Plant Physiol. (1966) 41, 1037-1043

Photoinhibition of Chloroplast Reactions.I. Kinetics and Action Spectra'

L. W. Jones2 and B. KokResearch Institute for Advanced Studies, 1450 South Rolling Road, Baltimore, Maryland

Received March 23, 1966.

Sumnmary. A study was made of photoinhibition of spinach chloroplast reactions.The kinetics and spectral characteristics of the photoinhibition over a range between230 and 700 my have been examined. The decline of activity due to preilluminationwas independent of wavelength, and dependent upon the number of quanta applied,not upon the rate of application. The effectiveness spectra of photoinhibition indicatethat active ultraviolet light is absorbed by a pigment which is not a normal light ab-sorber for photosynthesis and acts with a high quantum efficiency (> 0.1) forphotoinhibition.

Active visible light is absorbed by the pigments wlhich sensitize photosynthesis(chlorophyll, carotenoids). A verv low quantum efficiency (about 10-4) was observedfor the photoinhibition with visible light.

The action spectrum of the photoinhibition of dye reduction by chloroplasts andlyophylized Anacystis cells indicated that the damage caused by visible light is due toquanta absorbed by photosystem II. However, since system I might not be involvedin dye reduction, the spectra may reflect only damage to photosystem II.

Photoinhibition of photosvnthesis has been de-fined as the debilitating effect of high intensities ofvisible light upon the photosynthetic capability ofgreen organisms (17). The term is extended inthis study to include the effects of tultraviolet lighttupon the photosynthetic reactions of chloroplasts.The kinetics of photoinhibition in whole cells havebeen stLudied using visible (16, 19, 24, 27) and ultra-violet light (2, 13, 25). In both cases, secondarymetabolic effects prevented a quantitative analvsis.Chloroplasts are more stuitable for stuch a studv be-cause of their relative independence from nonpho-tosynthetic metabolic processes. Definitive dataconcerning the spectral characteristics of pigmentswhich sensitize photoinhibition are lacking, althoughattempts to collect these have been made (9). Thisstudy was undertaken to fill this gap in the under-standing of the damaging effects of large doses oflight of selected wavelengths uipon the photochemi-cal activity of chloroplasts.

1 This investigation was supported in part by theAerospace Medical Division [AF 41(609)-2369] andthe National Aeronautics and Space Administration(NASw-747).

2 Present address: University of Tennessee, BotanyDepartment, Knoxville, Tennessee.3 Abbreviations: DCMU, 3- (3,4-dichlorophenyl) -1,1-

dimethvlurea; DPIP, 2,6-dichlorophenolindophenol; PQ,plastoquinone; PMS, phenazinie methosulphate; PPNRphotosynthetic pyridine nucleotide reductase (ferredoxin).

Methods

Spinach chloroplasts were prepared as describedby Hill and Walker (12) from leaves grown in a

greenhouse or obtained in local markets.Except where otherwise noted, the same reaction

vessel was tused for preliminary exposure of theparticles to photoinhibitory light and the subsequentassay of activity. The microvessel was made froman altuminum block, 35 X 25 X 3 mm. A slot was

cuit 3 mm wide by 18 mm deep and covered at thesides with quartz coverslips. A 30 dul aliquot ofchloroplasts filled the slot 3.3 mm deep yielding a0.1 cm2 illuminated area with an optical path 3 mmin length. The small surface area increased the in-cident intensities attainable with available sources.The relatively large amount of alumintum in contactwith the suspension greatly increased the rate ofheat removal from the sample.

Light for photoinhibition was obtained from a2000-w Xenon arc lamp. For experiments tusingfilters to isolate desired wavelength bands, a seriesof quartz lenses were uised to collimate the beam.The beam was passed through 6.5 cm of H,O, thefilters, and then focussed onto the microvessel. In-cident light energy, as well as the fraction absorbedby the chloroplasts, was measured with a thermopileplaced directly behind the vessel. The thermopile

1037

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PLANT PHYSIOLOGY

was calibrated at 328 my uising a ferrioxalate actin-ometer fluiid (10) in tlle same microvessel that wasuised in all experiments. The rear quartz windlowof the microvessel was frosted in an attempt to re-dulce the effect of scattering in the fractional ab-sorption (a) measLurement. In other experiments,the Xenon arc was focuissed on the entrance slit ofa 500 mm focal length B and L grating (blazed at300 m,u) monochromator. The optical frame of theentranice condenser lens was focusedI at the frontsuirface of the microvessel. For measuirements ofincident energy, the microvessel was removed andthe thermopile placed in the same position.

