Plant Physiol. (1968) 43. 606-612

Studies on Electron-Transport Reactions of Photosynthesisin Plastome Mutants of OenotheraDavid C. Fork and Ulrich W. Heber'

l)epartment of Plant Biology, Carnegie Institution of Washington, Stanford. California 94305

Received November 6, 1967.

A bstract. Fluoreseence characteristics and light-indueed absorbance changes of 5 plastomemutants of Oenothera, all having a defect in photosynthesis, were investigated to localize thesite of 'the block in their photosynthetic mechanism and 'to relate mutational changes in theplastome to specific biochemical events in photosynthesis. In 4 of the mutants examinedphotosystem 2 was largely, or completely, nonfunctional. Excitation of -system 2 did not causereduction of oxidized cytoohrome f in these mutants. The system-2 dependent absorbancechange at 518 mu seen in normal 'leaves was absent in the mutants. Moreover, the mutantshad a high initial fluaorescence in the presence and in the ab'sence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which did not change during illumination, indicating that the reaction centers ofsystem 2 were affected by the mutations. Photosystem 1 functioned normally.A fifth mutant had an impaired photosystem 1. Even high intensity far-red light did not

lead to an aocumul-ation of oxidized cytochrome f as was seen in normal plants. Photosystem 2was functioning, as evidenced by the fast reduction of the primary system-2 oxidant, and bNthe characteristics of the 518-m., absorbance change.

Because 1 of the 2 photosystems is functional in 'all mutants, and because they all havethe enzymes of the photossynthetic carbon cycle, it appears that the effect of the mutationis specific. The results suggest that the plastome controls reactions within the electron-transportchain of photosynthesis.

Genetic information has been shown to residenot onlly in the nucletus but also in other parits ofthe cell. The genetic information contained in thechloroplasts is cailled the plastome (20). A numberoif geneticallly weill-defined plastome mutants ofOenothera were 'recently investigated (15) in an

attempt to determine the cause of photosyn'thetic(leficiencies observed in these planits. Alll the mui-

tants appeared to have t-he enzymes needed for thereduction of carbon dioxide, and for the regenera-

tion of the carbon dioxide acceptor, 'but in spite ofthis they photoreduced little o,r noGCO2. The resultsof the work of Halllier (14) suggested th,a,t electronitransport might be imipaired in some way. We haveinvestigated electron-transport reactions in an at-tempt to ilocalize the site of deficiency. The resultso'btained ifrom studies on time courses of fluores-cence 'as wellll -as on 1'ight-induced absorbance changesindicated that 4 of the tmuttan,ts -have an impairedphotosystem 2, antd another appears ito have a blocknear system 1. The resuilts demonstrate a relation-ship between the plastome anId electron transportof photosynthesis.

1 On leave from the Institute of Botany. Universitvof Dusseldorf, Germanv.

606

Materials and Methods

The photosyntheticallly-deficient plastome mu-tants used in this investigation were isolated fromOenothera hookeri (plastome Ia, Ty, Th) andOenoth era suaveolens (plastome IIa, Thy) by WAr.Stubbe. They are propagated by cross-pollinatingflowers from normal and imutant tissue. As theplastid type iis transferre!d 'both by the pollen andby the embryo sac in Oenothera, the offspring willbe heteroplastic. Somatic segregation of mutantand normal plastids then leads to variegation. Oftenvariegated leaves have mutant tissue on 1 side ofthe midrib and normal on the other. The develop-ment of mutant tissue requires support from normalgreen parts.

For the present work the different mutantplastid types were, combined with the genomes ofOenothera hookeri or of the hybrid albicans Xhookeri and with the normal plastid types I and IV(fo,r differenit plastid types cf. 23, 24, 25).

All of the mutants {are characterized by a re-duced rate or complete lack of photosynthesis. Leafsections containing only mutant tissue are palegreen compared to normal parts. The mutants Iyand IIy have about 50 % less total chlorophyll thanthe normal, and mutants Iac, IIct, and I8, about

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FORK AND HEBER-PHOTOSYNTHESIS IN PLASTOME MUTANTS OF OENOTHERA

25 % less. The chilorophyll a to b ratios are aboutthe same for al!l the mutants and for normal tissuie(14). The multant pa,rts of the plants are photo-sensitive and do. no-t survive prolonged exposure tosunlight. Their biochemical and cytogenetic prop-erties have been described (7, 14, 15).

