacted at all levels. Besides reducingelectron transport activity, high temperature and high PARdecreased the irradiance at which activity became saturated(Fig. 1). Saturating PAR declined from approximately 800 to300 ,umol m-2 s-' after thylakoids were treated at 22 and42°C, respectively, in darkness; from 400 to 200 ,umol m-2s- after thylakoids were treated at 22 and 42°C, respectively,with 1000 ,umol m-2 s-' PAR; and from 300 to 100 sAmol m-2s-' after thylakoids were treated at 22 and 42°C with 2000umol m-2 s-' PAR. The consequence of these effects was amarked reduction in quantum yields from each increment ofheat and radiation (Table II). High quantum yields of non-stressed thylakoids were decreased by high temperature andan additional amount by high PAR at each temperature level.Whole-chain electron transport was slowed more than PSII

+ PSI activity by high temperature (Table III). High temper-ature caused greater injury than high PAR to whole-chainactivity; as before, however, the latter stress decreased activitysubstantially at the low temperature and slightly at the hightemperature. Whole-chain rate responses were nearly similarwhether thylakoids were subjected to high temperature and

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40

TIME(mln)

Figure 2. Photosynthetic whole-chain electron transport (A), PSII +PSI (B), and coupling (C) activities of wheat thylakoids after treatmentat 220C and 0 Amol m-2 s-1 PAR (100% control), 220C and 2000,umol m-2 S-1 PAR (0), 420C and 0 gmol m-2 S-1 PAR (0) or 420Cand 2000 zmol m-2 s-' PAR (U) for six time periods. Thylakoidpreparations were subjected to simultaneous temperature and PARtreatments for indicated periods, and activity was measured at 220Cand 1600 ,mol m-2 s'1 PAR as described in "Materials and Methods."Activity of control without treatments was 245 and 321 Amol mg-'Chl h-1 for whole-chain electron transport and PSII + PSI, respec-tively. Vertical lines through data points are standard errors; valuesthat are not shown are smaller than symbols.

high PAR simultaneously or to high temperature preceded or

followed by high PAR.Activity of PSII + PSI, in contrast to whole-chain transport,

was damaged only slightly more by high temperature than byhigh PAR (Table III). Injury was most severe when the twostresses were combined. The order in which the stresses were

imposed had little differential effect on PSII + PSI activityexcept at 42°C and 2000 Omol m-2 s-' PAR. In that case,activity was damaged slightly but significantly more by im-posing the two stresses simultaneously than by imposing themsequentially in either order.Whole-chain electron transport was damaged earlier by

high temperature than by high PAR (Fig. 2A). Rates decreased68% when thylakoids were heated in darkness at 42°C for 2min compared with 36% when they were illuminated with2000 ,mol m-2 s-' PAR at 22°C for 10 min. The increase in

injury by high PAR at 42°C that was observed after 10 minin other studies also occurred after each time interval in thisstudy.

Injury to PSII + PSI over time by high temperature, unlikethat to whole-chain activity, was gradual instead of abrupt(Fig. 2B). High PAR alone caused less injury than hightemperature but again increased the severity ofdamage whenapplied with heat at all intervals over the 10-min period.Coupling decreased precipitously-85% after 2 min and

100% after 10 min-at 42°C (Fig. 2C). High PAR decreasedcoupling only slightly at 22°C until 10 min, when a markedloss of activity occurred. Even less activity was lost from highPAR at 42°C because of the severe damage inflicted by thehigh temperature treatment.Thylakoid partial reactions that included PSII and water

oxidizing activity (H20 --+MV, H20 -- DCPIP) were inhib-ited more than other reactions by high temperature (TableIV). High PAR was less deleterious but caused most injury tothe same reactions and exacerbated their response to hightemperature. The PSII reaction with PSI (CTL -- MV) orwithout PSI (DPC -- DCPIP) was slowed more than PSIIuncoupled activity (DPC -- SIMO), but high PAR had littleeffect on any of the reactions either singly or jointly with hightemperature. All three measures ofPSI activity (DHQ -- MV,DAD -- MV, DCPIP -- MV) were very stable to hightemperature and high PAR.