Photosynthetic activity was assayedl in a dualbeam spectrophotometer which recorded the ODof the sample duiring actinic illuimination (22).Light intenisity (lutring the assay was varied by neui-tral denisity filters in the actinic light beam. Themeasuiring beam was 600 mju for DPIP (en, =

20,000/cm pH 7.6) and(l at 340 m,u for NADPH(Fr,l = 6200/cm)23.

In a ty-pical experinment, 30 ,Au of chloroplast sus-pensioni containinig 1 jg of chlorophyll in Tris-HCll)lffer (0.05 Nt, pH 7.6) was pipette(d inlto the micro-vessel at room temperatture. The suispension wasirradiatedl with the (lesired (losage of inhibitory light,thein 10 /AJ of the appropriate assay mixtuire wereadded aind activity measuired in the spectrophotome-ter. A new sample was use(l for each exposuireperiod. Uniless otherwise stated, the final reactionmixtutre of 40 /A total volume for the DPIP reduc-tion assay containe(l, in 1umoles: 1.5 Tris-HCl (pH7.6) anid 0.01 DPIP. TI'he 40 Al reaction mixture forthe assay of NADP reduction containedl, in umoles:1 Tris-HCl (pH 7.6); 0.02 NADP; 0.050 ADP;0.070 \gClI; 0.050 K,HPO4: and satuirating PPNR(35 ,ul/ml of final mixtuire).

II experiments concerning the effect of O>, a1-ml sample was placed in a quiartz Thuinberg cuIv-ette. O., wXas removed before irradiation by adding10 ,umoles gluicose and 100 jug gluicose oxi(lase,'-uicklv evactuating the cuivette an(l flulshing withiitrogen. After repeating the evacuiation an(d flush-ng 10 times, the vessel was placed in the (larkat room temperatuire for 15 minutes before irradia-tion. After irradiation the vessel was opened andthe dye added and mixed aerobically.

Preparations of Anacvstis nidtl(ans were madefrom 2- to 4-day old cultuires which were grown at250 uindler fluiorescent light of abouit 1000 ft-c in-tensity and were bubbled with 1 % CO., in air. Lyo-phylization proceduires followe(d closelv those uisedby Schwartz for Chloreci(l (26). Cells from thecuiltulre were washed twice in distille(l water, stis-pended in a minimuim of distilled water an(d frozeniin a (dry ice-acetone bath. Lyophylization usuiallytook 1 to 2 hours. For use, 1 mg of the dried prep-aration was restuspended in 1 ml of reaction medium(0.5 m sucrose 0.05 M Tris-HCI and 0.01 M NaCl atpH 7.2). Irradiation and the assay of dye redutctionactivity wxere carried ouit as for chloroplasts.

Results

Before stLdyivng the kinetics and wavelength (de-pen(lence of photoinhibitionl some characteristics ofphotoinhibition tln(ler the conditions uisedI in theseexperiments were determinedl.

Photoinhibition of chloroplast reactionis has beenreported to occuir at liquiid nitrogen temperatulre, l)utat a rate abouit $ times slower than at room tempera-tulre (21). The lower rate of photoinhibition atliqii(l nitrogen temperatuire was thouight to he cauisedby the difference in light path duie to the ice crystalsin the suspension. Experimeints run at 230 and(I 3'usinig light of (lifferent wavelength regions werefouind to have 01)0's of from 1.0 to 1.06 (table 1).Photoinhibition therefore, (loes not involve a tem-peratuire-dependent (lark reaction.

Table I. Tenmperatlure Indepcndence of Plhotoin/lilbitionof Chlioroplasts in Variouis Fazvlenytli Re.(i(lnsAssav: DPIP reduction in saturating, red actinic

light at room temperature. NNumbers givein are half-times of decay in seconds, derived from semilog plotsof the decay ctirves. Wavelength regions wvere isolatedby filters.

Wavelength region

230-390 mnL360-525 nll>570

230

1.07.0I.:,

30

1.27.07.5

Q1o

1.061.01.0

The time couirse of photoinhibition in 'U\ and(visible light is independent of 02 concenitr-ation(table II). The mechanism of photoinhibitiontherefore, cannot be a simple photooxidation involv-ing molecular O., This lack of )., depenideiiceclearly (listingiiishes photoinhibition from the bleach-ing of the chlorophyll which is (lependenit uipon O.concentration (6, 27).