For the experiments ithe mutant leaf tissue wascluit away so as 'to avoid perielinal chimeras ornormal tissue. Some experiments were performedcomparing -the response of mutant tissue on 1 sideof a leaf with that of noirmal tissue on the otherside of the same lIeaf. However, since the experi-ments with different leaves, normall or mutant,generally agreed very wel,l we did not often haveto use controils from other parts of the same leaf.or even from the same volant.

For measurements of absorbance changes thesections of mutant or normal tissue were held onthe surface of a lucite light pipe which was uisedin tihe apparatus described previously (17). Mono-chromatic measuring light havinfg a half-4band wid'thof 1 m,u was obtain'ed from a Bausch and Lombmonooh'romator having a grating wit'h 1200 lines/mm. The actinic light was filtereld throu,gh 27 mmo'f wq'ter andl combinaitions of colored glass andinterference-type fi,lters. Red light was obtainedby combining Cailflex C (Balzers, heat reflector)with RG 2 (Schott). Far-red light was obtainedwith Cailfilex C and RG 8, or Calf,lex C combinedwith a BalIzers 'interference filter having a peaktrans-mission of 709 m,t, half-band wid,th 15 m,t.The intensities uised were measu'red with a sil;oonphotocell calibrated with a thermopi'le.

M\ easuirements of fluorescence were made wziththe same apparatuxs. slightly modified, so that lightwhich excited fluorescence was incident onI thesame side of the tleaf which faced the photomulti-plier (EMI 9558B). A combination of interferenceaniid colored glass filters wuith a peak transmissionof 684 nip, half-band width 15 mnu, was placed ifnfront of the photomniultiplier to transmit flutorescentanld absoirb actinic light. Fluorescence was excitedlby a broad band of bluie 'light which was obtainedby uising Calflex C in combination with Corningfilters 4305 and, 5562. This band had a peaktransmission nlear 440 mjL and a half band of 85 mu.Flulorescence was also excited by a bro'ad band ofg,reen light havinlg a peak near 520 m,u, half-bandwid,th 54 m,u. The latter was obtained using Callf'lexC in combination with Corning filters 9782, 4015and Schott BG 18.

When needed, 'leaves were treated with 3- (3,4-dichlorophenyl)-1,1-di,methylurea (DCMU) by float-ing sections on a 10-4 M solution for 12 hours.

All experiments were done at room temperature(220) with air as the gas phase.

Results

Mutants Having Impaired Functioning of Photo-system 2. Reactions of the f-Type Cytochrome.

0

403

353

-20

-3 420

400 450 500 550Wavelengfh,mp

FIG. 1. Light-mintus-dark difference spectra forleaves of muitant 1T of Oenothera. TPhe spectrum withcircles gives the response obtained after 3 seconds ofillumination and the spectrum witih triangles after 0.1second of illumination with light of 709 mju (14.2 nein-stein cm-2 sec-1)

To test for system-2 activity in the mutants weexamined tthe -reactions of ithe f-type cyt'ochromesince it has been well d-ocumented (2, 3, 9, 13, 19)that this compound fuinctions as a red-ox carrier inthe electron-transport chain between the 2 photo-chemicail systems. It iis oxidized by system 1 andreduced by system 2. The light-minuis-dark differ-ence spectrtum for mutant IT (fig 1) is character-istic of that produced ulfpon excitation of an f-typecytoc,hrome and had a large nega'tive Soret bandslightly below 420 mt, 'a pos,iti-ve band near 403 mn

E0N

a

0u

c-a-0

-0

a

-ca

Normal709on

4~~~~~s ~~~~a

OffI

Mutont I &709 on

651 on Off

,&I/, = +5xl0-3 _i d,

709 on Off709on 4

651ion t 651on

;+f b 651 off _orm ; et $

* 709off 651 off709off

0 5 10 15 20 0 5 10 15 20Time,sec

FIG. 2. A comparison of the kinetics of light-inducedabsorbance changes at 420 m,u caused by oxidation ofthe f-type cytochrome in leaves of normal Ocuiothcr-aand mutant Ij. The wavelength of the actinic lightwas 709 m,c (14.2 neinstein cMu2 sec-1 for traces a andb and 7.2 neinstein cm-2 sec-1 for traces c, d. and e).The intensity of the 6l- m,u light was 2.8 neinsteincm-2 sec1.

