ActionSpectrafor Evolutionby Chloroplasts Substrate ... · The light ex-posures were on only long enough to permit the spike to reach its highest value. The height of the 02 pro-duction

Action Spectra for 02 Evolution by Chloroplasts with & without Added Substrate,for Regeneration of 02 Evolving Ability by Far-red, & for 02 Uptake1

D. C. ForkDepartment of Plant Biology, Carnegie Institution of Washington, Stanford, California

Recent studies (3, 6, 10, 12) have shown thatphotosynthesis requires two photochemical reactionsdriven by different pigments. Furthermore, it hasbeen suggested (3, 10, 12) that these two light re-actions occur in series. Absorption by chlorophyllb and by chloroplhyll a,7, (so-called accessory pig-ments) are thought to bring about oxidation of waterand evolution of O., with a reduction of an acceptor[X, or perhaps plastoquinone (12)]. A secondphotochemlical step, mediatefl by a long-wavelengthform of chlorophyll a, causes reduction of TPN (oran artificial Hill oxidlant) with the electrons comingfrom reduced X.

If some oxidized X were available, O. evolutionwould be possible without a Hill oxidant and with-out the functioning of the long-wavelengtlh chloro-phyll reaction. (It may be recalled in this regardthat a limited O., evolution upon illumination ofchloroplasts suspended only in sucrose solutions hasbeen known for over 80 years.) Such a recurring,but small, evolution of 02 by illuminated chloroplastswas also observed in the present study. The assump-tion that this 02 evolution in the absence of a Hilloxidant is a reflection of the activity of the acces-sory-pigment reaction can be tested by comparingthe action spectra for O. evolution before and afterthe addition of a Hill oxidant. The action spectrumdetermined in the absence of an oxidant should haveits peaks at wavelengths corresponding to the ab-sorption of chlorophyll b and chlorophyll a,70. Inthe presence of a Hill oxidant the action spectrum for02 evolution would be shifted to longer wavelengthsand reflect the functioning of both pigment systems.

It is of interest, moreover, to investigate howchloroplasts which lack a Hill oxidant are able tocontinue to evolve limited amounts of 02 over aperiod of several hours and to determine what fac-tors influence this 02 evolution.

A similar study has been made recently by de-Kouchkovsky (personal communication) who usedmaize chloroplasts.

Materials & MethodsSwiss chard (Beta zvulgaris L. var. cicla) used

in these experiments was grown in a garden at Stan-ford. Mature leaves were picked, rinsed with dis-

1 Received Aug. 1, 1962.

tilled water, and chilled. Preparation of chloro-plasts was done in a cold room at 3 to 40 in dimgreen light. Leaf blades free of large midribs wereground in a solution (hereafter called grindingsolution) containing 0.4 M sucrose, 0.01 WI NaCl ant0.05 ^i K,HPO,-KH,PO4 buffer (pH 6.9). Theslurry was strained through eight layers of cheese-cloth and centrifuged at 200 X g for 2 minutes. Thesupernatant fluid was centrifuged at 1000 X gfor 8 minutes to sediment unwashed whole chloro-plasts.

02 changes in chloroplast preparations weremeasured by a Pt electrode covered by a thin film ofteflon that was permeable to 02 but protected theelectrode surface from the deleterious effect of pro-teins and other substances in the chloroplast prepa-ration. M\oreover, the teflon-covered Pt electrodepermits the measurement of 02 exchange in solu-tions such as quinone and potassium ferricyanidewlhich wouldl otherwise react at the bare Pt electrode.

The electrode assembly used in these studies wasadlapted from a bare Pt electrode describedl by Frenchet al. (7). The use of Ag-Ag2O as a referenceelectrode in 0.5 N KOH under a plastic film was sug-gested by Clark et al. (2) and by Carritt and Kan-wisher (1). The electrode shown in figure 1 com-hines features of the Clark (or Kanwisher-Carritt)menmbrane-covered electrode with the Blinks-Haxoelectrode principle (8). The base was clear lucitean(l was designed so it could be positione(l repro-(lucibly under the beam from a monochromator.The Pt electrode, 1 X 15.3 mmll, wvas set flush into thelucite. A rectangular Ag-Ag2O reference electrodle,22.6 X 28.2 mm, with the center cut out was madefronm pure silver 0.8 mm thick, and was mounted sothat it was also flush \ith the Pt electrode. Thelucite was grooved underneath the Ag-Ag.O electrodeto make a pool for the KOH. The area of the Ag-Ag.O electrode was about 75 times that of the Ptelectrode. Both electrodes were coveredl with a 6.4A thick film of teflon held down by a rubber ring.A thin film of KOH over the surface of the elec-tro(les was trapped under the teflon. C(c-nnectionbetween the KOH pool and the KOH under the tel'-lon film was achieved through channels cut at inter-vals in the space between the electrodes as shown iIIthe insert of figure 1. Leads soldered to the under-sides of the electrodes were brought out under therubber ring, sealed with beeswax, and attached toposts on the base.

323

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324

Cover

Gasket

Dialysismembrane

Cells

C=~ Rubber ring

Teflon

FIG. 1. TIefloni-covered Pt electrode used for nicas-uring rate of 0° production by chloroplasts. It is de-scribed in the text.

