Chiamydomonas reinhardtii Counteracts Photoinhibition'

Activation of a Reserve Pool of Photosystem 11 in Chiamydomonas reinhardtii Counteracts Photoinhibition'

Patrick

J.

Neale*

and

Anastasios

Melis

Departmentof Plant Biology, University ofCalifornia, Berkeley, California 94720

ABSTRACT

The effect of strong irradiance

(Q000

micromole photons per square meter persecond) onPSII heterogeneity in intact cells of Chiamydomonasreinhardtii was investigated. Low light (LL, 15 micromole photons per square meter per second) grown C. rein- hardtii arephotoinhibited upon exposure to strong irradiance, and the loss ofphotosynthetic functioning is due to damage to PSII.

Underphysiological growth conditions, PSII is distributed into two pools. The large antenna size(PSIIa)centers accountfor about 70% of all PSII in thethylakoid membrane and are responsible forplastoquinone reduction (OB-reducing centers). The smaller antenna(PSII)accountfor theremainder of PSII and exist in a statenot yetabletophotoreduceplastoquinone(0B-nonreducing

centers). The exposure of C. reinhardtii cells to 60 minutes of strongirradiancedisabled about half of theprimarycharge sep- arationbetweenP680 and pheophytin. The

PSII,content

remained the same orslightlyincreased duringstrong-irradiance treatment, whereasthe photochemical activity ofPSIIadecreased by 80%.

Analysis of fluorescence induction transientsdisplayed by intact cellsindicated that strong irradiance ledtoaconversion ofPSlI,6

froma0.-nonreducingto aQO-reducingstate.Parallel measure- ments of the rateof oxygen evolution revealed that photosyn- thetic electron transport was maintained at high rates, despite the lossofactivity byamajority ofPSIIa.Theresults suggest that PSlI, inC. reinhardtii may serve as a reserve pool of PSII that augments photosynthetic electron-transport rates during expo- sure tostrong irradianceand partiallycompensates forthe ad- verseeffect ofphotoinhibitiononPSII,.

Lightenergydrivesphotosynthesisbutexcesslightispoten- tially damaging to the photosynthetic apparatus. The latter phenomenoniscalledphotoinhibitionandis manifestedas a

loss in chloroplast electron-transportcapacity (forrecent re-

viewssee38, 39). Though plants differgreatlyintheir sensi- tivity to photoinhibition, current evidence indicates wide- spread occurrence ofphotoinhibition inboth terrestrial and aquaticenvironments(2, 33). In mostcases,the loss of PSII function isresponsiblefordecreasedelectron-transportactiv- ity,though damagetothe PSIcomplexhas also beenreported (39).

Recentinvestigationsfromseveral laboratorieshavesought

toestablish thesingleorseveralsites oflight-dependentdam- agewithin thePSIIcomplex.Onegroupofstudies has focused This researchwassupported bythe U.S.DepartmentofAgricul- ture,competitive researchgrantnumber88-37264-3915to P. J. N.

andNational Science FoundationgrantDCB-88 15977 to A. M.

on the primary electron-transport events within the PSII reactioncentercomplex, i.e.

P680*

Pheo

QA

-* P680'Pheo

QA

P680+Pheo QA- (1)

When isolated spinach thylakoids were illuminated with strong-irradiance (2500 Amol.m-2

s-')

at0C, both the

QA2

(320nmabsorbance change) and Pheo (685 nm absorbance change)signalwere loweredin parallel(8, 9, 12), suggesting inhibition of primary photochemistry. The PSII primary chargeseparation wasalso inhibited when intact cells of the green alga,

Chiamydomonas reinhardtii,

were exposed to strongirradiance

(12).

Therapidlossofthepheophytin pho- toreduction during photoinhibition has also been detected using EPRdifference spectroscopy (46). These results indi- cated thatphotoinhibitorydamageaffectedtheabilityofPSII toformtheP680+Pheo- chargeseparation.

An alternative proposal (22, 37) is that photoinhibition results from light-dependent damage to the plastoquinone bindingsite, whichis located on the 32kD'Dl' polypeptide ofPSII.Accordingtothe latter model, onlyelectrontransfer from QA to the boundplastoquinone,QB,isdisrupted, whereas nodamage occurs to the watersplittingenzyme or thereaction center. Supportfor this proposal was found in the rapid in vivo

turnover

of

Dl

in

C. reinhardtii

(22, 37).

Subsequently,

it hasbeen accepted that the functional componentsofthe PSII reaction center(Mn, Z, P680, Pheo, QA andQB) are all boundto the

Dl/D2

heterodimer inanalogywith the struc- ture ofthecrystallized bacterial reaction center(32, 44). In the contextofthe lattermodelofPSII,enhancedturnover of

Dl

would be alikely consequenceofprocesses thatrepaira damaged PSII reaction center (3, 28). The precise events leadingto thelossofPSII primarychargeseparation remain tobe resolved(36, 43).

Thereisconsiderableexperimentalsupportfor theconcept ofheterogeneity among PSII reaction centers both in the functional antennasize and electron-transport

properties

on thereducingsideof QA (5).TheconceptofPSII

heterogeneity

was originally introduced to explain the biphasic nature of PSII activity, measured either by fluorescenceinduction ki- 2Abbreviations: QA, primary quinoneelectron acceptorofPSII;

F,, nonvariable fluorescenceyield;

Fe,,

initialplateau of fluorescence yieldin the fluorescenceinduction curve;Fp,peakfluorescence in the fluorescence induction curve;Fm,maximumfluorescence; F,,variable fluorescence=

Fm-F1,;

P680,reactioncenterofPSII;Pheo,pheophy- tin;Z,secondary PSIIdonor;QBsecondaryquinoneelectron acceptor ofPSII; LHC,light-harvestingcomplex;PS

II-QB1-nonreducing,

PSII centerwithimpaired QA-QBinteraction.