Initial fluorescence (Fo) at 685 nm increased slightly from22 to 42°C when thylakoids were treated in darkness, but theeffect was somewhat ameliorated by light (Table V). Maxi-mum fluorescence (Fmax) at 685 nm was decreased greatly byhigh temperature and slightly by high PAR. As a consequence,variable fluorescence (Fv = Fm. - F0) was quenched more byhigh temperature than by high PAR and was quenched mostwhen the two stresses were simultaneously imposed. The ratioFv/Fma. fell from 0.70 to 0.50 at 22°C and from 0.30 to 0.26at 42°C as PAR increased from 0 to 2000 ,umol m-2 s-' PAR.The Fo at 740 nm was not changed by the treatments; how-ever, Fm. and Fv were slightly decreased by high temperature.The ratio of Fv/Fmax at 740 nm decreased from 0.67 to 0.61at 22°C and from 0.42 to 0.32 at 42°C as PAR increased fromthe lowest to the highest level.

Peptide band patterns in electrophoretograms of thylakoidpreparations were not visibly altered by temperature and PARtreatments (data not shown). The apparent enduring associa-tion of major proteins with membranes was supported by thenonappearance of peptide bands in electrophoretograms ofthe supernatant fraction of treated thylakoid preparations.

Lability of thylakoids to high temperature decreased asplants advanced from seedling to jointing developmentalstages and then increased during anthesis and maturation(Table VI). Sensitivity to high PAR was more constant duringvegetative growth but also increased during reproductivegrowth. Injury was greater from high temperature than fromhigh PAR at all stages and was always most severe when thetwo stresses were combined.

DISCUSSION

Thermal injury of plants and thylakoids was as stronglyexacerbated by high PAR in this study as was injury from

t

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Table IV. Photosynthetic Partial Reactions of Wheat Thylakoids after Treatment with TwoTemperatures and Three PAR Intensities

Thylakoid preparations were subjected to simultaneous temperature and PAR treatments for 10 min,and reaction activities were measured at 220C and 1600 Mmol m-2 s-' PAR as described in "Materialsand Methods."

PAR (Mmol m-2 S-1)

Reaction 0 1000 2000

220C (Control) 420C 220C 420C 220C 420CMrmol mg-' Chl h 1 SE Activity vs control (% ± SE)

1. H2O MV 273±10 15±1 81 ±3 7±1 66±2 7±12. CTL ,MV 310 ± 14 64 ± 4 87 ± 6 50 ± 2 82 ± 6 40 ± 23. H20- DCPIP 302±30 17±2 79±9 11 ±1 68±3 7±14. DPC -DCPIP 250±22 60±9 87±9 47±6 74±5 36±65. DPC> SIMO 194 ± 7 72 ± 8 97 ± 4 67 ± 7 94 ± 2 68 ± 16. DHQ- MV 296±7 97±5 98±5 94±3 97±1 88±67. DAD -MV 435±11 98±3 99±4 93±6 94±6 92±68. DCPIP - MV 366 ± 15 99 ± 3 99 ± 5 95 ± 3 94 ± 2 92 ± 4

Table V. Initial (Fo) and Maximum (Fm,J) Chl Fluorescence of Wheat Thylakoids after Treatment withTwo Temperatures and Three PAR Intensities

Thylakoid preparations were subjected to simultaneous temperature and PAR treatments for 10 min,and Chi fluorescence was measured at 220C and 100 ,mol m-2 s-1 PAR as described in "Materials andMethods."