6)07

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68PLANT PhIYSIOLOGY

and ain a band at 552 mpl. The negative 475 and

positive 518-m,u bands seen in the difference spec-trutm for the initial deflection produced tupon illu-mination are discuissed in another section of theresull ts. It is clear that the a bandl of cvtochronmef is suiperimposed on other large positive changesin thie green region which w1ill not be discussedhere. The shoulder aroundi(l 435 m/ may be cauisedlly oxidation of P700 (or of a b-type cytochrome).

'The alntagonistic effects of far-red and red lightoni the oxidation state of cytochrome f (measturedat 420 m,u) in a normal leaf are shown in figuire 2.

Far-red light (709 m/) cauise( rap.d loxidationi ofthe cytoc,hrome (seen in trace a as a downwardl(leflection). Dark reductioni of the cYtocbhromeafter 709-m/ light was sloNv. Trace b of figuiresho.w,s the effect of tuirning off 709-mM lighit but atthe same time turning Onl 651-mtu lighit. In thiscase rapid reduction of cytochrome was seen since651-mM light excited systemii 2 effectively. Theintensity of 651 m/t was low enoulgh so that thisbeam alone did not prodluce an appreciable deflectioni(cf. trace b la.bellled "651 off"). The acceleratingeffect of switching 651 for 709-mu light upoIn t'hereduiction of tihe cytocfhrome in normal leaves was

abolishedl after inicuibatioIn in DCMU. AMoreover,after this treatment 651-mnu light alone producedslow oxidation of the cytochrome similar to thatprodluced by 709-mM light.

The accelerating effect of 651-mn light oni thereduction of cytochrome f coultld not be lenlon-strated for muitant To. Figure 2 (trace c) i'l;luis-trates that in mutant 1I, as in ithe normal lIlant,illutminatioon with 709-mtt light l)roduced a fast oxi-dationi wvhich was followed by a slow re(duiction inI

the (lark. Illuimination of multant Th wvith weak651-m,u light alonie p>rolhced a sloxv oxidlation(trace d) and wheni 651-im/.L was switched fuor709-m/, light (trace c) there was no reduiction an(l

the cytochriome remained oxidized. System 2 thuisappears not to fulnictioni in this muttanit. The sanmegeneral behcavior was foulind for multtanlts Ia, Iy,and Ily indicating that in these niiitanits photosystenm2 is also essentially nonfunctional. Differencespectra oitainled by illuminiation of these muiitaintswitlh re(l o,r far-red light were almost identical aId(exhibited the same chlaracterist.cs as those givenin figulre 1 for multant lb. Miitant ly had, in

addition, a smalil positive peak at 430 mM suggestingreducetion of a cytochrome of the b type.

Absorbanec Chan'lcs (it 58,ip.. Absolrbancechaniges seen at 518 ma, in normiial Ocnothcera leaves(trace a fig 3) are complex butt typical of tho.seprodutce(l by other green planits containing chloro-phyll b (11, 12, 27). Illtumination with a broa(lband of red lig,ht whic,h excited both system 1 aniid2 produiced, initially, a fast rise which was followedby a Ilowver, and larger, inicrease to a steady state.Leaves poisoned witih DCAMU still ha(l the fast, butsmaller, initial increase andl the steadv-state cihangeNvas ailniost completely inhibhited (trace b). \ctioiu

E

-o0

-o

a

0

0 5 10 0 5 10Time,sec

FIG. 3. A comparison of the kinetics of the light-ilduce(d ahblsorhance changes at 548 mn, in leaves ofniormcal Ocnothera anid mutanits ITy and hIa. Actinicillumination was provided 1w- a broald band (620-80 mi)of red light of about 2.6 X 10' ergs cm l-2 eeC .

spectra donie (11) on the green alga Ulva lobatahave shown tha,t the fast, iniitiall increase withor withou1t DCMU as well as the low steady-statechange persisting in DCTMU are sensitized by sys-tem 1. The slow, larger absorhance inkcrease is

sensitized by system 2.Mutant Ily had a 518-m,m change like that of

normal plants whose system-2 activity had beenblocked by treatment wi,th DCMU. This plant hadidentical 518-m,u absorbance changes in the presenceor absence o.f DCMTU (traces c and d) indicatingthat system 1 alone is functional in this plant.Very similar responses at 518 mp, were found forsonie other muititants (Iy, 1h and la). The re-

spouses of mulftant Ila are discussedI in a latersectiOn1 .

