photosynthetic properties of mutant strain of chlamydomonas of ... ·...

Plant Physlol. (1969,) 44. 1001-1012

Photosynthetic Properties of ac-31, a Mutant Strain of Chlamydomonasreinhardi Devoid of Chloroplast Membrane Stacking'Ursula W. Goodenough, Judith J. Armstrong, and R. P. Levine

The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

Received February 27, 1969.

Abstract. A pale-green mutant strain of Chlamydomonas reinhardi, ac-31, is characterizedby the absence of any stacking of its chloroplast membranes. The capacity for photosyntheticelectron transport, phosphorylation, and CO, fixation in ac-31 is substantial, and it is con-cluded that these photosynthetic activities occur within the single membrane. The photo-synthetic capacities of wild type and ac-31 as a function of increasing light intensity areoompared. Saturation is attained at higher light intensities in ac-31, and the kinetics of the2 sets of curves are distinctly different. The possibility that energy transfer is enhanoed bymembrane stacking is suggested by these results. The repeatedly-observed correlation be-tween reduced stacking and disfunctional Photosystem II activities is discussed in view of theobservation that ac-31 has no stacking but retains a functional Photosystem II.

InI the p)rece(ling paper ('13) it was (lemonstratedthat the chl oroplasts of l)hotosynthetic mutant strainsof C. reinihardi exhibit normal disc formation butaltered l)atterlls of disc association or "stacking".In 1 clalss of niitanit strains, whllicih iclucldes (tic-I i5.c1-l4l,. and F-34.;. preponideranice of "unstacked".single discs is found. These strains all lack anactive cytochrome 559, a component of the electrontransport chain that lies close to Photosystem II(PS II), and none exhibits significant Hill activitvwith oxidants such as 2.6-dichliorophenolindophenol(DPIP), p-benzoquinone, or potassium ferricyanide.In contrast. the other photosynthetic mutant strainsexamiiined are all capable of Hill activity with theseoxidanits. and nione shiows a l)rel)onderance of siiglecli scs.

The observ-ed correlation of defective Hill activitywith defectiv-e disc association was of interest illlight of a recenlt )ail)er by Hoianin and Schimid ( 19),whZllere it is shown thait chfloroplasts from yellow-greensectors of the variegated tobacco mutant, NC 95,lack both 1-1 ill activity and chloroplast membranestacking. These authmor, p)1(popse that 's 11iacti%'itvIS (lependeilt on tilhe cosely-a;lCked associatioll ofstacked elIlnebranes, and suiggest that stacking createsa lvidroplhtbic en\ i ronutnent where the oxidation 4wvater can occur.

Irn tluiis paper we (describe the ultrastrulcture and(SoMe of tile plotosynthetic properties (of aic- a,apale-green strain of C. reinhardi. The chloroplastdiscs of ac-31 exhibit Ino stacking whatever, and vetPS II activity is very high on a chlorophyll basisand substantial on a cell basis. Other parameters

I Supported by research grants G(M-12336 from theN.I.H. and GB-5005X from the N.S.F., by a pre-doctoral fellowship (GM-24-306) awarded to U. W.Goodenough by the N.I.H., and by training grant GM-0707 from the N.I.H.

of plhoto5ynthetic activity have also been mieasured.and mlost of these are judged to b)e intact. Weconclude that stackinig is Ilot re(luired for photo-synthetic electr-oli transport. phlo)tosynIthetic CO.fivi;tioll. or !pho>tosvynthestic phospliorvlati]m. 'Ibt thatit mafiy possibly play a role in the trainisfcr of excital-tion energy.

Materials and Methods

Cniltmfre of t1le Organism. Cultures of wild-type,ac-,I and ac-I strains of C. reinhardi were grownnlder conditions described in the previous paper

( 13). Cells gro-vn in minimiial salt mle(liumi areireferred to as mninimial-qrown, and cells growvn in anacetate-supplenmented imiinimiial imie(liumiii are referredto as acetate-grown. The \vild-type strain describedin the lprecedlillg paper has since been recloned. andthe chlorophyll a:b ratio of the clone used in thepr"xsent experilmlents is somiewvhat lowver than that ofthle earlier clone. Th'Ile photosynthetic and finie-structural properties of the 2 cloneis arc otherl sesimlilar.

Electron AlicfrCsC(opv. I'rocedures uise(d for speci-n1eji pl-cparati!m:I-ale as previously (lescIribed ( 13).Trlhe plates for Figs. 1 -4 \vere photogIraplIed atoriginal uiagnifications oif 42,500>. Measurementsof nureinbralne dianieters were made oII plates of thesame11 maLgnification, using either- a Nikoi .Shadvow-grajph (MIodel C) or a Joyce-Loeb recording micro-denisitometer.