Chlorophyll bleaching an(d photoinhibition aredifferent processes. Figulre 1 shows a comparisonof the relative rates of the 2 reactions uinder idlenti-cal aerobic exposutre conditions. The decrease ofchlorophvll absorption follows a logarithmic (lecline,similar to the NADP reduiction activity, blut at arate abouit 50 times slower both in red anid blue light.Also, as was earlier observed with vwhole cells (16,17), there is an initial lag period in the b1eachingprocess, duiring which the normal photochemicalactivity of the chloroplast is completely lost. Suich

Tab!e II. O., Indcepndcnce of Photoinhibition in !'Isileand UV Light

Values are rates of DPIP reduction after exposureexpressed as percent of the rate observed before exposure.

Preillumination

60 sec i; > 400 m,u30 sec X < 400 m,u

+02

36 %22

02

40 %20

1038

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JONES AND KOK-PHOTOINHIBITION. I. KINETICS AND ACTION SPECTRA

30 .

0.2 t O Cl lohn'K ~~~~~~20ECN~~~~~

U-) 0N \l )

tionl(closed circes, skluares) andchip bleaching D

0 N,uNI0~~~~~~~~~~~N

NADP reduction Np 0)

0.05 E

0 2 4 6 8 minEXPOSURE TIME

FIG. 1. Comparisoni of the decay of NADP reduc-tion (closed circles, squares) and chlorophyll bleaching(open circles, triangles) during exposure to identicalsources of strong visible light. Circles: blue light of360-520 nit, NADP decay curve from line 2, table ost;triangles and squares: red light of > 570 mit, NADPdecay curve from line 5, table III. Assay: NADP asdescribed in Metlods. Chlorophyll bleaching measuredin intact chlloroplasts as decrease of absorbance at 678met.Both assahs using same preparation of chloroplasts atsame concentrationg (1 utgChI in 30 Itl).

data support the contention that chlorophyll bleach-ing is a secondary reaction proceeding only afterthe photosynthetic capacity is lust.

Red light(F> 5l70mr) causes a logarithmetic de-crease in the absorption at 490 ml with a half-timeonly slightly longer than that of the bleaching at678mf . Thus, light absorbed by chlorophyll affectsthe bleaching of carotenoids and chlorophyll almostequally. This effect should be accounted for in pro-posals concerning the photoprotective function ofcarotenoi'ds.

No evidence was found for the formation of asoltble inhibitory suibstance upon irradiation withUV light. Fresh chloroplasts added to an irradi-ated inactive sample were fully active. Thus, theeffect of UV light is localized in a strcture-bouindchloroplast constituent. Forti and jagendorf (7)fouind the same result in their study of the effectof visible light on photophosphorylation.

The manifestation of photoinhibition dependsuipon the rate of the photochemical reaction used toassay the activity of the photoinhibited chloroplasts.If the subsequent assay was made uising weak light,the decline in activity brought abouit by exposure tothe actinic beam began immediately and proceededat a rate independent of the actinic intensity. Ifthe suibsequent assay of chloroplast activity wasmade with intensities above saturation, the declinein activity due to photoinhibition began only after

a characteristic lag period and proceededI at a ratedependent tupon the intensity: higher assav intensi-ties showing higher activity for identical samples ofphotoinhibited chloroplasts. TI'he lag period at hiighintensities has been reported for photoinhibition ofO. evolution in whole algal cells (17). All experi-ments described in the following sectiolns were car-ried otut tusing weak intensities to assay photosyn-thetic activity, and as suich, describe the decrease inthe quantum yield.

Kinetics and Quantum Yield(s) of Photoinhi-bition. The rate of decline in activity of photosyn-thetic reactions has a logarithmic character withrespect to time of exposure to the photoinhibitivelight. This decline, studied previotusly by others, isin accord with the general exponential attenllationlaw:

Rt = R,, e-" Iwhere R. is the initial rate, Rt is the rate after ex-posuire to photoinhibitive light duiring time t; and cis a proportionality constant.