Chloroplasts wvere spread in a thin layer on theteflon directly above the Pt electrode and( coveredwith a piece of moistened dialysis membrane held inplace by a rubber gasket and cover (fig 1). Fltuidcirculated through the cover and over the membraneas shown in figure 2. A centrifugal pulI) xN ith ahold-up voltime of about 3 nml dIriven by a synchron-ous motor provided a flow rate of 410 iil/nmin. Gasconcentration w,as adjustefd and held constant by agas exchanger made of 4 cm glass tubing partly filledwith sections of snmall tubing. The idea of usinga flowing system came originally from P'rofessor'Jack Myers (7). This systema provided a constantflow of fluid of constant 02 tension over the chloro-plasts, and( thus changes of 02 tension at the elec-trode may be regarded as resulting from chloroplastactivity. About 50 ml of fluid was required to fillthis circulating systenm.

The Ag20 coating was formed originally hy pol-arizing the Pt electrode at -0.8 v with reference tothe Ag electrode and leaving it in the air for a day.The resulting Ag-Ag9O reference electrode vas thenready for use. Since the teflon membrane is perme-able to C02, the electrode assemlbly, when not in use,was kept in a (lesiccator over NaOH pellets to pre-vent CO, fronm neutralizing the KOH in the elec-trode.

When in use, the Pt electro(de as ppolarizel at

PLANT PHYSIOLOGY

-0.8 v by a circuit with 1.35 v Mallory (RM-42RT)mercury batteries which also powered a balancingcircuit. Voltage across a 100-ohm resistor in serieswith the electrode was measured as described previ-ously (7) by a Beckman model 14 chopper amplifier.

The electrode without the cover showed a lightresponse and caused the recorder pen to go in thesame direction as that caused by 02 production. Thissignal apparently resulted from a light reaction atthe Ag-Ag2O electrode. When the cover was inplace the Ag-Ag9O electrode was sha(le(d and there

Pt was no longer a response to light witlh the highestintensities used in these experiments.

The experiments were conducted at room temper-ature (19-210) using unwashed whole chloroplasts

Ag-Ag2O suspen(led in grinding solution.

Results7'lie Tine Coutrse of 0. Ezvolution tIt 630 niu.

Figure 3 shows curves of O. evolution I) chloro-plasts exposed to the samle intensity of 650 my lightun(ler anaerobic and( aerobic condlitionis. The ana-

LightIl | Teflon covered11 Pt. electrode

Sync. motor pumpFi(.. 2. Circulating and gas exchange system used

with the tefloni-covered Pt electrode. It is described inthe text.



FORK-ACTION SPECTRA FOR 02 EVOLUTION BY CHLOROPLASTS

60

40

Ii 20

CO0 6

x

0 60

0i

20

0

I

undsolu0.01(pBlastB.(99.

Anaerobic A

Daki-650mu1-4Dark-4-650m * Dark *1650m4-Dark

Aerobic B

5 10 15 20 25

Time,minutes

FIG. 3. 02 exchange with illuminated chloroplastser anaerobic (A) and aerobic (B) conditions. Thettion used in the circulating system was 0.4 M sucrose,M NaCl, and 0.05 M K2HPO4-KH2PO4 buffer

I 6.9). A dark interval of 17 minutes separated theexposure of part A from the first exposure in partDuring this time air, instead of purified nitrogen.995 %, N,), was bubbled in the gas exchanger. The

dashed line traces the course of the dark base line whichdrifts upward in B since a complete equilibrium has notyet been obtained in the fluid bubbled with air. Deflec-tions above the base line correspond to net 02 evolutionand deflections below to net 02 uptake. Intensity of the650 m,u beam = 803 ergs cm 2sec-1.

erobic curve (part A, fig 3) shows a high initialrate (spike) followed by a rapid decrease to a steady-state net 02 evolution after 3 minutes. The subse-quent spike after 3 minutes of darkness was smallerthan the initial one and smaller also than a later one

following 10 minutes of darkness.

1-5

0.

c 4

.20.

I

50 _

O 1 I, I10 2 4 6 8 10 12 14 16

Time,minFIG. 4. 02 production spikes from brief exposures

to 650 m,u given at dark intervals after a previous 4-minute exposure to this 650 m,u beam. The light ex-

posures were on only long enough to permit the spiketo reach its highest value. The height of the 02 pro-duction spike produced by the initial 4-minute exposurewas 62 units. The intensity of the 650 my. was 803ergs cm-2sec-1. Gas phase, N,.

Figure 3B compares the effect of exposing chloro-plasts to the same intensity of 650 myA light underaerobic conditions. Upon illumination, the 02-pro-duction spike is seen again. However, the rate of02 production declines in the light reaching a steady-state net 02 uptake after about three minutes. Thechloroplasts show an 02 gulp when the light is turn-ed off. The dark base line, which slowly drifts up-wards, is attained again after about three minutes.

Chloroplasts retained their ability to evolve 02

70.