Chlorophyll Detecrninations. Total chlorophyll.chlorophvlls a and b, and cell nunmber wvere deter-niined as in the preceding paper (13).

Photosvynthetic Capacity. Photosynithetic electronitransport reactions w\ere assayed using a model 14Carx- recordinig spectrophotometer (15, 33) withchloroplast fragments prepared from cells disrupted

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1002

by sonication (25). Cyclic and non-cyclic photo-synthetic phosphorylation were mleastured as previ-ouislv described ( II, 14) with chloroplast fragmentslpreella eci from cell,s (lisirpte(d by grind(inig withsand ( 14).

The fixation of carbon dioxide by photosynthesisandl pllotoredtiction in whole cells wras measured in2 wvavs, eithier as the incorporationi of '4C-labeledNa H1CO2, or bv a titrimiietric method which measuresthe pHr chanige accompanying the uptake of HCO3-by whole cells in the light.

The rate of 14CO. fixaition by photosynthesis wasmeasuired at 40.000 lux and 250 usinlg cells that hadbeen waslhed once aand resuspeinded in minimialmedium. The cell suspension, placed in a smiiallflask, was agitated by a continuous stream of air for5 min in the light before the additioon of NaH14CO3.Samples of 0.1 ml were taken at 1 min intervals for5 mmin an(d plated to stainless steel plaanchets con-taining 0.1 mil of a miixtulr-e of concentrated HCI antdgZI-lacial acetic acid (4 :1). The saimiples wvere driedand counte( with the aid of a gas-flow counlter.Cor-r-ectioni for light-independent 1 4CO., fixation wasobtained froml control experiments run in an idlenticalmanner but in the dark.

Tlhe fixation of 1-CO. by pllotoreduction wasmeasured in a similar manner except that the cellswere agitated wvith pre-ptirified hydrogen that hadbeen passed through a Deoxocartridge. The cellsuspension was incubated in the dark for 15 min inorder to activate the hldrogenase, whereuipon thelight was turned on. NaHi4CO3 was added after5 mmin, and samples were taken and treated as de-scribed above for photosynthetic CO., fixation. Thecell suspension contained 10 pot 3,(3,4-dichloro-phenyl) 1,1-dimethyl urea (DCMU) in order toinhibit oxygen evolution. The rate-versus-intensitycurves for the fixation of CO.. by photoredtuctionNvere obtained using this method.

Rate-versus-intensity curves for photosyntheticCO., fixation were obtained by a titrimiietric methodwlhich gives rates comparable to those obtained bydetermining the rate of 14CO., incorporation, andwlhich has the advantage of being both economicaland rapid wNhen nmany measurements are to be made.Cells were wvashed and resuspended in 2.5 mMNaHCO,. Photosynthetic CO2 fixation was meas-tured by means of a Radiometer titrimeter assemblycomlprising a MAodel 28 pH meter, Model 11 titrator,

PHtYSIOLOGY

at type SB2RC titrigralph, and a type SBl ia syringeburette. The electro(le was an Arthur I1. ThomcasCompany coml)inationl pH electrode 4858-L160. fIlu-1mlination was lprov,i(de(l 1)V aL 100)) \watt tunlgstenprojection lam), and tlle temperature was ialiln-tained at 250. The titrimi%eter was used in the pHstat mode wvithi 0.1 mt HCI as the titrant. The pI-Iwas maintained at 7.8, anid the titrigraph p)rovideda continuouis recor(l of the amount of titraant ilee(ledto m<aintain tlis pH in the reactioin vessel. Tllus adirect record of the uptake of HCO,- by cells wvasobtainied. A relatively conistanit, siall dcark 1rate wvasrecorded which wvas the saame in the preselnce orabsence of cells, andl rates in the lighit x-ere correctedfor this dark rate.

Rate-versus-iutcusity Curves. The rate-vcrsius-intensity curves for CO., fixation by photosynthesisand photoreductioln ere obtained by mleasuirinig thesereactions at different lighlt intenisities froml a 1)000watt tungsten projection lamp. Full intensity was136,500 lux, and low-er intenisities were obtainedwvith the aid of neutral denlsitv filters or neuitraldensity screens. Two nil reactioni miiixtures wereused containiniig cells equivalent to 12.5 lug chloro-phyll per mfl. The cells were washed and r-estis-pended in the appropriate mlle(liulli (minlimal mlediuillmfor photoreduction mieasturemlents and 2.5 mIl'NaHCO, for measuremients of photosynthetic CO.,fixation). The concentration of the cell suspensionwas adjusted to cells equivalent to 12.5 ,ug clhloro-phvll per ml. and the suspension (about 100 ml) wasplaced in the dark where it was agitated with anmagnetic stirrer. A different 2 nml sample from thissuspension was used for eachi measuremenlt made,atnd certain points wAere checked at the end of theexperinment to insure that the cells had not chan-edin their photosyntlhetic capacity during the time thatthey Nvere kept suspended in the dark. At the con-clusion of 1 such experiment, the cells remainingin the suspension were fixed and examined with theelectron microscope, and they were found to beidentical to cells harvested in thle light.