Table III. Validity of Ixt Law for Photoinhibition ofDye Reduiction and NADP Reduction by Red (, >570 mi), Blue (360 to 520 mu) and UV Light

(328 min, half-band 15 min)Chloroplasts containing 1 ,ug of chlorophyll were irra-

diated in a 30 ul volume of 0.05 Ai Tris buffer (pH 7.6).After irradiation the remainder of the reaction mixturewas added in 10 ,ul (reaction mixture as in Methods).Actinic light intensitv was identical in all assays and-vvas less than 20 % saturating. Saturated rate of NADPreduction, 162 itmoles/mg Chl hr.

The value of a , was calculated according to e(qua-tion II.

,uEin/mimInc. Abs

AbsQuanta/Chli/min

Quanta/t1/2 Chl a tmin for tI/ 2 uEin-1

NADP ReductionBlue photoinhibitory light a = 47.5 %64.5 31.0 27,600 0.021 58033.0 16.0 14,400 0.055 7901.9 0.9 810 0.9 7300.25 0.12 110 6.1 670

Red photoinhibitory light a - 23.5 %71.0 16.7 15,000 0.082 12303.9 0.92 830 1.67 13800.56 0.13 117 10.0 1170

DCPIP ReductionBlue photoinhibitory light a = 52 %70.0 36.5 32,800 0.095 312035.0 18.2 16,400 0.2 32801.5 0.78 700 3.7 26000.22 0.115 105 10.9 1150

Red photoinhibitory light a = 21.5 %126.0 27.0 24,500 0.08 195068.0 14.6 13,200 0.155 20509.3 2.0 1,800 1.25 22502.0 0.43 390 4.8 1860

328 mA photoinhibitory light a = 29 %0.097 0.0280 25 1.7 42.50.056 0.0168 15 3.1 46.50.034 0.0102 9.2 4.8 44.20.0175 0.00525 4.7 8.5 40.0

0.510.380.400.45

0.120.110.12

0.100.100.120.29

0.070.0650.060.07

3.63.453.63.85

1039

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1040 PLA-NT

CUJRVE Joules t11/2 Ix t1cm2 sec. sec.0.3 A 0.615 13 8.0

o 0.400 19 760 0.193465 8.7

* 0.115 64 7.3

0.2-

IN

ao 1/2 R0

0.0 20 40 60 80 sec 100

EXPOSURE TIMEFIG. 2. Decay of dye reduction activity of chloro-

plasts during exposure to white light of various intensi-ties. Dye reduction assayed as in Methods. Light fromXenon arc filtered through 4 layers of glass, 37 cm ofwater and w ire screens. Intensity menasured xN-ith cali-brated thermopile.

To test for the reciprocity of time and intensityfor photoinhibition the experiments shown in figure2 were performed. Decay cuirves were measuiredwith intensities of white photoinhibitory light vary-ing over a 6-fold range. The data show a good fitto a straight line on a semilog plot. In spite of the6-fold difference in intensity, there was no differ-ence, within experimental error, in the amount oftotal energy necessary to (lecrease the rate of dyeredtuction to one-half the initial rate.

Resuilts of fuirther experiments uising photoin-hibitory light of selected wavelength regions and avider variation of dosage are shown in table III.The protocol tused in these experiments was i(lenticalwith that given in figuire 2, except that the incidentintensity is expressed in MuEinsteins per minutte. Thevaluie of energy per quantuim was chosen for thewvavelength for which the producit of the filtertransmissioni ancI the chloroplast absorption wasmaximal. Reciprocity of intensity an(I time is evi-(lent in all wavelength regions. For the range ofintensities and times for which this reciprocity holds,e(juationi I now may be modifiedl to:

R, = Rn c-c0at IIwhere I is the incident light intensity, a, the frac-tional absorption of the suispension aindI 0, the quan-tuim vield of the photoinhibition. A plot of a + vs.wavelength then is the action spectruim of photo-inhibition.

If the absorption of the suspension (aI) isknown, it becomes possible to calciclate the quiantumyield of photoinhibition: a plot of ' vs. wavelengthnow V-iel(Is the cutiantuim yield spectrulm. This quian-

PHYSIOLOGY

ttum yield is relative uinless the conceintratioins of theabsorbing moieties are known. The qtuanituim irnu-ber, thuis, is the number of qutanta absorbed perchlorophyll molecule necessary to (lecrease the ac-tivity of the chloroplasts to 37 % (1/c) of the orig-inal value.