60-

501

40k

30k

20

10

4

-i

C

0

.0I-

0

6-

I4

DO 500 600Wavelength.mm

700

0 200 400 600650m. light intensity, ergs cm-2 sec-1

FIG. 5 (upper). Action spectrum for the productionof the 02 spike by Swiss chard chloroplasts. The cir-culating solution used is described in the legend of figure3. The action spectrum was determined by using equalincident quanta (477 ergs cm-2sec-1 at 650 m,u). Atpoints below 580 mu the light intensities were limitedby the output from the monochromator. These pointswere adjusted upwards appropriately to correspond withother points in the action spectrum. Temp = 19.50,gas phase, N2.

FIG. 6 (lower). Relative height of the 02 productionspike as a function of 650 m,u intensity for chloroplastsused for the action spectrum given in figure 5.

325

I I

480mI

I I I

Darkl--650aw--i--Dark-4-6-SO.i, + - Dark -4- 0--k

u l



PLANT PHYSIOLOGY

for manlyl hours even in somiie cases after being lefton the electrode overnight in the (lark.

Tlhc Recover)v of the O.-czolving Capacity in theJ)Dark. Chloroplasts wtere exposed to 650 m,u lightfor 4 miiniutes. In the following (lark perio(l shortexl)osures to 650 mi wvere given just long enougl(about five sec) to allow the spike of O., pro(ductionto reaclh its highest point. Tllis slhort exposureampl)le(l the recovery without retar(ling it appreci-

aibly. Pigure 4 shows thlat the 0., pro(dution spikehias recoxere(d lhalf of its originatl hleight after aboutl.5 miiniutes in the dlark undler anaerobic conditions.It can be seen that the build-up continuiies for a con-si(ler-able tilmie in the (lark, and thlt a nma.ximum wN-asnlot attaine(l after 16 minulttes.

7'/le .qctioii Spectri-1{1 for 0., EvZolUtioimi. Theaction spectrum for the productioii of the 02 spikewas measuredl anaerobically for chloroplasts sllS-p)eil(le(l in grind(inig solution w\ hiclh loes nlot conitainia Hill oxi(laInt. For this purpose a 650 llt-referencebweami was turned oln. as (lescrile(l above. just longellouIghi to allow- the milaximilumil rise in the 02. spike,about five to teIn seconIls. This was repeated after 1

iniute (lark intervals until a constant r-espoimse wal.sattaine(l. A silmiilar five to ten seconld( exxposure toto the monoclihrolimator beam wtas thieni given, againljust loIng ellough for the peak rate to be reachedl.After at 1 iminute dark interval shior-t exposures tothe 650 mit-reference beam wx ere gixven as before,

anld w-ere follo-wed by an exposuire to the milonlo-chromator beam set at a different wavelength. Theratio: 0. spike produced by exposure to the nmono-chromllator beam to the O., spike from prece(ding 650itu-referenice beaml wvas usedl to plot the action

spectrumiii shown in figure 5. Peaks occur aro(I650 anld( 480 itiju which corresponld( to regions ofniiaximllUImt albsorption by chlorophyll l. \ sihoul(lercan he seenl at about 680 lit which would Correspon(lto chlorophyll a1 absorption. A check of the 0., pro-(luction Sl)ike vs. 650 miti intensitv ,howevd that, at650 iti, arnd at the intelnsity utsed for the actioin spec-trumiii measurements, the response fell on the linleai1-region of the curve (fig 6).

Effect of Lighit on the Recovrc'rY fof2-ez'o'inljC(apacitv'. The 02 production broutglt about by tlle650 mit beam was ilnfluenced by a prexious light ex-p)osIre. To stud(l thils effect, sholrt exposures to650 mitA liglht were giv-en an1 anaerobic pre)arati(inat 1-miultte initervals unitil a constant response -wa.>attaine(d (ftig 7 upper left ). A 2-mlilnute exposure tothe (50 mitA beam was theln giv-en. This exiostirc-w-as followvedl bv a 1-mlinute dark inter-xal whereupona far-red liglht (730 mit) wvas turlne( Ontorf 3 minutte-s.Near the end( of the far-red exposure. the 650 mubeam xas briefly superimiipose(d oni the far-red. Thleeffectiveness of 730 mit in increasing the 650 mituO2-spike may be exl)ressed as tlle ratio of the hei-hltof the 6(50 mit O.-spike (during aan exploRure to 730

I I:L ZE E0 0

) L) 1I650mp I -x I

I I a a

IE 730muj

20K

_a aE E0 0LO Lo

~( (0

650mp

~~EE0Z. E 0 CD

0 LO) (0_x I-

650mp 14 Dark - 650mpO Dark -

-Y -Y

aC

5 10-II I ~~~~~~~I

15Time, minutes

FT(;. 7. TIhle effect of 730 miu liglht (Ot the 650 mit-O, production spike in Sxx-iss clhard chloroplasts suslende(l i

the solutioII clescribed for figure 3. Thle loxx-er part of the figure is a (lark control. See text for details. Iltelisityof 650nm,i 803 ergs cmi --sec-I and the 730 mu = 459 ergs Cllm --'2ec-. C,as phase, N.

40

L-

a

-c

0r--U

x

CA

cm

+

r4

40

20[

0

0

II

326




Cj 60 Dark 480m; 'Dark |- 73OmP Dark

@ |-~~~~~~~480m&j-.