Results

Chloroplast Ultrastruletutre. The fine structureof the chloroplasts of wild-type and certain mutantstrains of C. reinhar-di i; describe(d in detail in theprevious paper (13). Figs. 1 to 4 of the present

FIG. 1. Chloroplast from minimal-grown ac-31, illustrating the maximal degree of disc association observed inthe strain. In certain places (e.g. at arrow) an appearance of membranle c-ntact is given, but this is in fact asectioning effect (see text). S, chloroplast stroma containinlg chloroplast ribasomles; Cy, cytoplasm containing cyto-plasmiiic ribosonmes; St, starchi. 69,750 X.

FIG. 2. Chloroplast from acetate-grown ac-31, illustrating a region of nm.axilmal l)ro.xinity between dliscs (arrow)at higher magnification. A gap of - 20 A separates the two membranes. 111,565X.

FIG. 3. Chloroplast from acetate-grown ac-31. Disc membranes tend to )2 furtlher apart than in miinimial-grown(Figs. 1 and 4) cells. 69,750x.

FItc. 4 Chloroplast from miniimal-grown ac-31, showing a more representative field than Fig. 1. Discs approacheach other in limited regions, but are generally at least 100 Ak apart. 69,750X.

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GOODENOUGH ET AL.---STACKING AND PHOTOSYNTHESIS 1003

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GOODENOtUGH ET AL.-STACKING AND PHOTOSYNTHESIS

paper depict clhloroplasts of ac-er. Most morplho-logical features of tlle ac-,3I chiloroplast are compar-able to the wild type, hbut the chloroplast membranesnever come togetlher to formi a stack. Fig. 3 is atypical field fronm acetate-growvn cells, anid the chloro-lplast discs are seen to be far apart in most places.In a typical field from minimial-growni cells (Fig. 4),the membranes tendl to lie closer together than inacetate-grown cells (Fig. 3). Fig. 1 shows a chloro-plast from a mininmal-grown cell whlich exhibits themlimaiiiuim anmotunt of nmembrane association ever

founid in ac-31. Hlere the membranes lie very closetogether for long distances, but they never touch.In certain places (e.g. Fig. 1, arrow), the membranesappear to make conltact, but these regions are pro-dulced whlen 2 adjacent minembranes go out of theplane of the section: they have been repeatedly,shown by microdensitometer tracings to be widerand less dense than a 2-disc stack.

It shotuld be stressed that a field sutch as thatshown in Fig. 1 was intentionally chosen to indicatethe most extreme deg ree of membrane associationin ac-1I, and that the discs are not commonly so

uniformly close together. Nonetlheless, it is evidentin all the nmicrograplhs that, at least in limited re-

gionls, the miicmibbranes *of adjacent discs often getverv close to one anotlher, separated onlv by a

narroNv gap. Such a gap is shown at higlher magni-fication in Fig. 2 (arrow). Careful microdensi-tometer measturements have been made which indicatethat the miniimum gap distance (i.e. the space be-tween the apposing surfaces of 2 adjacent mem-branes) is of the order of 17 to 23 A. Even at thisminimum. width, the membranes would seem to betoo far apart to permit any photosynthetic electrontransport between them, and since the gaps are

visibly continuous with the aqueous stroma, thereis no reason to suppose that they constitute a hvdro-phobic environment.

The 17 to 23 A gap observed in ac-3i should notbe confused with the "A" space which sometimesappears between the stacked membranes of higherplants and wlhich is reportedly 19 A wide (36).Spaces betweeln stacked mem1branes are not observedin wild-type C. reinhardi, nor in any of the othermutant strains we have studied (13): the width ofa stacked region (116-122 A) is consistently twicethe wvidth of a single memibrane (59-62 A). Sinceour fixation procedure for ac-3I is identical to the

proceduri-e uisedl for all the otlher straiis, it seemsunlikelv that we have l)reserved al"A" space in

ac--I alone."Chlorophyll Conitent1. TIn table I, thle chloroplhyll

content of ac-3I is coml)ared with wvild type. It isevident that minimiial-growin cells have a lower chloro-plhyll conitent tlhani acetate-grown cells in both strains,and that ac-3s is chlorophyll-deficient compared towild type under both growtth coniditionis. The clhloro-phyll deficiency of ac-3i is equiivalenit to that of ac-I,

anotlher pale-green mutant strain of C. reinhardihaving 0.8 to 1.3 uig chlorop)hyll/10 cells, and(l yetac-i is capable of normal stacking ( 13); thus. apigment deficiency does not, per se, prodtuce stackingaberrations. It should be stre-sed that botlh ac-I

anid ac-3'i contain a substanitial amount of chloro-plh-ll in comparison withl many pigment-deficientstrains: yellow strains *of C. reiiihaardi, for example,have chlorophyll contents of 0.03 to 0.08 uig chloro-phll/10 cells (21, 28), or 10 to 15 fold less thaneither ac-I or ac-3I.