1196

netics or by the reduction of the primary quinone, Q. (31).

The biphasic naturewassuggestedto be dueto thepresence

oftwodistinct populations of PSII, namelyPSII,,and

PSII#.

Their different kinetics were explained by different sizes of the light harvestingantenna(31). In higher plant chloroplasts, PSII,,accountsfor75 to 80% of PSIIcenters, contains about 250 Chl (a and b) molecules in itsantenna, and is located in the grana region of the thylakoid membrane, while PSIIp

accountsfor the remaining 20to25%, hasasmallerantenna size of only about 120 Chl molecules, and is located in the stroma-exposed regions of the thylakoid membrane (1, 30).

In C. reinhardtii grown at moderate irradiances, the

PSII,,

comprises about 45% of total PSIIcontent(34).

Anothertypeof PSII heterogeneity has been reported, i.e.

the PSII reducing side heterogeneity (24, 25, 29, 45). This typeofheterogeneitywasidentifiedby theinabilityofasmall fractionof PSII centers to transfer electrons from Q. to QB.

These PSIIcentershave been termed

QLrnonreducing

and^are normally inactive in theprocess ofplastoquinone reduction (7, 15, 24, 25, 29). One convenient methodtoquantify these QBrnonreducingcentersis from the PSII fluorescence induc- tion kinetics measured with thylakoid membranes in the absence of the PSII inhibitor DCMU (7, 29). Because QB- nonreducingcentersareunabletotransfer electrons from Q- toQB, electrons accumulate on Q,promptlyupon illumina- tion and this is reflected in the initial fluorescence yield increase fromF,,toF,, (13). Inmaturechloroplasts, thekinet- ics ofthe Qj,nonreducing centers are identical to those of PSII,1(7, 29), suggesting that there isastrongoverlapbetween

Q,-nonreducingcentersandPSIIg.

In thepresentstudy, the dynamics of PSII heterogeneityof low-light (LL) grown C. reinhardtii were examined during

exposureof cellstostrongirradiance.The loss ofPSIIreaction centeractivitywasmonitoredusing absorbance changemeas- urements at 685 nm (Pheophotoreduction). Inaddition, we

investigated the effect ofstrong-irradiance exposure on the pool sizeofQB-nonreducingtheQ,-reducing forms estimated from fluorescence kinetics. These changes were correlated with the properties oflight-saturated oxygen-evolution in C.

reinhardtii.Theresultssuggestthatundercertainstresscon-

ditions, Q8-nonreducing centers maybe converted to a QB- reducing form and, thus, become active in the process of plastoquinone reduction. At thesametime, strong-irradiance

exposure leads to a loss ofPSII,, primary charge separation activity. The 'activation' ofPSII3appearsto counterthe loss of^amajorfraction of PSII primary chargeseparation dueto photoinhibition.

MATERIALS AND METHODS

Chlamvdomonas reinhardtii strain CC- 124 (mt-) (Duke Universityculturecollection) wasgrown in an ammonium/

phosphate/trace-metalmedia whichwascontinuously stirred and bubbled with 3%CO2.Experimental culturesweregrown in 1 L culture flasks which had 5 cm pathlength in the directionofillumination from^{a warm}white fluorescentlight

source. Neutral density screens were used to obtain a low- light growth intensity (LL) of 15 ,mol m-2s-'. Cultureswere

used in midlog phase (0.5 -1.0 x 10'cells mL-'). Growth

ratewas ca. 0.5d-' undertheseconditions (25°C).

Strong-irradiance treatments were administered using a halogenlightsourcewitha 15cmthickwaterheatfilterat an intensity of2000 ,tmol m-2 s-'. Before exposure, cells were concentrated by settlingfor 30 min, after which supernatant wasremovedinsufficientquantitytoobtainanapproximate 10-foldhighercellconcentration. Analiquotof 10 mLfrom theconcentrated cell suspensionwas thenexposedto strong irradiance, during which cellswerevigorously stirred with a continuous stream of 3% CO2 in air. In othertreatments, a largerlight sourceand incubation chamberwas usedsothat preconcentration of cells was not necessary. Similar results wereobtainedwith eitherset up.Temperature wasmaintained at 25°Cusinga waterbath.

Apolarographic, Clark-typeelectrodewasusedtomeasure 02 evolution.Illuminationwasprovidedbyahalogenprojec- torlamp.Theincidentintensityto thesamplewasvariedby neutraldensity filters. After the treatmentperiod, cells were either useddirectlyformeasurementsofoxygenevolutionor prepared for otherassays as described below. Beforetransfer tothechamberoftheoxygenelectrode, cellswereresuspended in fresh growth media (kept under 3% CO2 in air) to the original culture density.Acirculatingwaterbath(Lauda LM6) was used to maintain both mediaand electrode chamberat the culturetemperature (25°C). Initial 02 concentration was loweredto 20% saturation by bubblingwith N2 for approxi- mately 20 s. The rate of02 evolution was then measured underanirradiance of 1500,umol m-2 s-' (HL) foraperiod of3 min, followed by atransitionto 230

,umol

m-2s-' (ML) inwhich therateof02 evolutionwas againmonitored fora period of 3 min. Gross

02

evolution rates were computed aftercorrection forthesubsequently determinedrateofdark

02 uptake.