40

-c

x

200

0

0 5 10Time,minutes

FIG. 8. Time courses of 02 production in 480 m,light (883 ergs cm-2sec-1) for Swiss chard chloroplastssuspended in solution given in figure 3. 730 m,u back-ground light (459 ergs cm-2sec-1) also increases the0., production spike from 480 m,u light. Gas phase, N9.See text for details.

X2.0

1.8 73OmlU

background0

1.6 -

0

1.4 -

0

(0

0 So 100 150Time, sec

FIG. 9. The effect of 730 mA exposure time on the0Dproduction spike brought about by a 650mct lightsuperimposed on 730 m,u. After 730 m,u light had beenon for the time indicated, a 650 m,u beam was turned onlong enough for the spike to reach its highest value.Both beams were then turned off. The ordinate is

myA divided by the height of the 650 mu 02-spike be-fore an exposure to 730 myA (in this case 1.37). Acontrol, with an equivalent dark period placed be-tween the 650 m,t exposures, (lower part of fig 7)gives a ratio of 0.77.

A similar time-course curve for 02 productionand enhancement of the height of the spike is alsoobserved when a 480 m,u beam is substitute(d for 650m,u (fig 8, compare with fig 7).

Figure 9 shows the increase in the 650 mn 02-evolution spike brought about by a previous expos-ure to 730 mn for varying times. Since the build-upby 730 n,/ light is relatively slow, a simultaneous ex-posure to 730 my and to 650 my light does not givenoticeable enchancement. The effect of the inten-sity of 730 niu light on the increase of the 650 nmi 02-spike is shown in figure 10. With the 1-miinute ex-

1.4 l

.io00~~~~~~-o 1.3

On

fi 1.2L3

0~

N /0

(._0

0 100 300 500Light intensity(730mp), ergs cm-2 sec'

FIG. 10. The effect of 730 mju intensity on the 0.,-production spike from 650 m,u light (803 ergscm -2sec- 1). The 730 mu beam was on for 1 minutebefore the 650 m,t beam was superimposed on 730 m,A.See figure 9 for a description of the ratio used in theordinate and the method of presentation of the twolight beams. The point at zero 730 m, intensity indi-cates the increased 0,-production spike brought aboutby substituting a dark period for a 730 mAt exposure.

posure used, the effect was half saturatedI at about240 ergs cm-2 sec-1. At higher intensities there isan appreciable amount of 02 evolution sustained in730 myA light and a corresponding drop in its effec-tiveness in causing increasedl 02 production from650 m/A.

The Actiont Spectrum for the Light-indu(ced Re-cozery. The action spectrum for the effectiveness

expressed as the ratio: height of the 650 mu O., spikeafter a 730 mu exposure (or dark period for dark con-trol) /height of the 650 mA 02 spike before a 730 muexposure. The intensity of the 730 m,u beam was 570ergs cm-2sec'- while the 650 mAt beam was 803 ergscm-2sec-1. Temperature 20.1°. Gas phase, N2*

327



PLANT PHYSIOLOGY

increasing 0, production from a 650 nw chromatic exposure, gave a ratio of increased 630)lotted from the same set of measuremients mitA 0° production equal to 1.03.,ed for figure 5. A point on the or(linate A Comiiparison of Dark and of Light-indticed Re-I1 correspon(ls to the ratio: height of the coverv. An obvious question is whether the slowion spike from the 650 nw-reference beaimi (lark reczovery of 0 -evolving capacity and the moir-enin after a previous exposure to mono- rapid light-induced recovery are similar or ditffe entight) divided by the height of the O, pro- processes. Do they both eventuallv give rise to thieke from the 650 mi-reference beam. The same level or are they additive? These (questiOn.<this action spectrum occurs around 730 were investigated by observing the response to shlortfar-red peak positions which were fountd exposures (5-10 sec) of a 650 in-reference beamlieasurements of this action spectrum oc- given at varying dark intervals after a 730 inw ex-713, 720, 725, and 729 mn. The position posure (fig 12). 650 nwA exposures wvere given at

peak is not certain, but it is clear that 0, 5, 20, 40. and 80 seconds after a previous 1 nuin-light can also bring about a response ute exposure to 730 nitu. A one minute dark perio(lthat of far-red light. A dark control for was substituted for the 730 nmi/ light in the (lark coll-vith a (larkperiod substitute(l for anmonio- trol. The recovery- brought about by 730 nm re-, , , mni ainis after the lighlt is turnied off an(I is added to

that brought about by a (lark period. This suggeststhat a period of (larkniess or ain exposure to 730 nwimlight prodluces the samze effect.|A Low Intensity Plotooxridatize Process anid itsActioni Spectrumi. 0, pro(luctioni by these clhloro-plasts was inhibite(d by DCMIU2. Figure 13slhowsthe effect of adding this herbicide to an aerobic cir-culatinig solutioll. The 6150 nwiA liglht was again turni-K ted on 36seconds after adding the DCMU. TheO.spike is smiialler than before and a rapid dr1-ol) in 0,*s^4 evolution is seel ( luriiig thle( 50 litk exposu re. A