Actionl (lad Quantum1tt7iNumnber Spcctra( ()f Phloto-inhzibitiont. The uniformity of the kinetics throuigh-ouit the spectruim, the high intensity of the Xenono arclamp, an(d the small cross-sectional area of the mi-crovessel (0.09 cm2) made it possible to measuirethe relative sensitivity of the chloroplasts to m11on1o-chromatic light of high puirity. However, the ther-mal (or (lark) decline in activity of the chloroplastsduring the relatively long exposure times (4-6 mill.)prohibited the measuiremenit of complete decay curvesat each wavelenigth.

AIn abbreviated method therefore, was uise(l. Ateach wavelength, control samples and photoinhibitedsamples were alternately assayed. From the ob-served ratio R,/Rt, we determined a4¢ and(I ll/(equiation II) giving the spectra shown in figuires3 and 4. WNre obtained the photoinhibition actionispectruim for wavelengths between 230 andcl 710 my(15 mi, half-band width) shown in figuire 3 with asingle preparation of chloroplasts. The 'UV spec-trum, ruin with higher resolution (9 mj half-bandwidth), is also presented in figuire 3 (inset ). Itshowxs a peak betweeni 250 and 260 my, a shouldleraround 280 m,u, and a pronounced minimulm at about240 m/.k. Below 240 m,u the sensitivity rose verysharply, buit light intensities suifficient for quianitita-tive measurements at these short wavelengths werenot available.

A more detaile(d action spectrum in the visibleregion is shown in figuire 4. Becauise of the verylow sensitivity of the chloroplasts to visible lightprecise data were difficult to obtain, even with theuse of interference filters instead of the moniochrom-ator. However, the spectra clearly implicate chloro-

4 0

10~~~~~~~~~~~~~~

2

200 250 300 m4

WAVELENGTH

FIG. 3: Relative inhibitorv activitv of selected wave-lengtlhs of light upon dx e reduction bv isolated chloro-plasts. Procedlure aind assay as described in text.

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JONES AND KOK-PHOTOINHIBITION.

40c

20c

0.'

Q.C

O.C

400 500 600 mM 700WAVELENGTH

FIG. 4. Photoinhllibitioni action and relative quantumnumlllber spectra. As figure 3, except moniochromaticlight isolated by interference and blocking filters. Dif-ferent symbols are different experiments. Quanta ab-sorbed per chlorophyll molecuile for t1/2 calculated as

in table III.

phyll as the sensitizing pigment. Plotted above theaction spectruim is the number of quanta absorbedper chlorophyll molecuile to give a one-half decreasein dye redtuction rate. The quiantuim requiirementappears to be constant over the visible spectruim,averaging aroun(l 3000 quianta/ chlorophyll for tl /2,in agreement with the data in table III (1/p = 4-5000/Chl).

Photoinhibition in Lyophylized i(cystis nidulans

Cells. The data of figure 4 shows a rise of quantumnumber at long wavelength. This suiggests thatthe red drop of the quianttum yield of photosynthesisfouind by Emerson and Lewis (5)) might also beevident in the spectrtum of photoinhibition. Therapi(lly diminishing absorption of chloroplasts atthese wavelengths made accturate measuirements ofthe light absorption of the clhloroplasts in the micro-vessel impractical. To explore this point fuirther,the actioin and relative qulantuim ntumber spectra ofphotoinhibition in visible light were determined forthe Hill reaction exhibited by lyophylized cells ofAnacystis niduil(ans. The pigmenits of this blue-green alga lend themselves to a clear separation ofthe 2 pigment systems and( their associated photo-acts ( 15)).

Lyophylized Ana(icystis preparationis (lid notevolve or consume 02 appreciably as measuired polar-

ographically uinless a reduceable suibstrate was pres-

ent in the light. \Vith K3Fe (CN)6) as an oxi(laint,the preparations showed 30 to 40 % of the 0, evolu-

tion of whole cells at all light intensities. The maxi-

mulm rate observed was 18 cell voluTmes per hour.

Similar rates of dye redutiction were foutnd spectro-

I. KINETICS AND ACTION SPECTRA 1041

photometrically. Howex er, the ivophylized prepara-tion also redtucedl the dye in the dark at a rate of 5 to10 % of the maximuim reduiction rate in the light.The dark reduiction continuied for loIng periods oftime an(d persisted after exposuires to light. At-tempts to annihilate the dark reductioni by proceduirevariations failed. No simuiltaneous uiptake of O., inthe' dark was fouind. Perhaps the (lye substitutedfor 0., as an electron acceptor in the (lisrupted res-piratory system of the lyophylized cells. All ratesof dye photoreduction were corrected for this darkreduiction. Significant NADP reduictionl, as seen byBlack et al. (4), couild not be obtained in the samereaction mixtuire uised for NADP reduictioin in.chloroplasts (8).