685 nm Fluorescence 740 nm FluorescenceTemperature PAR

Fo F. Fo F.0c pmol m 2 s-' Relative units ± SE22 0 5.0±0.2 16.7±0.6 2.1 ±0.5 6.3± 1.0

1000 5.9±0.9 12.6± 1.0 2.1 ±0.5 5.5± 1.12000 5.9 ± 1.5 11.9 ± 1.4 2.2 ± 0.5 5.7 ± 1.7

42 0 6.3 ± 1.0 9.0 ± 1.0 2.1 ± 0.5 3.6 ± 0.71000 5.5 ± 1.0 7.4 ± 0.6 1.9 ± 0.6 3.1 ± 0.72000 5.3 ± 1.4 7.2 ± 0.8 1.9 ± 1.0 2.8 ± 0.7

other stresses in previous studies (9, 23, 24, 28). Interactiveeffects of high PAR on thermal injury, however, were com-pletely unlike the stabilizing effects of low PAR in otherstudies (13, 27, 30). The PAR levels that we used simulatedambient intensity and clearly intensified thermal injury.Moreover, the injury inflicted by high temperature and highPAR was complementary whether the stresses were imposedsimultaneously or sequentially, suggesting the possibility of acommon locus for thermal inhibition and photoinhibition.

Environmental factors that diminish carbon fixation rein-force injurious effects of other factors at the whole plant level(4). Cold and drought stress, for instance, induce stomatalclosure, thus reducing CO2 fixation and resulting in a hyper-sensitive reaction to light (15). Present evidence, however,shows that high temperature interacts very differently thancold and drought with high PAR. High stomatal conductanceafter all temperature-PAR treatments suggested strongly thatthe interaction between these two stresses occurred in chlo-roplasts instead of in stomata.High temperature injury is primarily localized near the PSII

reaction center (5). It involves possible dissociation of light-harvesting pigments from the reaction center (2), loss ofwater-splitting activity (26), and uncoupling of photophosphoryla-

tion (19). Photoinhibition shares PSII as the primary site ofinjury, but transduction of excitation energy, the water split-ting complex, and photophosphorylation have not been im-plicated in previous studies (22).

Their conspicuous responses in the present study indicatedclearly that water oxidation followed by coupling are mostlabile to high temperature and also to high PAR and theirinteraction. Early kinetics of time-course effects were sugges-tive of coupling, but thylakoid partial reaction results showedthat water oxidation was even more sensitive. Fluorescencetransients of treated thylakoids also indicated inhibition oc-curred near PSII, probably on the photooxidizing side, andthe ratio Fv/Fmax, reflected a corresponding decline in effi-ciency of photochemistry (7). Lability of the PSII reactioncenter to high temperature (8, 19, 26) was greatly diminishedand PAR had little effect when the water oxidizing complexwas not involved. Even without the water oxidizing complex,however, PSII was markedly more labile than PSI to hightemperature (5). Release of proteins from thylakoid mem-branes did not appear to be a factor in injury, probablybecause the highest temperature, 42°C, was lower than that atwhich most release occurs (29).

Several other common sites are consistent with individual

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ENHANCEMENT OF THERMAL INJURY BY HIGH LIGHT INTENSITY

Table VI. PSII + PSI Electron Transport Activity of Thylakoids from Wheat at Six DevelopmentalStages after Treatment with Two Temperatures and Three PAR Intensities

Thylakoid preparations were extracted from composited leaf blades of wheat at indicated develop-mental stages and subjected to simultaneous temperature and PAR treatments for 10 min. Reactionrate was measured at 220C and 1600 Amol m-2 s-1 PAR as described in "Materials and Methods."Activities of controls (100%) treated at 220C and 0 MAmol m-2 s-' were 240, 250, 267, 248, 230, and225 umol 02 mg-' Chl h-' at seedling, tillering, jointing, anthesis, and 2 and 4 weeks after anthesis,respectively. Percentage values are relative to rates after treatment at 220C and 0 pmol m-2 s-' PARat each stage.