In addition to being chlorophyll-deficient. ac- /?iis also somiiewlat chloroplhyll b-deficient compared towild type (table I) or to ac-i. This b-deficiencyis, however, miodest in comparison with certainb-deficient (18, 30) or b-less (1) mutant strains ofhigher plants, and vet the higher plants are allcapable of substantial membrane stacking ( 12, 18, 30).

Growth Rates. Growth curves for wild tvpe.ac-I and ac-3 on minimal medium are given in

Fig. 5. The ac-si growth rate is slow, even com-

pared with the comparably chlorophyll-deficient ac-I.Growth rates on acetate-supplemented media are. ofcourse, muich higher for all 3 strains (not shown).

Photosynthetic Capacity. The fact that ac-3Ican grow on minimal medium indicates in itself thatthe strain is capable of photosynthesis. An analvsis

Weier et al. (36) have proposed that the "A" spacecontains chlorophyll and other hydrophobic components,and that these are normally extracted during ethanoldehydration such that the space is no longer observablewith the electron microscope. Our rapid dehydrationprocedures (total time in ethanol<10 min) extract littlechlorophyll, the tissue is still very green when it issectioned, and no "A" space is observed. We thus can-

not agree that the "A" space corresponds to the presence

of chlorophyll.Table I. Chlorophyll (chl) Contenits of Wild-typPe and ac-31 Straints of C. reinhardi

GrowthStrain conditions Total chl Chl a Chl b Chl b Chl a:chl b

.ug/106 cells ig/106 cells glg/10" cells % ratioWild type Acetate 4.2 ± 0.51 2.6 ± 0.3 1.7 ± 0.09 40 1.7 +- 0.08Wild type Minimal 2.5 ± 0.3 1.7 e 0.3 0.86 ± 004 34 1.9 ±- 0.2ac-31 Acetate 1.1 ± 0.07 0.84 ± 0.3 0.25 ± 0.09 23 3.4 ±t 0.4ac-31 Minimal 0.70 ± 0.08 0.49 ± 0.05 0.16 ± 0.03 23 3.0 ± (0.41 Mean + standard deviationi for 3 independent experiments.

1005



PLANT PHIYSIOLOG\'

'able II. Carbon Dioxide Fixation by Photosynthesisand Photoreduction by Wild-type and

ac-31 C. reinhardi

Photosynthetic CO, fixatioin was measured at 250in 2 ml reaction mixtures of minimal medium containingcells equivalent to 12.5 Mug chlorophyll/ml. After equili-

.-1:13 hr bration for 5 min in the light, 0.2 ml of NaH14CO3(0.5 ,ac per mole per ml ) was added. Samples weretaken at 1 min intervals.

The fixation of CO., by plhotor-eductioin was imieasuredat 250 in 2 ml reactioni mixtures of minimal mediumcontaining cells equivalent to 12.5 ug chlorophyll/ml and10 /uM DCMU. Following equilibration in the dark withhydrogen gas, the light was turned on, and 0.2 ml of

1: 22hr NaH14CO3 (0.5 ,uc per mole per ml) was added after- Z5 min. Samples were taken at 1 min intervals.

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04E

2

10 30 50 70Hrs. after innoculation

FIG. 5. Growth curves for wild-type, ac-31, and ac-ion minimal medium in the presetnce of 5 % CO, in air.Doublitig times for each straini are id(licated.

of tills photosynthetic cal)acity has beenI miiade byineastiring the strain's rates of CO., fixationi byphotosynthesis anid photoredtictioli (table II), ratesof photosynthetic electron transport (table ITT ), and

Strain Photosynthesis Photoreduction

Wild type 1151 0.3362 27.51 0.081'ac-31 184 0.202 122 0.095

,moles CO., fixed per hr per mg chlorophyll.2 Asmoles CO., fixed per hr per 10"; cells.

rates of photosynthetic cyclic aInd iioni-cyclic phos-phorylation, (table IN'). The valuies given vereobtained froni acetate-grown cells; thev are coini-parable whleniobt:liled1 fromininiiilal1-growil Cells.