^The rationale for theO2 evolution measurement

isdiscussedfurther below.

The in vivo fluorescence of intact C. reinhardtii cells was induced withgreen lightdefined by a CorningCS 4-96 and CS 3-69 filter. Control cells maintained inLLgrowth condi- tionsweredarkadapted 20sbeforemeasurement.Additional dark adaptation (5 min)was used after cells receivedstrong irradianceexposure priortofluorescencemeasurements. The fluorescence probing illumination had a flux of 24 to 100 ,umol m-2s-' asindicated.

Material for thedetermination of PSIIcontent wasrapidly cooledto0°Cinanice-brine mixture, pelletedat5000g for5 min andthepelletswerekeptfrozenat-80°Cuntil analysis.

Frozencells wereresuspendedinahypotonicbuffer contain- ing 20mmTris-HCl(pH 7.8), 35mmNaCl,and 2mMMgC12.

Brokenthylakoidswereobtainedbysonicatingthecellsin ice for 1 minpulsed mode(50% duty cycle, power= 5, Branson sonifier model 200). The concentration ofthe primary elec- tron acceptor, Pheo, ofPSII was determinedfrom thelight- minus-darkabsorbancedifferencechange at 685 nm (A A685) asdescribed (12, 21).Thylakoidswerediluted with additional hypotonic bufferto aChla +b concentration ofabout 10

/LM

and 2

OM

methyl viologen, 2

jtM

indigodisulphonate, 2 mM MnCl2, 0.05% (v/v) Triton X-100. Sufficient dithionite to lower the redox potential to -490 mV was added to the reaction mixture just beforemeasurement. The Pheo photo- reduction wasinduced by blue (Corning CS 4-96)excitation light at an intensity of 600 ,umol m-2 s-'. The measuring

beam had ahalf-bandwidth of 1 nm. The absorbance differ- ence measurements were corrected for the effect of particle flattening (40), and an extinctioncoefficientof 65mm-'*cm-' was applied (12). Under these conditions, the concentration of PSII is equal to that measured by the quantitation of QA (light-minus-dark absorbance change at 320 nm) ( 12, 18).

The fluorescence induction curve ofPSII was measured using thylakoids freshly isolated from C. reinhardtiias previ- ously described (34). Fluorescence was induced using broad bandgreenlight transmittedby acombinationofCorningCS 4-96 and CS 3-69 filters at anintensity of 25 ,umol m2 s-'.

Theproportion ofPSII1 wasdetermined from kinetic analysis ofthe area growth over thefluorescence curve measured in the presence of DCMU (20

ltM)

as previously described (29, 30).

Thylakoid membrane proteins were resolved by SDS- PAGE usingthe discontinuousbuffer system of Laemmli (23) modified as follows. The reservoir buffer contained 0.025 M Tris and 0.25 Mglycine(pH 8.3),the pHofthestackingbuffer andresolvingbufferwaslowered to 6.7 and8.7,respectively.

Polyacrylamide slabs (1.5 mmx 16 cm) were preparedusing a5%stackinggel and a 12.5 to 22%linear gradient resolving gel. The sampleswere solubilized in 100 mm Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 3% ,B-mercaptoethanol, and incubated at 60°C for 15 min. The gels were loaded on an equal Chlbasis (Chla+b of12nmol) andelectrophoresiswas performed witha constantcurrent of16mAfor16 hat20°C.

TheLHC-IIand Dl proteins ofthe C.reinhardtii thylakoids resolved by SDS-PAGE were identified by immunoblotting (Western blot analysis). Electrophoretic transfer to nitrocel- lulose and subsequent incubationswith rabbit antibodiesto maizeLHC-II(gift ofDr. R. Malkin)andDl (gift ofDr. G.

Schuster)and with

alkaline-phosphate conjugated

goat-anti- rabbitantibodywasperformedasin(19).Colordevelopment of the blots wasperformedasdescribed previously (1 1).

RESULTS

Loss ofPrimaryCharge Separation Activity during Strong-IrradianceTreatment

Theabsorbance change at 685 nm due to Pheo photore- ductionwasusedtoquantitatethePSIIreactioncentersactive inprimary chargeseparationin

Chlamydomonas

reinhardtii thylakoid membranes. Control LL-grown cellscontained an averageof2.0 mmol of

photochemically

competentPSIIper mol Chi a+b (Chl:Pheo = 500). The LL-grown cells were sensitiveto

photoinhibition.

After 60 min exposuretostrong irradianceless thanhalfofthe PSIIreactioncenters wereable to form a stable

primary

charge

separation (Fig. 1).

The decrease in

photoreducible

Pheo content occurred continu- ouslyduring

strong-irradiance

treatment

(Fig. 2).