* smialler O. gulp is als) seen wh-lieni thle light is ttiluie(5

SOs50 6 50 750 off. Subsequent expostures to6(50nwly lighlt restult inWavelength(in mu1)of exposure

anlet uptake ofO._. The light-dependent U.,uiptakeused prior to 650muj exposure becomes reproducible about five minutes after poison-

ing antI the0., gulpdisap)pears.These effects are interpreted as follows: wbile

the light is on 0, isproduce(l fromii astubstratethlatis rapidly used up. At the sametimle a respiratorystilmiulation is inducedh! the formation of a pro(luct_of the light reaction.\Vheln the light isturned1off/o

the respiratory stimulationpersistsunltil this photoprodtuct isused up,thu s giving the02. gulp. DCM\Ulpoisons the0., evolution butn ot theplhotostimulatedresp)iration whichcanlnowVhe measure(l without in--I 730mi

w0Z terference by 0, ev'olt tion.exposure ....................................Theaction spectrum1for O, ui)take was tleterminl-

e(t for this chloroplast suspeInsioin by the l)rocedlureo for the automatic recor(ling of action spectra using-Imin doris-'s<

quaccll unu bers of incl d(le nt quanta (7). Figure 1 4shows that the peak in the re(l regioni for thlis effectoccurs at 690m. . 0., uptake as a function of inl-

204060 80 ioo 120 140 tensity of 690 mnt is given in figure 15. Sinice thleTime,sec

curve is half saturated at about 250 ergs Cm- 2-sec- I

(p)Thme.ao spectlty e action spectrum wvas dletermiinied by using a quan-usppligh epeation inctr the ab-rouc tumi-i flux equivalent to that sho\vin by'the position ofUupon exposure to650 mit light.The data the arrow (67.2 ergs CmI- 2sec-onn the saturationiure and for figure 5 were obtained from the curve. Upon addition of triclhloroacetic acid fTCArimient. The values for the ordinate wvere to the circulating solution to a final conicentrationl ofas described in the text. 1..2c, the chloroplasts turnedd olive l)rownand 0

2(lower). 09-evolution spikes obtained from uptake xvas abolished at the light intensity used for

a 650 nls beam given a"t varyinig dark intervals after a1-miinuite 730 mit exp-osure (Ior (lark period for dlark con-trol).)I.tensity of the 730 iAba,eam 459 ergs

cm~-2sem~ ~ 650niy 8903 ergs ,e- C

2 D C MU, 3-3(3.4-dichloropheniyl)-1,1-dimethylurea,

xxaskinddly supplied by Dr. H. J. Thorne, E. 1.I) u Pont

&Coo..,WNiilminiigtont.

of lighit inbeam was fthat was usof figure 102 producti(given 1 rchroimiatic 1ductioIn spilred peak irniju. The iin other mcurre(l at 7of the blueblue-greensimilar to 1figure 11, v

1.4

c 1.2

E

1.000

01

C0

-00.

E0

CF01

0

01ona

1.4_

1.2

1.00

FIG. I11of a previotion spikefor this figisame experdetermined

FIG. 12

328




8

6k

2k~

O L012 4 6 8 10 12 14 16 18

Time, minutesFIG. 13. Swiss chard chloroplasts exposed to 650 m,A light before and after the addition of DCMU. The

final concentration of the DCMU (added dissolved in 0.2 ml 95 % ethanol) was 2.7 X 10- M. The same intensity650 mni beam (803 ergs cm-2sec-') was used for all exposures. Gas phase, air.

0~

600 650 700 750

Waveleng+h,mpFIG. 14. The action spectrum of O2 uptake for the

aged (24 hr) chloroplasts which had been treated withDCMU as described in figure 13. See text for details.

the action spectrum. There remained, however, a

large 0O uptake when the TCA-treated chloroplastswere exposed to bright white light. This 02 uptakemay be a result of photooxidation reactions similarto those described by Franck and French (5) where-as the present photooxidation process is measurableat low intensity (i.e. with a quantum yield roughlycomparable to that of photosynthesis). This processis believed to be that described as respiratory stimu-lation in Porphyridium by French and Fork (6).

The Effect of Potassium Ferricyanide on 0^ Pro-duictiont. A comparison was made between the 0,

production of chloroplasts to which no Hill oxidanthad been supplied and the 02 production after the ad-

I

_N0k

600

Light intensity, ergs cm-2 sec'

FIG. 15. 02 uptake as a function of 690 m, lightintensity for the chloroplast suspension treated withDCMU as described in figure 13.

c0e-ux'C

N0

0

0

-& 1650 Dark- 650 [KDark 650+Dark 650 Dark-.a mp mP MP muIn

AddDCMU

lIi--lI I I I I

329

4_

)



PLANT PHYSIOLOGY

60F

40F

201

0

1

Dark Dark65Ompi 1-73Omi -4 Dark - I 650mp k 730mp -- Dark

650m; 1-1 I 650m j I

160

120

80

40

0

I

0 5 25 30

Time, minutesFIG. 10. The effect of l)otassium ferricyanide on the shape of the time course curves of O., evolutioni and on

the relative rate of 0., evolution in the steady state. Potassium ferricyalnide (final concentration of 4.3 X 10-; M-)

\\as adde(l to the circulating solution durinig the 17-miniute dark interval. Gas phase, N.,. The initetnsity of the650 iii,A heamii used was = 803 ergs cm- 2sec-' the 730 m,A beam = 459 ergs cml-2sec -.