Developmental StageTemperature PAR 2 Weeks 4 Weeks

Seedling Tillering Jointing Anthesis after anthesis after anthesis

°C Mromm-2S -1 % Activity ± SE

22 0 100 100 100 100 100 1001000 87 ± 1.0 85 ± 2.0 85 ± 1.8 71 ± 2.5 77 ± 2.6 53 ± 4.02000 76 ± 1.2 78 ± 1.2 79 ± 1.1 69 ± 2.6 74 ± 4.0 48 ± 3.0

42 0 63 ± 0.3 70 ± 2.1 67 ± 2.2 56 ± 1.7 37 ± 1.6 36 ± 3.01000 45 ± 0.3 64 ± 2.0 60 ± 2.1 45 ± 0.6 29 ± 1.4 33 ± 3.02000 40 ± 0.4 53 ± 1.0 58 ± 2.0 38 ± 2.4 27 ± 1.4 31 ± 3.0

effects of high temperature and high PAR reported previouslyand interactive effects reported here. At the whole plant level,as discussed above, high temperature is unlike cold anddrought and does not induce stomatal closure to hypersensi-tize reactions to high PAR (4, 15). At the thylakoid level,however, diminution of the light-induced proton gradientfrom heat (30) and loss of the protective effect of nonphoto-chemical fluorescence quenching from high light flux density(17) may occur together. Either of the latter effects mightdestabilize the water-splitting complex (30) or disrupt themechanism for dissipating excitation energy (17).

Photoinhibition injury to the second electron acceptingplastoquinone of PSII (QB)-binding protein, a favored hypoth-esis (16), might also aggravate thermal injury to PSII or thewater splitting complex. The 32-kD apoprotein is present atlow substrate levels, turns over rapidly, and is only synthesizedin light, but light also increases its susceptibility to proteolysis(18). Early thermal injury in the time course studies (Fig. 2)and sequential studies (Table II) slowed electron transportand, according to the hypothesis, should have amelioratedphotoinhibition injury to QB-binding protein. Any such pro-tective effect, however, was not evident. Similar responsesoccurred in other studies in which stress predisposed insteadof protected photosynthesis from photoinhibition (9, 23, 24,28).Wheat in major production regions and many other species

grow vegetatively during cool, moist periods and reproduc-tively during hot, dry periods with high solar intensity (1).The latter developmental stage is especially important because90 to 95% of the economic yield of wheat comes fromphotosynthesis at that time. Our results show that this criticalstage coincides with a marked increase in lability ofthylakoidsto prevailing conditions. The association could well have acause-and-effect relationship with the structural changes inthylakoids during senescence (6) and their acceleration byhigh temperature (1). The decline in ability to recover fromhigh temperature and high PAR during aging (25) wouldlikely accentuate the consequences in vivo.

LITERATURE CITED

1. Al-Khatib K, Paulsen GM (1984) Mode of high temperatureinjury to wheat during grain development. Physiol Plant 61:363-368

2. Armond PA, Schreiber U, Bjorkman 0 (1978) Photosyntheticacclimation to temperature in the desert shrub, Larrea divari-cata. II. Light-harvesting efficiency and electron transport.Plant Physiol 61: 411-415

3. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Poly-phenoloxidase in Beta vulgaris. Plant Physiol 24: 1-15

4. Barenyi B, Krause GH (1985) Inhibition of photosynthetic re-actions by light. A study with isolated spinach chloroplasts.Planta 163: 218-226

5. Berry J, Bjorkman 0 (1980) Photosynthetic response and adap-tation to temperature in higher plants. Annu Rev Plant Physiol31: 491-543

6. Butler RD, Simon EW (1970) Ultrastructural aspects of senes-cence in plants. Adv Gerontol Res 3: 73-129

7. Butler WL (1978) Energy distribution in the photochemicalapparatus of photosynthesis. Annu Rev Plant Physiol 29: 345-378

8. Cheniae GM (1970) Photosystem II and 02 evolution. Annu RevPlant Physiol 21: 467-498

9. Downton WJS (1983) Osmotic adjustment during water stressprotects the photosynthetic apparatus against photoinhibition.Plant Sci Lett 30: 137-143

10. Heber U, Tyankova L, Santarius KA (1973) Effects of freezingon biological membranes in vivo and in vitro. Biochem BiophysActa 291: 23-37