The Pheo measurementsdefined the decrease in total ca- pacityfor PSII

primary

photochemistry;however,additional information was sought on the relative sensitivity ofPSIIaand

PSII#

to strong-irradiance treatment. The kinetics of PSII photochemistryweredefined fromthefluorescenceinduction curve of isolated thylakoid membranes in the presence of DCMU.A

comparison

of thecurvesobservedfor control and photoinhibited thylakoids (30 min

treatment)

is

given

in

10 20 30 40 50 60 70

Time

^,

s

Figure1. Light-inducedabsorbance change at 685 nm in isolated C.

reinhardtii thylakoids attributed to the photoreduction of the primary PSII acceptor, Pheo. Thylakoids (10 gMChi a + b) were suspended inthe presenceof 2 mm MnCI2, 2zlM methyl viologen,2

lM

indigodi- sulfonate, and 0.05% (v/v) Triton X-100. The redox potential of the degassed suspension was adjusted to -490 mV with sodium dithio- nite. Blue actinic illuminationof600,mol m-2s-1defined by a Corning CS 4-96wasturned 'on'attime=20 s, and turned 'off' at time= 60 s. The uppertrace is from thylakoids isolated from cells of C.

reinhardtiiafter 60 min exposure to strong irradiance. The lower trace is fromthylakoids isolated from control cells of C.reinhardtii.

110

°

00

L.

ioo'

o

90

0 g

0 7080

Ul)

°.- 70 Co 60

< 50 40 L

0 20 40

Time, min

60 Figure 2. Amplitudeof the absorbance changeat685nm(AA685)as afunction ofstrong-irradiance exposureof C. reinhardtii cells. The amplitude of the absorbancechangeof control cells(see Fig. 1)is taken as 100%. Treated cellswereincubated for upto 60 min at 2000 umol m-2 s-1. Each point represents the average of 4 to6 exposures(e.g. Fig.1) ofonepreparation.

Figure3A. Afterstrong-irradiancetreatment, Fmofthylakoid membraneswasdramatically lowered,butF,wasonly slightly lower(cf. 8-10). The kinetics of the area increase over the fluorescence induction curve of

thylakoid

membranes from control and

strong-irradiance

treated cellswere

analyzed

on

semi-logarithmic plots

(Fig.

3B). The

proportion

of

PSIIp

in LL-grown control C. reinhardtii averaged 30% ofthe total PSII in thethylakoidmembrane

(intercept

of the slowphase with theordinate at zero time in Fig. 3B

[31]).

The strong-

V._

0_ ciA)

):3

a) e

ED

46-

LL LJ

1 /

~Control

'30 min incubation

n Il

0

E

o 2.0

E

E 1.6-

- 1.2

\0.8

W1

0.4 Cl)

0-

0.0 0.2 0.4 0.6

Time, s

Figure3. Fluorescence induction kineticsof C. reinhardtii. A, Time courseof fluorescence inductionby weak green actiniclight(25/Amol m-2s-1)inthepresenceofDCMU (20AM),forthylakoid membranes isolatedfrom either control cellsof C. reinhardtii,orfrom cellsafter 30minincubation at2000Amol m-2 S-1strong irradiance;B, semi- logarithmic kineticanalysisof theareagrowthoverthefluorescence induction curve[area (t)]for thylakoids isolated from control cellsor from cells incubatedfor30minunder strong irradiance. Theslopeof the slow(exponential) component associated with PSIl6is indicated by a dashed line. The extrapolation of this line to the zero time intercept indicates the relative (In of the) proportion of PSIlY. The intercept for the30minincubated cells is closer to origin,indicating asignificantlylarger relativeproportion of PSII afterstrong-irradiance incubation.

irradiancetreatmentresulted in amorethandoublingin the relative proportion of

PSIIy,

which then accounted for the majority of the fluorescence emitting PSII (Fig. 3B). To quantitate the effect ofstrong-irradiance treatment on the pool sizes ofPSII,,andPSIIg, therelativeproportionof each PSII was applied to the total functional PSII measured by Pheophotoreduction in each membranepreparation (Fig. 4).

Thisanalysisshowedthat

PSIIa

wasrapidlyphotoinhibitedby strong irradiance, whereas the

PSII#

content remained more or less constant. Such results are consistent with previous measurementsofstrongirradiancetreatedspinach thylakoids (9, 27), and show that photoinhibition damage is directed mainlyatPSII,.

Thylakoid membrane proteins of control and strong-irra- diance treatedC. reinhardtiiwereanalyzed using SDS-PAGE and immunoblot techniques in order to test whether any significant amount ofprotein degradation occurred during strong-irradianceexposure. Ofparticularinterestwasthethy- lakoid membranecontentofthe DI protein,thedegradation of which has been correlated with the loss of PSII activity duringstrong-irradiance exposure(37). Intheanalysis of the thylakoidmembranesof theC. reinhardtii usedinthisstudy, the proteins displayed the same electrophoretic pattern and the same staining intensity(loadedonequal Chlbasis) inde-

30 60 30

Incubation time, min

Figure4. Strong-irradiance effects on absolute pool sizes of total PSII (0), PSIIL(A), and

PSII,

(0) in the thylakoid membrane of C.

reinhardtfi. Cells were incubated for up to 60 min under strong- irradiance and then returned to growth irradiancefor recovery. The total PSII active in primary charge separation was estimated from light-induced absorbancechange measurementsat685nm(e.g.Fig.

1).ThePSII and PSII componentswereestimated using the kinetic analysis of the fluorescence inductioncurve(e.g. Fig.3). Eachpoint represents a separate thylakoid preparation with independent ab- sorbancechange andfluorescence inductionmeasurements.