(litioll of potassium ferricyanide. The result obtalil-e(l in such an experimiient is shoxxn in figure 16.The left part of the figure slhows O. exchanges ob-taiiie(l whlen. circulating only the grindinig solution.The first exposure to 650 ma gave a time course of0, pr-oduction very similar to those (lescribe(l earlier.The ability of a previous 730 mu exposure to increasethe 650 mii,u O, spike is also seen. During a (larkiniter-val of 17 miiinutes potassium ferricvanide wvas

a(l(le(l to the circulating solution. A subsequent ex-

posure to the same 650 miU beam ipro(luce(l a steadly-state rate of O, evolution which was about 25 timleslighier than that obtained previously. Moreover,

the timne course of O, evolution at 650 m,u in the pres-

enice of ferricvanide lacks the 02-production spike.pre\-ious exposuure to 730 il no longer stimlulatespro(luction in the 650 mig beam. In somiie experi-

menfts with ferricvanii(le the time course for pro-

(Iuctioni exhibited a long-term in(luction effect witha protracted O., spike. In these cases, however,stea(lv-state 0, pl)ro(luction xwas attained after abouttlhree miniutes in the light.

/itc Jctioii Spectr-ion1 for 0., Evzolittion w,cit/iPotossilsstii Ferricv oaii(c. The actioil spectrumii for

0, evolution with ferricvanide was obtained by usingautomiiatic proce(lures mentionle(l above and( is shownin figul-e 17. It lhas a main red peak at 678 iiiu anda broad slhoulder in the 640 to 650 region. A checkof O, production as a functioni of intensity of 675imu liglht sho\\wed that the actioIn spectrum wvas dle-terimiine(I well wsithlini the linear region of the satura-tioni curve.

O., P)rod nctionl fr-omll (h1iloro opl/st Frog in outs.\VNaslhe(d chloroplasts were rupturled osmnoticallyA byresuspending in 0.01 m NaiCl and 0.05 -m phosphatebuffer for 10 miiiniutes. Centrifugation at 18,800 X glfor 15 miniuites sedimiienite(d the fragimielits. The etf-

c0

--a0

L._

aC\i0

0

cr

550 600 650Wavelength, mp

700

FIG. 17. The action spectruml for O., evolutionl inI

Swiss chard chloroplasts to which potassium ferricyanidlelhad been added. The ferricyanide concentration xxas thesame as that used in figure 16. At 675 n1/lL the quantumflux \ as 365 ergs cm 2sec Gas phase, N-.

I-

CAc

0

0

cr

13v30



FORK-ACTION SPECTRA FOR 02. EVOLUTION BY CHLOROPLASTS

fects described in figure 3 for whole chloroplastswere observed with fragments which also retainedan ability to evolve 02 for many hours after isolation.

Discussion

It is clear that illuminated chloroplasts are ableto evolve O. even though no Hill oxidants are pro-vided. The cotmipoinents responsible for this 02 evo-lutioni miust be bound to the chloroplasts since a con-tinual dialysis of water-soluble substances takes placeinto the circulatiing medium which passes over theclhloroplasts on the electrode.

This endogeinous evolution of 02 apparently- re-sults largely fromii the functioning of chlorophyll bsince peaks at 650 and 480 miy in the action spectrumfor the 02-production spike were measured usingchloroplasts without an added Hill-oxidant. Theshoul(ler in this action spectrum at 680 m/l indicatesthat at least one form of chlorophyll a may be active.The proximity of the 0)-evolving step in photo-synthesis to the accessory pigmnent system has beenpostulated recently by Witt andl co-workers (12), byLosada et al. ('10) and by Duysens' group (3).Witt's scheiie, derived from studies on absorptionchaniges. suggests that electrons from water reducean unknown substance, X, (plastoquinone) andl thatthe accessory pignment system is closely connectedto this reductionl. The oxidation of this reducedcomiipoun(l ( X- ), in turn, ultimately dependls uponthe activationi of chlorophyll a whereby electrons,remloved froni X -. ultimatel- reduce TPN (or anartificial electron acceptor such as ferricyanide).

Hill (9) (liscoveretd that substantial quantities ofO2 couldl be produced from chloroplasts suppliedlwith an appropriate hydrogen acceptor. In thepresenit experimiients, likewise, the addition of ferri-cyanide to the circulating solution allowed steady-state 02 pro(luctioni to proceed at a rate w-hiclh wasabout 25 times faster than the low endogenous 0OprQduction observed under anaerobic conditions.Not only was the rate of 02 production higlher withferricvanide but also the action spectrum for O. pro-duction had a major peak at 678 miA in a region ofabsorption by chlorophyll a and a shoulder arounid640 to 650 itn corresponding to chlorophyll b absorp-tioIn. This actioni spectrum may be comparedl witha norimial action spectrum for O., evolution by greenplants (cf UTlz'a taeniiata of Haxo & Blinks, 8).

The 0-production spike seen initially upon il-luminationl would depend upon the amount of anatural electron acceptor which is present in anoxidized foriii. If no endogenous (or artificial)Hill oxidlanit is present, the unknown componentwvould accumiiulate in the reduced state in the lightanid the rate of O., production would decrease. Thetinme course of 0.. evolution in chloroplasts withoutadded ferricyanide has, initially, a burst of 02 pro-duction which drops rapidly in the light to a lowsteady-state 02 evolution (anaerobic chloroplasts).This small net 0. evolution could be supported bya slow endogenous re-oxidation of the unknown re-duced compound.