11. Hoagland DR, Arnon DI (1950) The water-culture method forgrowing plants without soil. Calif Agric Exp Stn Cir 347

12. Izawa S (1980) Acceptors and donors for chloroplast electrontransport. Methods Enzymol 69: 413-434

13. Kislyuk IM (1979) Protecting and injurious effects of light onphotosynthetic apparatus during and after heat treatment ofleaves. Photosynthetica 13: 386-391

14. Krause GH, Santarius KA (1975) Relative thermostability of thechloroplast envelope. Planta 127: 285-299

15. Kyle DJ, Ohad I (1986) The mechanism of photoinhibition inhigher plants and green algae. In LA Staehelin, CJ Arntzen,eds, Photosynthesis III. Encyclopedia of Plant Physiology (NewSeries), Vol 19. Springer-Verlag, Berlin, pp 468-475

16. Kyle DJ, Ohad I, Arntzen CJ (1984) Membrane protein damageand repairs: Selective loss of a quinone-protein function inchloroplast membranes. Proc Natl Acad Sci USA 81: 4070-4074

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AL-KHATIB AND PAULSEN

17. Laasch H (1987) Non-photochemical quenching of chlorophylla fluorescence in isolated chloroplasts under conditions ofstressed photosynthesis. Planta 171: 220-226

18. Mattoo AK, Hoffman-Falk H, Marder JB, Edelman M (1984)Regulation of protein metabolism: Coupling of photosyntheticelectron transport to in vivo degradation of the rapidly metab-olized 32-kilodalton protein of the chloroplast membranes.Proc Natl Acad Sci USA 81: 1380-1384

19. Mukohata Y, Yagi T, Higashida M (1973) Biophysical studieson subcellular particles. VI. Photosynthetic activities in isolatedspinach chloroplasts after transient warming. Plant Cell Physiol14: 111-118

20. Ort DR, Izawa S (1974) Studies on the energy-coupling sites ofphotophosphorylation. V. Phosphorylation efficiencies (P/e2)associated with aerobic photooxidation of artificial electrondonors. Plant Physiol 53: 370-376

21. Ortiz W, Lam E, Chollar S, Munt D, Malkin R (1985) Topog-raphy of the protein complexes of the chloroplast thylakoidmembrane. Plant Physiol 77: 389-397

22. Powles SB (1984) Photoinhibition of photosynthesis induced byvisible light. Annu Rev Plant Physiol 35: 15-44

23. Powles SB, Berry JA, Bjorkman 0 (1983) Interaction betweenlight and chilling temperature on the inhibition of photosyn-thesis in chilling-sensitive plants. Plant Cell Environ 6: 117-123

24. Powles SB, Bjorkman 0 (1983) Photoinhibition of photosyn-thesis: Effect on chlorophyll fluorescence at 77 K in intactleaves and in chloroplast membranes of Nerium oleander.Planta 156: 97-107

25. Powles SB, Osmond CB (1979) Photoinhibition ofintact attachedleaves of C3 plants illuminated in the absence of both carbondioxide and of photorespiration. Plant Physiol 64: 982-988

26. Santarius KA (1975) Sites of heat sensitivity in chloroplasts anddifferential inactivation of cyclic and noncyclic photophos-phorylation by heating. J Therm Biol 1: 101-107

27. Schreiber U, Berry JA (1977) Heat-induced changes of chloro-phyll fluoresence in intact leaves correlated with damage of thephotosynthetic apparatus. Planta 136: 233-238

28. Strand M, Oquist G (1985) Inhibition of photosynthesis byfreezing temperatures and high light levels in cold-acclimatedseedlings of Scots pine (Pinus sylvestris). I. Effects on the light-limited and light-saturated rates of CO2 assimilation. PhysiolPlant 64: 425-430

29. Volger H, Santarius KA (1981) Release of membrane proteinsin relation to heat injury of spinach chloroplasts. Physiol Plant51: 195-200

30. Weis E (1982) Influence of light on the heat sensitivity of thephotosynthetic apparatus in isolated spinach chloroplasts.Plant Physiol 70: 1530-1534

1 048 Plant Physiol. Vol. 90, 1989

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