SDS-PAGE

WESTERN BLOT

kDa r1 2 3- 4 1-2 3 4 1 2 3 4

92.5- 66.2- 45.0- 31.0-

21.5- ::3:

14.4- ,:

.WI.. "o___

COOMASSIE Di

TREATMENT 0 30 30 30 0 30 30 30 RECOVERY - 0 1560 - 0 15 60

LHC-JI

0 30 30 30 (min)

- 0 1560(min) Figure 5. Polyacrylamide gel electrophoresis and immunoblot analy- sis ofthylakoid membrane proteins from C. reinhardtii. Left panel, thylakoid membrane proteinswereresolvedby electrophoresis(SDS- PAGE) and stained withCoomassie brilliant blue. Thylakoid mem- braneswereloadedon anequalChI basis,with atotalof 12 nmol of Chla+bper lane. Lane1, control cells; lane2,cellsexposedto30 minstrong irradiance; lane3, cells exposed to 30 min strong irradi- ancefollowedby 15minrecoveryatgrowth irradiance; lane4,asfor lane3 but 60minrecovery.Center panel, SDS-PAGE prepared and samples loadedasfor left panel, followed by electrophoretic transfer tonitrocellulose and immunological detection of the D1 polypeptide (Western blot). Right panel, SDS-PAGE prepared and samplesloaded asfor leftpanel, followed byWesternBlotforthePSIIlight harvesting Chi binding polypeptides(LHC-11).

pendentoftreatment(Fig. 5, left panel). Inparticular, there was noobservablechangein theprotein bands in the 25 to 34 kD region where the Dl and PSII light harvesting Chl- binding(LHC-II) polypeptidesareexpectedtobefound(Fig.

5, left panel). The content of Dl and LHC-II polypeptides

=II,IM77 ^-

a.. aim,

was detected by probing with specific antibodies. Again, the amountsof the Dl polypeptide inthe thylakoid membrane wasindependent ofstrong-irradiancetreatment as evidenced by the equal amounts of antibody binding to Dl (Fig. 5, center panel). Thylakoids that were isolated from strong- irradiance treated cells had about half the primary charge separation activity of control cells(cf Fig. 4). Moreover, no evidence ofa netchange inDl wasfoundover the recovery period (Fig. 5). The content of the PSII light harvesting polypeptidesin thethylakoidmembrane alsodid not change during strong-irradiancetreatment andsubsequentrecovery (Fig. 5, right panel). Thus, photoinhibition occurs without any netlossin antigenicallycompetentDl orLHC-II poly- peptides. Similar conclusions have been drawnby other in- vestigators (42, 46).

Strong-IrradianceEffectsonPSIIReducingSide Heterogeneity

Thefluorescenceinductioncurve ofintact cells ofC. rein- hardtiiin the absence ofPSII herbicides (Fig. 6) resembles that ofother green algae and higher plants. Fluorescence increases from a nonvariable yield(F,,) to an initial plateau

(F,,),

followedby asecondincrease to a peak

(F,)

(Fig. 6A).

At alowerintensityof excitation,thereismuch less secondary rise, although the initial increase continues to be a constant proportion of

F,,

(Fig. 6B). Both in vivo and in vitro studies haveshown that thefluorescenceyieldincrease fromF,,to

F,,

arises fromPSII reactioncenterswhicharephotochemically competent but unable to transferelectrons efficiently from

Q.4-

to QB (7, 29). These centers have been termed

QB-

nonreducing

(cf.

Lavergne [24]). Previous work has shown thatthese

QB-nonreducing

centers have a

PSII#-type

ofan- tenna(18, 29).Theslow,exponentialkinetics of the F0to F,, rise (Fig. 6B) show that the Q-nonreducing centers in C.

r , F~~~~~~p

a)-.--

C-)

t

<

a)

0 0.25 0.50 0.75 1.00

Time, s

Figure 6. Fluorescence induction kinetics of intact C. reinhardtii cells suspended intheabsenceof PSII herbicides. The actiniclightcame onatzerotime.A,Actinicirradiancewas96 Mmolm-2S-1greenlight whichinducedtheFo,Fp,,toFpsequence;(B)actinic irradiancewas 24 ,umolm-2 S-1 sothatonly Foto Fp, amplitude is induced. Curve (B)isplottedwithanordinatescalefactor of 4 tocompensatefor the loweractinic excitation. Note that the same Foto Fp,fluorescence yieldincreaseoccursinboth(A)and(B).

reinhardtiialso have the characteristics of

PSII,.

Theincrease of fluorescence emission from the PQ-reducingPSII centers

(QB-reducing)

is

delayed

untilreduction ofthe

plastoquinone

pool, upon which electrons accumulate on QA and fluores- cence undergoes a further increase from

F,,

to

F,.

Total variable fluorescence, Fv= Fm- F,,was calculated usingF,m measured in the presence of 10 MM DCMU (Fig. 3). The fluorescence emitted from the Qrnonreducing centers was thuscalculated to be about 15% of total variablefluorescence.

The amplitude of the exponential fluorescence increase from F,toF, was lowered uponstrong-irradiance treatment, suggestingthat therelative concentration ofQB-nonreducing PSII centers was lowered (Fig. 7). The lowering occurred rapidly, only 20% of the exponential amplitude remained after 15 minofstrong-irradianceexposure(Fig. 7). The drop in the F,, to

F,,

yieldwasconfirmedbyindependent measure- ments of the F,,to

F,,

amplitude with thylakoid membranes isolatedfrom control andstrong-irradiancetreated cells(data notshown). As was noted above(Fig. 4), such lowering in the amplitudeoftheinitial fluorescenceyield increase should not beattributedtophotoinhibitionof

PSII6,

which is resistant to damage (9, 27). The photoinhibition rate of PSII, has been reported to be several times slower than the rate at which PSIIa is damaged (8), and much slower than the rate of lowering ofthe

Fp,

amplitude. Instead, the lowering may be a manifestation ofthe conversion of

QB-nonreducing

centers into aQB-reducingstate (seealso below). TheF,,to

F,,

ampli- tude recovered very slowly when strong-irradiance treated cells were returned to normal growth irradiance (Fig. 7), suggestingthat

PSIIo

didnotrevert to a

QB-nonreducing

status.