The activity of the Anwcastis preparatioins showe(da broad pH optimuim between pH 6 and 8, the ac-tivity dropping to zero at pH 4 andl 10. Althouighdistille(l water or 0.1 xl phosphate buiffer did notsuistain fuill activity, the molarity of the suicrose illthe suispending me(iuim was not critical. No dif-ference was fouind between samples prepared at 00or at room temperatuire. If stored dry at -200 tinderniitrogeni, the activity of the preparation was maiin-taine(l for over a week.

The photoinhibition decay culrves observed withthis material after long perio(ds of exposure departedfrom the uisuial logarithmic (lecay fouind( with chloro-plasts; the effectiveness was slightly less than ex-pecte(l. A dark period between exposuire and assayalso tended to lessen the effectiveness of the inhibi-tory light. Perhaps a small buit significanit amoulntof the repair reaction, typical of whole cells (seeDiscuission) is still active in lyophylizedI cell prepa-rations. Aside from this slight deviation, the lyo-phylized cells showed the same relation.ship between

°12 --o

0.2"U

0.1

o 84 ,~ " tt .r

.0 , i A

400 500 600WAVE LENGTH

my 700

FIG. 5. Photoinihibition action and relative quantuimnumber spectra. As figure 4 except for the use of lyo-

phylized Anacystis cells. Procedures as described in text.

0

1-c

TC

0)9

fA

U U

O/*

II

. I

.12* 000

0AAA18[ \\ °/;0

A A

04 0

0~~~~~~

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1042 PLANT

the intenisity of visil)le preilluiminatioin light aindl thetime of exposulre as that fotund for chloroplasts.Action spectra of photoinhibition were ruin in amanner i(lentical to those of chloroplasts, aind anexample is shown in figulre 5 .

The action spectruim (a p) is very similar toaction spectra of photosynthesis for this (15) andother blue-green algae (11), the highest activity be-ing in the region where the accessory pigment phy-cocyanin absorbs most strongly. The maximtumeffectiveness (4 = 0.2a'a) is very close to thatfound for chloroplasts (4) = 0.1 /a, see fig 4) sincethe absorption of the Anacystis suspension uised wasabouit douible that of the chloroplast suispension. Therelative quiantum requirement (fig 5, top) shows abroad minimuim between about 550 m,u and 650 mnu,and a clear "red drop" at wavelengths above 650 m,uand below 550 m,u similar to O., evolution in Ana-cystis (15). The high requiirement at long wave-lengths and around 500 mjA indicates that chlorophylla and carotenoids are relatively inactive. Bothspectra show inicreasing sensitivity at 405 mu sim-ilar to the UV effect encouintered in chloroplasts.Pigment connected to system II thus seems to bepredominaint in the sensitization of photoinhibitionby visible light of dye reduction.

Discussion

It should be emphasized that in all experimentsreportecl here, exposuire to photoinhibitory light wascarrie(I ouit in the absence of exogenouis chloroplastoxidants. It is assuimed, therefore, that even thelowest inteinsities are effectively above light satuira-tion.

The uise of chloroplasts withouit suibstrate greatlysimplifies the study of photoinhibition. In wholecells, 2 processes compete with or oppose photoin-hibition: A) photosynthesis itself, which dlivertsquanta in weak and moderate intenisities and( B) atemperatuire dlependent restoration mechanism whichacts duiring and after exposure to visible light (16).No sutch repair mechanism could be found in spinachchloroplasts, but a small activity of this type wasobserved in the lyophylized Anacystis preparations.

The observed reciprocity of intensity and ex-posure time, found with chloroplasts in visible light,is not consistent with previously reported resuilts forwhole green algae (17) which indicated proportion-ality with the squiare of the intensity. This differ-ence can now be explained by the 2 opposing reac-tions in whole cells, lower light intensities appearingrelatively less effective. Photoinhibition by UVlight has been assumed to follow the reciprocity law(2, 13), but proof for this was again difficult be-cause of secondary metabolic effects in whole cells.