The rates in tables 11 to lV are compared withwild-type rates on both a chlorophyll and a cellbasis. Calculations made on a chlorophyll basisgive values for ac-3I that are high compared towild type, this is expected. since ac-3I is pigment-deficient and hence more cells or chloroplast frag-mernts must be used in the reaction mixture to attainchlorophyll levels similar to vild-type levels. Onithle othier hanid, calculatioIls o01 a cell basis are biasedin. the other direction, for giveni their small chloro-phyll enidowment, the p)hotosynthetic capacity ofac-3I cells is greater than the values calculated ona cell basis would indicate.

Table 111. Electrow Truaosport Iextiontws bly Clhloroplast Fragnients oif Wild-type (an1d ac-31 C. reinhardiFor the Hill reactiol with DPIPI', the cuvette in the sample comiipartimielnt oif the spectrophotonieter contained

chloroplast fragimienits ( 10 yg chlorop)hyll ) prepared by the sonicationl of cells, an(d the following in gmiioles: potassiumlphosphate, p)H 7.0, 20; KCI, 40; M-gCI.,, 5.0; and( 1)PTI, 0.1. 11 fuilnal volmile \vas 2.0 m1. TIle D)PIP was omitte(lfro,m the control c11vette in the i-cfrefcrcee coIatilopatmlt.

For tIme flill reaction wvith .N 1I)1, tiet civette conitainied cliloroplast fr-agmentits ( 10- 15 Ag chlor-ophiyll) preparedby the sonication o( cells, anIld the following in ummoles potassium phosphate, p11 7.0, 20; NKC, 40; MNgCl.,, 5.0;NADI, 0.5 and ferredoxin prepared froir wild-type (r*reinhmardi, 0.005. lalf a unit of ferredoxin-NADP reductase,prepared from wild-type C. reit-hardti, wvas also added. Thie final voluIme s;WAs 20 111i. terredoxin, ferredoxin-NADP reductase, anid NADP were oinitted fromi the conitr-ol cuvette.

For the photoreductioni of N.A [)P firoim the D)PI'P-ascorbate couple, thle rieactionl milixture conitainied, ill additioInto the components for the NAD)P Ilill reaction, the follo wing in Amoles: DPIP, 0.1; sodium ascorbate, pH 7.0, 10; andDCMU, 0.02. The conitrol cuvette conitainied everythinig but ferredoxin, ferredoxin-NADP reductase, and NADP.

All reactions were run at a light intensity of 20,000 lux and 25C.

Hill reaction NADP reductionStrain DPIP NNADP wvith DPIP-ascorbate couple

0.17120.058

1006

wilId type:10.5 hr

Wild type 84' 0.3982 78' 0.3702 36'ac-31 265 0.169 232 0.148 91

I ,moles photoreduced per hir per ,ug chlorophyll.2 ,umoles photoreduced per hr per 100 cells.



GOODENOUGH ET AL.-STACKING AND PHOTOSYNTHESIS

Table IV. Cyclic and Non-cyclic PhotosytWhetic Phosplhorylation by Wild-typc and ac-31 C. reinhardiThe reactions were run at 250 in 25 ml Erlenmeyer flasks. The reaction mixture (2 ml) contained chloroplast

fragments (40-80 ,ug chlorophyll) prepared from cells disrupted by grinding with sand, and the following in ,moles:glycylglycine buffer, pH 8.0, 40; MgCl2, 10; ADP, pH 7.5, 5; AMP, pH 7.5 in NaOH, 5; and potassium phosphatebuffer, pH 8.0, 10, containing 0.5 to 1.0 /Lcurie32Pi. For cyclic photosynthetic phosphorylation, the mixtures contained0.067 pLmole of phenazine methosulfate and 0.02 Amole of DCMU. For non-cyclic photosynthetic phosphorylation, 2,umoles of potassium ferricyanide were added. All reaction flasks were flushed continuously with nitrogen through-out the experiment. Reactions were terminated after 3 min of illumination (30,000 lux) by turning off the lightsand by adding 0.2 ml of 20 % trichloroacetic acid to each flask. Dark controls were run in flasks covered with blacktal)e.

Ncn-cvclicStrain Cyclic ATP formed Ferricyaniide reduced P/2 e-

Wild type 1541 0.7302 1021 0.3062 488' 1.472 0.42ac-31 1270 1.73 491 0.669 970 1.32 1.02

,umoles per hr per mg chlorophyll.2 unioles per hr per 106 cells.

4)

Light Intensity (per cent of 136,500 lux)

FIG. 6. Rate-versus-intensity curves for wild type ( I ------- 4 ), ac-31 ( 0 0 ), and ac-1( -----I ). The rate of photosynthetic CO2 fixation was measured againist light intensity (% of 136,000 lux).