Similarloweringin F, to

Fp,

^amplitudehas been observed in otherspecies of algaeandhigherplants uponstrong-irradiance exposure, but has not been attributed to changes in the functionalstatusofPSII

QB-nonreducing

centers(4, 10, 47).

Strong-Irradiance

Effects onPhotosyntheticRates Only

QB-reducing

centers participate in steady-state pho- tosynthetic electron-transport (oxygen evolution) in vivo,

TREATMENT RECOVERY

-o

1.0

0.8 a)

Ll7

0.6 0.4

E 0.2

01

0 10 20 30 10 20 30 40

Incubation time, min

Figure7. Amplitude ofthefluorescenceyieldincreasefrom F,toFp,

(pool size ofPSII Qs-nonreducing)measured on samples taken at varioustimesduringa30minstrong-irradianceincubation andsub- sequent'recovery'atnormalgrowthirradiance.The loweredF,toFp,

amplitude afterstrong-irradianceexposuresuggestsanactivationof

PS11#(0Q-nonreducing)inelectrontransfer fromOAtoQB.

while

QB-nonreducing

centers are not involved in oxygen evolution (15, 24). Hence, upon photoinhibition, it is ex- pected that rates of oxygen evolution should decrease in proportion with the loss ofPSII5, photochemical activity. We tested whether such arelationshipwasobserved during pho- toinhibition of intact cells. This was accomplished by com- paring the extentof photoinhibitionof

PSII,

with the rates of oxygen evolution measured under both medium and high actinic excitation. Photosynthetic02 evolutionwasmeasured for 2 to 3 min under intermediate-light (ML; 230 umol m-2

s-')

which saturates electron-transport through PSIIain LL grown cells(34). Short-term measurements at amuchhigher light intensity (HL; 1500 ,umol m-2 s-')were used to gauge electronflow through both PSIIandPSIIp.This HLintensity is more than six times greater than the ML to ensure that any electron transportbythe small antenna

PSII,

wouldbe saturated.

Itwasestablished that prior tostrong-irradiancetreatment, the rate ofphotosynthesis (oxygen evolution) underHLex- ceeded that underML by less than 10% (see legend, Fig. 8).

This observation confirmed that, in control cells,

PSII6

con- tributed littleto the processofH20 oxidationandplastoqui- nonephotoreduction. Figure 8showsthat,followingastrong irradiance treatment, there was a loss in oxygen evolution capacity(photoinhibition). After 60minofstrongirradiance only 40% ofthe initial activity, as measured by ML, had remained (Fig. 8, open circles). However, theextentof pho- toinhibition ofPSII(,wasconsistentlygreater than thatofML oxygenevolution (Fig. 8, triangles). After 60 minofstrong- irradianceexposure, only20% of active PSIIahadremained.

In contrast, the inhibition ofphotosynthesis was much less when measured by HL oxygenevolution than when measured byML oxygenevolution(Fig. 8, solid circles). After60 min

1004

:5 80

._g 60

to 40

0 20

0(

0

Incubotion time,min

Figure 8. Rates of photosynthetic activity (02 evolution) andconcen-

tration ofphotochemically activePSII,,centersas afunction ofstrong- irradiance treatment and subsequent recovery. All quantities are

plotted relativetocontrol values⁼100% (AL),(PSII,,)thePSII(,activity

asestimated fromphotoreduciblePheoandproportion ofPSII,,inthe fluorescenceinductioncurve(cf. Fig.2and Fig. 4). (0), (ML)rateof oxygenevolution at 230,umol m-2s-',controlcells had anaverage rateof675fmol 02cell- h-'.(-), (HL)rateofoxygenevolutionat 1500,molm-2 s-', control cells hadan averagerate atHL of 714 fmol⁰²cell-' h-'.

instrongirradiance,HLactivitydecreased only to63%ofthe initial activity. No change occurred in the Chl content per cell(about 5fmol Chla+b cell-') duringthestrong-irradiance incubation. These results suggest that the net loss of

PSII,

activity due to photoinhibition was partially compensated throughconversionofaportionofthe

PSII,

poolfrom aQB- nonreducing to Qs-reducing status. Because of such conver- sion,the decrease of oxygen evolution rate in HL was limited toabout halfofthe lossof

PSII,

activity.

Recovery from Photoinhibition

Recovery from photoinhibition of photosynthesis in C.

reinhardtii (26, 37) and other plants (16) has been reported tobecompletely dependent on chloroplastprotein synthesis.

This protein synthesis is presumably directed toward the apoproteins ofthePSIIreactioncenter(37, 42).However,the possibilityremained that conversion of

PSII0

toa

QB-reducing

statecould enhance recovery rates. Inthisregard, short-term

02

evolution measurements weremade

immediately

follow- ing 60 minstrong-irradiance treatment, or after 60 min strong irradiancefollowedby 10 to 60minincubation inlowgrowth irradiance (15 Amol mM2 s-'). The change in rates was com- pared to relative changes in concentration ofPSII. (Fig. 8, right panel). The recoveryof

PSII,a

activity wasslowduring incubation of photoinhibitedcells in the LL growthirradiance

(cJf

41). However,weobservedafastphase ofrecoveryduring which the increasein the ratesofMLand HLoxygen evolu- tionexceeded theincreasein totalPSIIaprimary chargesep- arationactivity (Fig. 8). Theshort-term(0-10 min) increase in 02 evolution rate was not inhibited by addition ofthe chloroplast protein translation inhibitors chloramphenicol (200

,ug

mL-')orlincomycin(20

,ug

mL-')(data notshown).