A re-oxidation of the unknown reduced compoundapparently continues in the dark since a sufficientlylong dark period renews the capacity of chloroplaststo produce the 02 spike again. Repeated brief ex-posures to 650 mt (shown in the upper, right handpart of fig 7) show this progressive build-up of thelheiglht of the O-production spikes after a previousexposure to light.

A (lark period, as well as an exposure to certainwavelengths of light, can bring about an increasedlO,-production spike. The action spectruml for theincrease of a 650 m,u 02-production spike showsthat far-red light (730 m,t) and blue-green light (ca.500 mIA) are both effective in this regard. As wasseen in figure 12, an exposure to 730 miu light anda (lark period are apparently additive in their effecton the 650 nm.0, spike.

The far-red peak around 730 miu in the actionspectruml suggests that 02 production might be in-fluence(d by a phytochrome system. If exposure to730 itA or a dark period brings the chloroplasts intoa state favorable for O. evolution, an exposure to650 myt, absorbed strongly by the 660 mit-phyto-chronme, would convert the chloroplasts back againto a con(lition w-here O., evolution is retarded. Thesharp drop in the rate of 0. evolution during a 6502mni exposure would be understandable if this mechan-ism were operating. However, the phvtochromesystem seemls not to be involved since the timecourse curve of O., evolution at 480 mn (nIot ab-sorbed by the reversible phytochrome system) alsoshows a decline in the rate of O., evolution (luringthe light period (fig 8). The stimulatory effect ofpretreatimient wvith 730 itA light was nevertheless seenwith subsequent 480 mni exposures.

At present, it would seem reasonable to attributethe stimiiulation of O2 production by far-re(d to a lightreaction x-hich in soimie way influences the re-oxida-tion of the unknown reduced componelnt discussedpreviously.

An O.,-prodluction spike was also seen upoln ini-tial illuiminationl of a chloroplast suspension underaerobic conditions (fig 3B). Here, however, a smlallnet 0. uptake was seen after steady state conditionshad been attaiine(l. In some experimlents no net 02exchange was observed. MIehler (11) lhas shovnthat O. can act as a Hill oxidanit. In this case O.,is both produced andl consumlied and no gas exclhangeis apparenit. In the present experiments dlarkeningof chloroplasts under aerobic condlitions resulted ina gulp of O._ This could result if an O., productionand an O.,-uptake reaction were bofh occurring inthe light. If 0, productioni wrere to stop morequickly upoIn darkeInilig than an O.,-uptake reaction,a gulp of O, w-ould be observedl. It seemls probablethat an O,-consuniing reactioin takes place in the lightconcurrently wvith O. production. The steady-statelevel of O., exchange w%vould dlepend upon which re-actioil predlominates. Under anaerobic condlitions anet O., production can be sustained, and Ino O. gulpoccurs upoI (larkeniing. Under aerobic conditions,ho\\ever, steady-state O., exchange varies from posi-

331



PLANT PHYSIOLOGY

tive to negative dlependling upon wliclh reaction isgreater.

A light-dependent 0. uptake is still seen after0. evolution has been poisonde(l by DC.MU (fig 13).This uptake of O., in contrast to the uptake observedby Mehler after ethanol-catalase hadl heen a(l(led asa trap for the H,O.. di(d not (lepelncl on the function-ing of the O.-evolving system. Green fragmlents ofred algal chloroplasts w\lhich nio longer evolved 0,after phvcobilin pigments had been leachedl out like-wvise showedl a light-dependent uptake of 0, (4).

O, uptake as a function )f 690 mn light intenisityin chloroplasts poisone(& with DCMIIU w-as linearonly at very low light initenisities. This 0.. uptakeat low light intensities was al)olishedl after chloro-plasts w-ere treated with triclhloroacetic acid, in con-trast to the 0. uptake seen in (lenatuire(l chloroplastsexpose(l to bright white liglht. The former processmay depend utpon enzymic reactions. tlle latter onphysical, photooxidatiN-e processes ( ).

The action spectrnmli tor tlle loxw light ilnteinsitv0.2-uptake reaction was complex. It had. howexver.a imiain peak at 690 mn' corresponding to the absorp-tion of a long--wavelength formii of chlorophyll a.

IThe tuiictioilini of the lows inteiisit\- 0. uptakereaction may dlependl upon a reduction of 0.) miiedi-ate(l largely b1 chlorophyll a. This reaction \wotuldrequire anI en(logeinous suppl)l of electrons frloIml areduced comiipoun(l other than that supplied b1 theaccessory pigmiient reaction I-X- in XV'itt's sclhemiie(12')1

SummarySwNiss chard ( Beta v'ld/arlis L. var. cicla ) chloro-

plasts (& chloroplast fragments) sutspeii(le(n in asucrose, NaCl solutioln in phosphate buffer w-ith noadded Hill oxidant retaine(l, for miany hlours, alimitedl capacity to evolve O., upon illuminiliation.After about three minuiltes in lig,ht a steady rate of0. production wNas stistainecl und(ler anaerobic condi-tions wlhichi was mluch lowN-er thaln the heiglht of aninitial 0. burst. tnd(ler aerobic conditions the O0burst was followed by a net 02) uiptake in the lightunder stea(dy state cond(litiolns, and an O. gulp wasobserved Up)Oil darkening.