The titrimetric miethod was used. Cells were washed and resuspenided at a chlorophyll concentration of 12.5 .g/mlin 2.5 nim sodium bicarbonate. Two ml were placed in the thermostated vessel of the titrimeter, and they wereagitated with a magnietic stirrer. The temperature was maintained at 250. 'Measurements were made in the darkand in the light for at least 5 imin. The values plotted in the figure have been corrected for the dark ulptake ofHCO,-. Light was provided by a 100 watt tungsten lamp.

0

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(N0I-)

0E

Even on a cell basis, the photosynthetic rates ofac-31 are at least half the r-ates of wild type. Ifchloroplast memiibrane stacking were required for anyof the reactions tested, the rates would be expectedto be nil.

An interesting feature of the photosynthesis ofac-3i is its high coupling ratio (P/2e) for non-cyclic photosynthetic phosphorylation and its highrates of cyclic phosphorylation compared to wild type.A possible explanation for this phenomenon is thatthe fuised discs of wild-type chliroplasts are somehow

more suibjected or sensitive to damlage dturing theprocess of cell disrtiption thlani are the free discs ofac-3i, and that this danmage somehow uincouples bothcyclic and non-cyclic phosphorylation from photo-synthetic electron transport. A more interestingpossibility is that stacking may somehow serve toregulate rates of photosynthetic phosphorylation.

Photosynthetic Efficiency. Fig. 6 gives plots ofphotosynthetic CO, fixation against increasing lightintensity (rate-versus-intensity cturves) for ac-3I,ac-ir, and wild type, and Fig. 7 shows the initial

1007

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PLANT PHYSIOLOGY

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.

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C 0

C0

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Light Intensity (Xof 136,500 lux)

FIG. 7. Rate-versus-intensity curves for wild type ( * A) and ac-31 ( f - 0 ). Eachset of points represents a separate experiment. The procedure was the same as described in the text and for Fig. 6.A, B, and C indicate the three different phases of the rate-versus-intensity curve for ac-31.

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GOODENOUGH ET AL.-STACKING AND PHOTOSYNTHESIS

portions of stuch curves for 5 different experiments,2 with wild type and 3 witlh ac-3I. Two features ofthese plots are of interest. One is that the rates ofCO., fixation for ac-,3I tend to saturate at somewhathighler light intensities than either ac-I or wild type.This difference is most readily explained by thesomewhat lower chlorophyll a :b ratios of ac-3Icompared to the other 2 strains (table I), sinceb-deficient strains of higher plants are known tosaturate at high light intensities (1, 18). In otherwords, ac-3i, by virtue of its b-deficiency, would beexpected to trap photons less effectivelv than eitherac-I or wild type.

If a chlorophyll b-deficiency were the only factorlimiting the efficiency of photosynthesis by ac-SI,then one would expect its rate-versus-intensity curveto resenmble the curves obtained for ac-I and wildtype except that the initial linear slope seen in ac-Iand wild type would be longer and less steep inac-3I. Tn fact, a second difference is noted, for itis evident in Fig. 6 that the initial slope for ac-?lis not linear, but has instead 2 distinct components.These componients are explicitly delineated in Fig. 7:a rapid initial phase (Fig 7, A) is followed by aless rapid second phase (Fig. 7, B) until saturationis approaclhed (Fig. 7. C). Suclh biphasic kineticsare observed in repeated experiments. as Fig. 7indicates, an(d thley are not evident in the rate-rL-crsut-intensity curves obtainied for the b-less mutant ofbarley (1), indicating that they are not the coi se-quence of a b-deficiency alonie. It would seem, then.that perhaps 2 factors are limiting the photosyn-thetic efficiency of ac-SI. At low light intensities.1 rate-limiting step appears to dictate the amountof plhotosyntlhesis the cell can carry out, whereas athiigher inten,ities. a second step becomes limitinguintil a final state of saturation is attained.

It was stat'ed earlier that the distance separatingadjacent disc membranes in ac--i is usually a largeone. but that the membralnes sometimes approaclheach other aind may come as close together as 17 to23 A. Distances of this magnitude, althouglh prob-ably too great to pernmit electron transport, are nottoo great to allow substantial transfer of excitationenergy by a meclhanism -of inductive resonance. Forexample, according to Forster theory (7), transferefficiency is still very high when the distance sepa-rating donor and acceptor nmolecules is in this range.We are led to propose, then, that 1 of the 2 stepsthat limits the photosynthetic efficiency of ac-3i ispossibly a less efficient transfer of excitation energyfrom existing sites of photon trapping to reactioncenters. We have in mind a model in which at leastsome transfer of excitation energy normally occursbetween donor and acceptor molecules that are locatedon or within 2 different membranes; that when thesemembranes are adjacent, i.e. stacked, energy transferbetween such molecules is very efficient, but thatwhen they are separated, as in ac-3I, this transferbecomes less efficient, and this somehow limits the

rate of photosynthesis. Clearly this model has onlycircumstantial support at present, but we feel it hassufficient interest to be put forth in this paper.