Both ML and HL02 evolution rates increased, and alarge difference remainedbetweenphotosyntheticrates at these two irradiances.Such anincreasein electron-transport rateswith- out acorresponding increasein numberofPSII5 abletocarry out a primary charge separation could be accounted for by continuing conversion of

PSII,

to a

QB-reducing

status, at least in theinitial period following strong-irradianceexposure.

This fastphaseofrecovery mayhave restored some photosyn- theticcapacity. However,full restoration ofPSIIchargesep- aration(which probablyinvolvesturnover ofcomponent PSII polypeptidesis required before photosynthetic rates recover completely[28]).

DISCUSSION

The quantitation of PSII

primary

charge separation, as measured by pheophytin photoreduction, is a

specific

assay fortheamountof damagethatoccurs

during photoinhibition.

Measurementsofpheophytin photoreductionin strong-irra- diance treated Chlamydomonas reinhardtii are consistent withprevious findingsoflossofPSIIprimaryphotochemistry upon photoinhibition (6, 9, 12, 35, 46). Also in agreement with earlier studies (9, 27), we found that photoinhibition adversely affected PSII,,whereas PSIIpappeared resistant to damage (Fig. 4). The maintenance ofPSIIpprimary charge separation activity during strong-irradiance treatment com- bined with the simultaneous decrease in theamplitude ofthe

NONAPPRESSED _APPRESSED

PSU/3 ACTIVATION PS1I13 PSIq PHOTOINHITO /> PS]IQ,

(0Q3-nonreducing) (()8-reducing) (functionol) (domaged) LHCII-

peripherol

Figure 9. A schematic of part of a proposed PSII repair cycle, adapted from Guenther and Melis (17). The wide, hatched arrows indicate those steps emphasized during strong-irradiance exposure.

F,, toF,,,amplitudesuggests that after strong-irradiancetreat- ment alarge proportion of

PSII#

arebeginningto contribute tothe electron-transportprocess.

Thequantitative contributionof

PSII,

canbeestimated by comparingthesize ofeachoxygen-evolvingPSIIpoolbefore andafterstrong-irradiancetreatment.Thevariation inoxygen evolution rates, relativeto theactivity ofPSII,, centers, pro- vides one way of estimating the changes in PSII pool sizes.

Strong-irradianceexposureof60 min leadtoadecline inQB- reducingcenters to63%oftheinitial activity.However,PSII0 declined to about 20% ofthe activity in the control. The remaining QB-reducing activity is attributed to centerswith the smaller

PSII#

configuration. We suggest that these

PSII"

QB-reducingcentersoriginated fromQB-nonreducingcenters upon conversion to a

QB-reducing

form. We estimate that after exposure to strong-irradiance up to 77% of

PSII,-QB-

nonreducing was converted to

PSIII-QB-reducing.

This esti- mateis basedonthecalculation ofhowmany centerswould have beenactivatedto aQB-reducingstatein orderto main- tain the observed rates of light-saturated oxygen evolution (Appendix).

Thus, it appears that as a result ofthe strong-irradiance treatment, and in spite of substantial activity loss by PSII, (photoinhibition),a newsetofPSIIcentersisnowengaged in 0 evolution. The exact nature ofthemechanism and regu- lation ofthe conversion of

PSIIg-Qu-nonreducing

to

PSII,-

QB-reducingis unknown andmorework isrequired. Depend- ing on the photosynthetic organism,

PSII,

may comprise between 20to45% ofthe total numberofPSII reactioncenter complexes (7, 30, 34). The results from this work suggest that

PSIIO

centers are, in part, a reserve pool of PSII, readily available tothe chloroplast in case ofcatastrophic photoinhibition.

This activationof

PSII,,

appears tobeaspecialaspectof the general role ofQB-nonreducing centers in a proposed PSII repaircycle (17, 18). A schematicdepicting the role ofthis activation in thegeneral PSII

repair

cycle is

given

in

Figure

9. Operation ofthe cycle can lead to the accumulation of

PSII,-QB-reducing

centersduring strong-irradianceexposure.

Activation may include movementof the complex from the stroma-exposed region of the thylakoid membrane to the grana partition region (18, 28). The process could occur rapidly during strong irradiance exposure because no addi- tional protein complexes need be

synthesized. Furthermore,

the peripheralLHC-II and the Q,rreducing

PSIIO

centers re- mainseparate,perhapsby^amechanism similartotherevers-

ibleseparation betweenLHC-II and PSII knownto occurin strong-irradiance (i.e. state

transitions,

reviewed in

[2]).

In- deed, state-transitionsprobably occurred

during

ourstrong- irradiancetreatmentsof C.reinhardtiiandmayhavereduced

thefunctional antennasize ofsomePSII,,centers.According to ourestimates, thisprocess made onlyminor contributions to the increase ofQB-reducing PSII,centers during strong- irradianceexposure(Appendix).