The observed rate law precludes heating of theenvironment as the cause of inhibition. In this casethe halftimes of photoinhibition wotuld be dependentupon light intensity and not the absolute dose ofquanta. A multiple hit mechanism, where 1 quian-

PHYSIOLOGY

ttum prepares a sensitive locusi for damage b) a sec-ond qtuantuim, wotuld also show a (leviation from thereciprocity lass uinless the half-life of this sensitiveloctus was long compare(d to the rate of quianitumabsorption. At the loss est intenisity in which wNeobserved validity of the reciprocit) law, each chloro-phyll on the average absorbed a quiaintum twice persecond; the life time of the sensitive locuis thuts wouildhave to be more thani 0.5 seconid. If one assuimesthe (lamage occuirs in a trappinig center (see below)and that there is one trap per 40 to 400 chlorophvlls,(system II or system I respectively) (20), eachtrap wsotld then receive a quiantuim abouit once every10 to 1 msec respectively, and the requiired life timeof the sensitive locuis wotuld thuis be correspon(linglyshorter. Even then, the dlamage cauised by the sec-ond quantuim wouldl have to occur with a very lossprobability (qtuantulm yield).

Quianta absorbed by the photosv'nthetic pigments,tin(ler circumstances When photosynthesis is takinlgplace, are transferred to a uiniqute pigment which isresponsible for the photochemistry of the system(reactioin center or trap). The lack of blelachingof the absorbing pigmenit duiring photoinhibitioii,the Q10 of one, and the concomitalnt decline of qulan-thim yield, satturatiing rate and maximuim flash vield(22) imply' that the inaictiv-atioIn occtirs in the reac-tion center or a relate(d moiety. Thuis, qutainta cauis-ing inactivation mtist also be tranisferred to thisreaction center rather than being (lispose(l of as heator fluorescenice in the pigment bed. Photoinhibitionby visible light thuis is caused by a secondary' photo-reactionl in the trapping ceinter which occurs with alow but definite probability' and(I which resuilts ininactivation of the reactioni center.

One can asslume that photoinhibition occurs withthe same quiantum yield, regardless of intensity, orthe presence of substrate. To just saturate _NADPphotoreduction by chloroplasts, each chlorophyllmolecule absorbs one qtuanitum every 4.5 second (as-stiminig a qtuantuim yield of 0.5 equation/Eimi anid amaximtum rate of 220 ,umoles mg Chl hr). On thisbasis, the lowest visible light intenisity used in ourinhibition experiments (2 quanta absorbed per Chl-sec, table III) wNas about 10 times light saturationi,whereas the highest initensit)y use(d (about 500 quantaabsorbed/Chl-sec) was 2500 times saturationi. Theextrapolatedl half-time of photoinhibition in just sat-urating visible light equials about 2 hours for .NADPre(luction or about 4 hours for dye redluction.

The red drop in the actionl spectra of both chloro-plasts and Anacystis ( fig 4, 5) requtires either: thatthe sensitizing pigments belong to photoact II, orthat a balanced influix of quanta between the 2photoacts is necessary for inhibition to occur, similarto the requirement for complete photosynthesis. Analternate explanation rests uipon the assumptionithat dye reduction occurs via photoact II only, sothat the data of figures 4 and 5 would pertain onlyto photoiinhibition of system II. This question willlie considered in more detail elsewhere (14).

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JONES AND KOK-PHOTOINHIBITION. I. KINETICS AND ACTION SPECTRA

As the absorption of the chlorophylls and carot-enoids, as vell as the action spectrum of photosyn-thesis decline below 430 m,u, the absorbing pigmentfor photoinhibition in UV light cannot be a photo-synthetic light absorber. The wavelength at whichthe transition occurs from 1 type of inhibition to theother appears to be arotund 420 m,u.

The action spectrum of photoinhibition in theUV region follows the absorption spectrum of manycompouinds fouind in the chloroplasts (notablv plas-toquiinone, linolenic acid, and perhaps a pteridineanalogiie). It also has a striking resemblance tothe action spectrum of the bleaching of Euglena cellswhich was attributed to the destruiction of a nucleo-protein (23). Identification of the pigment re-sponsible for the inactivation of photosynthesis isdifficult because most of the moieties absorbing inthis region are probably adversely effected duringexposuire. The nature of the sensitizer in UV lightwill be discussed in a suibsequent paper (14).

Literature Cited

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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.