Addition of potassiulmii ferricvanide to the clhloro-plasts allowed 0.. evolution to procee(l at a rateabout 25 tilmies hipgher in thle steady state than theendogenous rate.

A comparison Nas miia(le between the action spec-tra for 0.) evolution ftroImi chloroplasts w\7ith andwithout an added Hill oxi(lalnt (ferricvaniidle). Theaction spectrulmi for the evolution of O., 1w cliloro-plasts wvitthout ferricvan id(le followed the absorptionof light by chlorophyll 1) and(l hadl peaks at 650 and480 nw whlile the actioln spectrum for O. lproduc-tion in the presence of ferricvanide ha(l a peak at678 mly and( a shoul(ler alrotun(d 650 mi. Thlese re-stilts are inter-preted as ind(licatilng a close connectionbetween 0., evolution and the acces.sorx pii-mentsystem.

The pro(luction of 0. hy chloroplasts exl)osedl to650 nlm lighit w\vithout an1 added1 ill oxi(lalnt coul(dbe increase(l 1y a previous exposure to certain wave-

lengths of light. The action spectruml for the ef-fectiveness of light to increase the 02 productionupon excitation of chlorophyll b had a maximum inthe red at 730 m,u. AMoreover, a dark period aftera previous exposure to 650 m, light also resultedin a higher 0, pro(luction spike from a subsequlent650 mig exposure.

The stimulating effect of a (lark period(l & per-haps far-red light) on the O. production spike isinterpreted as being a result of a slow endogenotusre-oxiclation of a substance reduce(d upon oxidationof wvater. This effect of a dark perio(l and of 730mu light onl the 650 mn .0.-production spike -wasnot seen wvhen ferricvanide acted as an electronacceptor.

Chloroplasts treate(l w-ith 3-(3.4-dichlorophenyl )-1,1-dlimethylurea, whliclh suppresses O.. evolutioll ex-hibited a light-depen(lent uptake of 0... The actionspectrumii for this 0., uptake had a mllaill peak in there(l regioin at 690 n1it.

AcknowledgmentsThe author gratefully acknowledges thlC many stimll-

lating discussions and( valuable suggestions madle lvProfessor C. Stacy French. It was a l)leasure also t(owork with M1r. Richard WV. Hart w1io dlesigne(l anidconstructed the electrode assembly use(d in this work.

Literature Cited1. CARRITT, D. E. & J. NV. KANWISnER. 1959. \nI

electrode systemii for mleasurinig dissoix e(l oxygen.Anal. Chetmi. 31: 5-9.

2. CLARK. L. C., R. \NOIF, 1). CIRAN(GtER, & 7.. TAYIOR.1953. Continuous recordlillg of hlood oxygeni teln-siOns by polarograplhy. J. A\ppl. Ph!siol. 9 189.

3. I)DUYSENs, L. N. -M., J. AMIESZ, & B. M. K.AMP.1961. Two plhotochemical systeiils ill photos il-

thesis. Nature 190: 510-11.4. FORK, D. C. 1961. Light dependent oxygen npl)-

take in red algal cliloroplasts. Carnegie Inst. of\Vash. Yearbook No. 60: 369-70.

5. FRANCK, J. & C. S. FRENCH. 1941. Ph}oto(oxidation1processes in planlts. J. Geni. Phlsiol. 25: 309-24.

6. FRENCH, C. S. & I). C. FORK. 1901. Two primaryphotochemical reactions in photosynthesis drivenby different pigmenits. Preprinit of paper read at5th Int. Cong. of Biochemistry. Symposium N-IPreprint 73: 1-15. 'Moscow.

7. FRENCH, C. S., D. C. FORK, & J. S. B'R0\\N-. 1961.Measuremenit of photosynthetic aetion1 spectra.Carnegie Inst. of \NVaslh. Yearbook No. 60: 362-63.

8. HAxo, F. T. & L. R. BIANKS. 1950. PhotOSVII-thetic action spectra of marinie algae. .1. Geni.Physiol. 33: 389-422.

9. HIiI, R. 1937. Oxygen1 production by isolatedchloroplasts. Nature 139: 881-82.

10. LOSADA, 'M., F. R. NVHATLEY, & D. I. ARNTONN. 1961.Separation of two light reactionis in nonicclicphoto-phosphorylatioin of greeni plants. Nature190: 606-10.

11. MIEHLER, A. FT. 1951. Studies oni reactionis of il-lumiiinated cliloroplasts. I. _Mechanism ot the re-(Itiction of Oxygen anid other 1 ill reagents. A\rcb.Biocheimi. 33: 65 -77.

12. \iTT, H. 1T., \. 'MULTLFR, & l,. Rt \MIiR(,. 1961Oxidized cvtochrome & chlorophy ll t in idhno-synthlesis. Nature 192: 967-69.

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Documents

ActionSpectrafor Evolutionby Chloroplasts Substrate ... · The light ex-posures were on only long enough to permit the spike to reach its highest value. The height of the 02 pro-duction