The observations Onl the behavior of cytochromef ini mutant (and also mutants y, ITa, and Hly)as welil as those oIn ithe 5l8-mp. change discousssedat,bove aill suggest that svstem 2 is almost completelynonfuinctional in these plants. Ftirther evidencetfhat system 2 was not functioning was ob)tainedfrom their fluorescence behavior.

Timte Courses of Fluorescence in7 DCMU-TreatedLeaves. There is evidence (4, 5, 6, 8, 10) that thefluiorescence o,f chilorophyll in algae ancd higherplants originates mainly fro,m system 2 and th,at tlhefluorescence increase which occurs around 685 mn.duiring illumination reflects the oxidation state ofthe primar) pho,tooxidant (Q) of system (10).When Q is in the oxidized form uit quenches chlolro-phy ll flulo,rescence and whein in bhe reduced state

No DCMU Normal +DCMU+Off NOff

a1 b* a * b TOn On AI/, =10-2

Mufonf Hi',Off Off

On c On d

Off I* Mufant ll AI/x10-3'Xf" Off

4 ~~~~~~~4 fOn e On

608

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FORK AND HEBER-PHOTOSYNTHESIS IN PLASTOME MUTANTS OF OENOTHERA

100 Mutant fl(No DCMU)

Mutant Ul.(No DCMU)

75-

Normal (DCMU)0)° 50 -

0)

25 -

00 5 10 IS 20 25 30

Dark fime,secFIG. 4. Fluorescence at 6&4 mg produced immnedi-

ately upon illumination of niormal leav-es of Qenotheraand of mutanits HJy and 11a a-s a function of dark in-terval l)etween exposures to blue actinic light (267 ergscm-2 s-ec-i).

(QH) it l)roduices increased fluorescence. In thealbsence of DCMU, oxidation of QH may be bro-ughtaibout by excitation of system 1. QH may ailso beoxidized by a dark reaction that proceeds in thepresence or absence of DCMU.

In the normal leaf treated with D-CMU thefluorescence *at 684 mJL produced initially was low.Duiring ililumination the fluorescence rose rapidlyto a higher level as Q was reduced by system 2.Figure 4 showrs that in DCMU-treated normaileaves QH was half reoxidized ina the dark in about0.7 sec. A number of mutants ('Ia, Iy, Ib, and(Hy) were found to have a high initial fluorescenceat 684 m/ rcomlpared to normal plants whetherpoiso-ned with DCMU or not. Moreover, this highifluorescence seen initially did not increase furtherduring illumination and was not decreased even bya long dark interval (10 mm). Figure 4 showsthis wacakof dark reoxidation of QH for mutantII)Y.

A Muttant With Imipaired Functioning of Sys-tem i. FlAuorescence. Mutant 1ha was found tohave a fluorescence behavigorintermediate betweenmautant ly or I>l and the normal plant. Figure 4shows that for mutantIda QH was ha,lf reoxidizedin the dark in a(bout 0.6 sec. The fluorescencebehavior o-f this mutant was siimilar wi-th andwithout DCMU. Thus, unlike the other mutantsdescribed so far, mutant Ilae retains funrcFtionalsystaem-2 reaction centers because after Q is formedin a dark reaction it may be reduced again in theli.ght.

To es-timate how welil photosystem 2 was func-tioning the half time of the increase oif variablefiluorescence, whidh reflects the reduction of Q,was meassured at different light intensities. Therise, as judged from the half-time of QH formation,was found to be 30 to 50 % faster in DCMUAtreatedmutant Tla than in normal -leaves treated with

DCMU indicating that reduction of Q is at leaslt aseffective in this mutant as in normall leaves. How-ever, initiail f(luorescence after a 'long dark periodwas higher in tihe mutant than in the normal leavesand t'he mutant had somewhat less varia)ble ftluores-cence. Therefore, the number of system-2 reactioncenters may be somewhait reduced in mutant Ihawhen compared with the normal.

AbsorUance Change at 5i8 m,u. Further evi-dence for the functioning of system 2 in mutantIIa wa,s seen in the behavior of the 5l18-mMu absorrb-ance change. Trace e of figure 3 shows that thechange before poisoning with D'CMU consisted ofa fast, initial rise fol,lowed by a second, slower andsomewhat 'larger increase, characteri,stic of photo-system 2 acitivity. Thi,s second phase was smallerthan in the normal plant. Trace f shows the effectof incubating the leaves in DCMU. A's in thenormall plant the fast, initial transient was notinhibited, but even increased, and the second risewa's abolished.