In accordance with previous studies on this topic (7, 18, 29), we haveattributed the variable fluorescence yield from F,,to

Fj,

to asignificant fraction of PSIIcentersthatare

QB-

nonreducing.However,alternative explanations fortheorigin ofthe F,,toF,, transitionhave appearedin the literature. One hypothesis is that QAreverts to an'inactive' state, Qi, witha half time of 30sinthe dark. In the stateQi,Chl afluorescence isquenched moreefficientlythaninthe stateQA. Therefore, F,, correspondstothe

Qi

state, andF,,corresponds to the state QA (20). A second hypothesis is that the F,to F,, transition represents the activation of the oxygen evolution system.

When the oxygenevolvingsystemisdark-adapted, SoandS, areinhigh concentrationand quenchfluorescencemorethan the S2 and S3 states,whichare generated upon illumination (13). Wefavortheinterpretation thatF,otoF,,reflects electron accumulationonQA inreactioncentersimpaired in the QAto QBelectrontransport

(Qu-nonreducing

centers).Anumber of observationssupportthis interpretation.The amplitude of the

F,

to F,, component is similar to the proportion of

F,

ac- countedfor by

PSII#

^andthekinetics oftheF,,toF,,increase resemble the exponential kinetics ofPSIIp(Fig. 6), (14, 18, 29). Theamplitude of the fluorescence yield increase fromF,, toF,, in thylakoid membranesis unaffectedby the presence of the artificial electron acceptor ferricyanide or the PSII electron donorhydroxylamine(29). These'DCMU-like'

QB-

nonreducing centers have been isolated in stroma-exposed thylakoidmembranesfrom C. reinhardtii(PJNeale,AMelis, unpublished data), indicating that they are localized in a thylakoidmembranedomain which isspatiallyseparatefrom thatof theQB-reducingcenters.Furtherevidencethat the F,, to

F,,

transition corresponds to the

QB-nonreducing

centers hasbeenprovided by observing therecoverytimeof

Fp,,which

hasahalf-time on theorderof 2 s(7, 29). Thisreoxidation rate is too slow for these centers to make any significant contributiontosteady-state electrontransport.

Theavailability ofa reserve pool of PSII offersa number of advantagestoalgalcells in minimizingthedamagedueto

strong-irradiance exposure. Since the potential dropin elec- tron-transport ratesis partially offset by

PSII#,

the cell can

maintainasupplyofreducing equivalentsneededto synthe- sizenewreactioncenterproteins. Also,the smallantennasize of PSIId isanadvantagein protecting againstfurtherphoto- inhibitionof the newlyintegratedcenters. Insummary,these results suggestthat

PSII3

isalight-activated 'reserve' pool of PSII which may play an important role in sustaining plant growth andproductivityunderadverselightconditions in the naturalenvironment.

APPENDIX

Thefollowing procedure was used to estimate thechange in the poolsize of the

PSII#-QB-reducing

centersduetostrong- irradiancetreatment.Forthe purposeof

calculation,

thetotal PSII contentof control cells isset to 1.0. Sincethe PSII are

the main contributors electron transport in the control

cells, we set

PSHrQ,reducing(control)

⁼

PSIIj(control)

^(A

¹⁾

The number of Q,rreducing centers after strong-irradiance

treatment is estimated from the change in

PSII

electron

transport

PSIIQ,rcedijc

ing(treatment) ⁼

PSIIQxrc,d.

ng,(jcontrol)

X

PSII('e,rvpn

iranspor,(treatment) (A2) where, again,PSIIclciron lransporl isexpressed as a proportionof

theratein control cells. Itwasassumedthat onlyPSII, centers

are damaged during strong-irradiance exposure, so that the numberofPSIL. remaining after strong-irradiancetreatment is

PSII(treatment) = PSII(control) PSIIphiooinhiihitcd (A3)

where

PSIII)ph10,1in,hibiid

is the decreasein numberof PSIIs active

in primary chargeseparation. The numberof

PSIIO-QB-reduc-

ingafterstrong-irradiance treatmentis therefore

PSIIP QB reducing(treatment)

=

PSHQO,re.duic.ing(treatment) PSIIL(treatment)

(A4) Now consider ^a specific estimate based on our

PSII

quanti- tationsforC. reinhardtii. TherelativePSIL,contentof control cellswas0.70 (Fig.4). Electrontransportestimated from HL

0,

evolution, after exposure to strong irradiance, was 0.63 relative to control (Fig. 8). The strong-irradiance treatment

resulted (on average) in a lowering to 52% ofPSII able to

perform a primary charge separation (Figs. 2, 4, 8), so that

PSIphoo//inhibi(ed=

0.48. Therefore

PSIIQB-rcdiic,ing(treatment)

⁼ 0.70 x 0.63 = 0.45

PSIIL(treatment)

⁼ 0.70 0.48 = 0.22 Note that the

PSI1,,

content estimated this way results in a

somewhat larger

pool

size after strong-irradiance treatment than thatindicated in Figure 4. This is attributed to the fact that some(probably less than 10%) ofthe

PSII,,

^centershave been convertedto PSIIe by the processof^astrong-irradiance induced state-transition (2). Using equation A4 corrects for the minor contribution to the

PSII#-Qy-reducing

pool made by thisprocess. Finally,

PSII3

QB- reducing(treatment) = 0.45 0.22 = 0.23

Since the

PSII,

in the control cells was 0.30 of PSII, we

derived0.23/0.30 =0.77. Thus, weconcludethatupto 77%

of

PSII#

is in the

Qy-reducing

^state after strong-irradiance

exposure.

ACKNOWLEDGMENTS

The assistance ofPeter Morrisseyand Dr. Elena del Campillo is gratefully acknowledged.

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