In contrast to the differences observed for wildtype and ac-3i with respect to their rate-verslus-intensity cturves for CO., fixation by photosynthesis.the curves obtained for photoreduction were foundto be identical (not shown). It seems reasonableto assulme, therefore, that the factors affecting photo-synthetic efficiency in ac-gI do not alter a processthat depends on the operation of PS I alone.

Photorespiration. The apparent rate of photo-syn-ithesis caln be affected bv photorespiration (re-viewed in ref. 38). Since differences between wild-tvl)e and ac-?I photorespiration rates miglht accoutntfor some of the differences seen in rat'e-versus-inten-sitv curves ('39). it became important to test for thispossibility. Plhotorespiration rates are dependentboth on CO., tension and light intensity (38). WVetlherefore measured growth rates of ac- I and wildtvpe at high (5 % in air) and lowv (atmospheric)CO.. conc'entrations in the expectation that. if photo-respiration rates differed in tlle 2 strains, the ratioof the growtth rate on high CO.. to that on low CO.,for wild type wvotuld be diffel-rent fromii the ratioobtained for ac-3.1. In fact, the ratios were thesame. In a similar experiment. CO.. fixation bypllotosynthesis was measutred for the 2 strains bothait hih an(d low CO., conlcenltrationls and at high(136,5l 0fiix) and low (12.000 lux) light intensities.Again, the ratios obtained for wild tylpe and forac-., did not differ. Thus, plhotoresl)iration differ-clces are not thought to contribute to the differencesin the rate-versuts-intensity cturves seen in Figs. 6and 7.

Discussion

The ac-31 Mlliutationi. The close associatioil ofbiological membranes is a rare plhenomenon. foundonly in the clhloroplast. in the specialized "tight"junctions between certaini cells (6), alnd perhaps inthe mvelin sheatlh sturrotunding nerve cells. It isnot established whether chloroplast membranes actui-ally ftuse together or only become very closelvap-posed: in either case, the association is apparentlya strong one, for it is retained in sonicated andsand-ground chloroplast fragments of C. reinhardi(uinpublished observations) .

The ability of chloroplast membranes to form astack can thus be considered as one of their dis-tinctive characteristics, and this ability can evidentlybe lost as a con-equenc'e of the ac-3i mutation. Theability to stack may be a property of the membraneproteins themselves. In this case, the ac-3I muta-tion might result in an alteration of the chargeproperties, or perhaps the conformation, of theseproteins such that they can no longer interact withone another. Alternatively, stacking may be medi-ated by some component, a stacking "factor," which

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1010

aIcts as a kind of g"lue" to lold a(lbacentmembranestogether.

Tlhree allelic ranitha mutant strains of barleyhave been described that also appear tunable to formclosely-aggregated grana (37), and possibly thesestrains suiffer from a defect similar to that affectingac-p. Mfutations at the rant ha an(d ac-3I loci

exhibit 'Mendelian segyregation. and ac-3i has beenmal)l)e(l to linkage groul)p V of the C. reinhar(ligenome (4'). Thus a property that is p-artictilarlyassociated xwith chloroplast mlenibranes is apparently-

ndler at least partial nuclear coiltrol.Stacking and Photosynithesis. Stacking is found

in the chloroplasts of mlost algae an(l higher l)lants(reviewed in ref. 24). Imlportant exceptions are theblue-green, red, and brown algae (2, 3, 5, 10, 16, 27.29). rhe nmajor- light-hlarvesting pigmenlts in thesealgal groups are the phvcobilins and fucoxanthins.anld it is possible that their plresence effects iiodifi-cations of the membranie architecture (8. 9, 17). TInany case, the existence of chloroplasts that do notrequire stackinig for photosynthesis d(oes not negatethe hylothesis that stacking is imnlortant for thephotosynthesis of chloroplasts in wvhich stackingoccurs.

As mentionie(d earlier,, Homanin a.nd Schmid (19)have suggested that PS TI activity is dependent on

stacking in higher plants. Two published observa-tions (lo not suipport this stuggestion. Ohad et al.(28) claim that in regreening chloroplasts of a C.,-einhardi yellow mutant, the resumption of Hillactivity slighltly l)recedles the oniset of stacking, andIzaNva and Good (22) fitndI that sp)iniach chloroplastscan be so dlisruplted 1y exposuire to Tricinie anid lowNsalt that all membraine fusion disappears, and thatsuch preparations will redtuce Hill oxidants at very

high rates. The former authors suggest that theftsion of discs may be important in "quantum con-

version efficiency," uvhereas the latter suggest itmav' somellow be involved in CO. fixation.

The experinlents with ac- si reported in thispaper (lenionstrate that chloroplast membrane stack-itng in C. rcinhardi is not reqtuired for CO. fixationbv photosynthesis or by photoreduction. cyclic and

iion-cvclic photosynthetic phosphorvlation, the light-inllluced redtuctioni of T-lill oxidants from water, or0for the light-indu(tced redutction of NADP from wvateror from the (dye-ascorbate couple. Unless C. rein-hardi proves to be very different from aIhigherplant, then. we cannot agree with Homiianin andlSchmid's stuggestion that PS TI activity requires thehydrophobic environment provided by stacked mem-

branes, nor with the concept (35) that electrontransport and photophosphorylation occur withinchloroplast "partitions" or stacks.

Reduced Stacking and Defective PS II. In con-

trast to ac-3I, chloroplasts have been described inwhich stacking is markedly reduced, but in whichsubstantial stacking still occurs. These include thechlorophvll h-less nittant of harley (12). Mn-de-ficient C. -reinhardi (34). chloramphenicol-treated

PRYSIOLOGIV

C. reinhardi ( 20 ) and tile mutant strailns ac-2)(23,26) and c(C-115, a(c-141, and F-34 (13) of C.reinhardi. In every case. the cells are furthercharacterized by the absence or inactivity of com-ponents closely associated with PS II. In severalcases, moreover, the cells can be cured of their PS IIdisability (MAIn cani be (added back to Mn-deficientcells, for instanice). an(d nlormal patterns of stackingaire recovered at the sallme time that PS IT activityis restored (21.20, 23, 24).

Ani al)l)arent paradox thuts arises: defective PST1 anid a reductioni in s:acking frequently occurtogether. whereas an active PS Tl is founlid in ac- -IwNhich has no stacking at all. These observationscan be reconciled bv proposing that a disorganizedPS II has the effect of curtailing niormal membranestackinig. This sutggestioln carries the implicationthat components of PS 11 are localized oni or niearthe membratnle surface which niorinallvx miakes conltactwith adjacenlt miembranes, ,s olplosedl to the surfacefacing the intra-disc space. Thlius, a niormal PS TTorganization would allow, or e\veni facilitate. anieffective stackilng plrocess, Whereas ant abnormalorganization would inm;pe(le it. in(leed, the (jis-organization of PS 11 mighlt beconie .so severe thatstacking beconies p)llysically impossible, a situatiollthat perhaps arises in the vellow\-green chloroplastsof the N.C 95 tobacco miutant (19).

Stacking and Chlorophyll. It has been suggestedthat chlorophyll is packed within a gralnum to pre-veent its being inactivated 1y photooxidation (31 ).It has also been lpredicte(l that if chlorophyll ispresenft ini a gyreenl alga ot higher plani-t. gralla willalso be plresent (35, 36). Since chloroplasts of ac-.1contain substantial qtuantities of chlorophyll and nograna, and since this chlorophyll is clearly very"active," these proposals are not supported by ourresults.

The fact remainis that ac-pI is deficient in chloro-phyll. WXe do Inot believe that the missing chloro-phyll cor-responds to the missing inter-disc. "glue.since (IC- i has similarly low chlorophyll lev els andniormial stackiing (13). Howev-er, it is possible thatthe absence of stacking may secon darilv produice apigment deficiency, and that stacking is, at least inpart, "a device to pack more chlorophyll into thechloroplast" (32).

If this were the only "futinction" of chloroplastmembrane associations, onie would -expect that thegrowth rates of ac-3i and ac-i on a minimal mediunmwould be comparable, that is, that both strains wouldbe limited in their photosynthetic capacity to asimilar extent by their similar chlorophyll deficien-cies. In fact, ac-3I grows much more poorly thanac-I on minimal medium )(Fig. 5). We are thusdrawn to the conclusion that stacking does partici-pate in photosynthesis, but that its role is moresubtle than those that have previously been proposed.Other aspects of the photosynthesis of ac-3I, such asthe light-ind1uced pH change. the 520 nm absorbance



1011GOODENOUGH ET AL.-STACKING AND PHOTOSYNTHESIS

changes, control of photosynthetic phosphorylation,fluorescence phenomena, and particularly phenomenarelating to the transfer of excitation energy, are

currently being studied in this laboratory in anattempt to define this role more explicitly.

Meanwhile, we wish to stress that, at least inC. reinhardi, the complete apparatus for photosyn-thetic electron transport and for photosyntheticphosphorylation, and approximately one third of thecell's chlorophyll, appear to be built into the structureof the single chloroplast membrane.

Acknowledgments

We acknowledge the facilities, support, and encour-agement of Professor Keith R. Porter and the helpfuldiscussions with Professor Richard Cone.

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