Cytochromiie f. The 'beihavior of the f-typecytochrome suggested that mutant hIa had a blockin electron transport near system 1. In thli,s mutantwe were tunable to see accumulation of oxidizedcytochrome f with 709-m,u actinic light as in thenormal and mutant lb (cf. fig 2). In mutant HaIeven ia broad band of 'high intensity far-red lightgave no response until the leaves were incubatedin DCMU. After this treatmenit accumulation ofoxidized cytoch'rome f occurred both in high andlow iintensity far-red ilight and the difference spec-trum had negative bands at 553, 420 and a positiveband at 405 mr,. Figure 5 compares the rate ofcytocthrome f oxidaltion as a futnction of intensity

02

o OC\

_W 2 4 6 8 10K104Intensity,ergs cm 2 sec'

FIG. 5. The rate of decrease of absorbance at 420mA (unoorrected for differences in the chlorophyllcontent of the leaves) caused by oxidation of the f-typecytochrome in normal leaves of Oeitothera and mutantsIIhy and 11a as a function of the intenisity of a broadband of far-red light (675-800 m,u). The leaves weretreated with DCMU as described in the text.

609

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PLANT PHYSIOLOGY

of far-red light for normal Oeuothera, mutant Ilyancd Ila after treatment with DCMU. It is clearfrom this figure that the efficiency of cytochromeoxidation in mutant IIa is very low in comparisonwvith normal OQnothera or muttant IIly. The dif-ferences apparent in figure 5 between mtttant hIaonl the 1 hand and normal Oenothera, or mutant Ihy,on the ot-her were even mulch more pronounced ifelectroIn flow from photosystem 2 was not blockedby DC'MU. Then excitation of system 2 by far-redlight (5) was sufficient to couinterac.t oxidation ofcytochrome f in multaint Tla buit not in other plantshaving normal system-i activity.

The quanituim requ1irements for cytochrome oxi-(lation were 10 for mutant ily, 16 for multant ly,30 for normal Ocnot hera, and 170 for muitanit TIa.Stuch measurements in highly-scattering leaves are

sullbject to relativelly large errors especially in thestrongly-absorbing 420-mu region (1), but theirrelation shows again impaired functioning of photo-sy-stem in muitant ITa.

Discussion

Mutants Hazing Impaired Photosysteml-2 Ac-tivity. The experiment's described in the first sec-

tion of the Results demonstrate that mutants la,Ty, 1h and ITy have a 1block in photosystem 2 whichis responsible for their inabiliity to perform photo-sy nthesis at a significant rate. The rate of l-ight-(lependenit 14CO. fixation by these mlu'tants was lessthan 1 % of t'he rate of normail ;leaves (15) exceptfor mutant Ia which, like multant Ila, couild almostcompensate respiration (Hallier, personall commtni-cation).

The initiall flu1rescesnce of unt,reated or DCMIU-poisoned multants (as txypified by mutant Ily, fig 4)was high and did not significantly increase d.uringillumination. Dark 'intervailis or illumination withfar-red light did not lower this high initial fluores-cence. This was even true for mutant Ia. Asthe ilight-induced rise in filuorescence reflects thetrapping of energy by 'the reaction centers of system2 (10), these ceniters welre all (multant ly) or

nearly all (mutant 1a) nonfuinctional (reduiced and

inecapable of reactiiig back in the (lark) or perhapsabsent altogether.

The inability of the photooxidant of system 2to function as ain efficient trap for energy absorbedby the 'light-harvesting pigment molectules was alsoseen in the behav,ior of the 518-m,u absorbancechan'ge. That part of the change produced upon

excitation of systemn 2 in normal leaves (11) was

absent in the 'mutants. In addition, cytochrome foxi(lized bv far-red lighit was not, as in the normalplant, reduced UpoIn excitation of system 2. Thuis,in these respects, the uinlpoisoned mutants behavedjust 1'ike normal plants treated with DCTMU.

It appears that the effect of the mutation was

specific for photosystem 2. Cytochrome f oxida-

tion was as efficient in the mutant as in normalleaves indicating that system 1 is functioning nor-mally. Experiments with isolated chlloroplasts fromthe mutants also demionstrated that system 1 isfunctioning normally since high rates of ATPformation in a cyclic type of photophosphorylationwere observed 'using PMS as a cofactor (14).Apart from a lower pginent content and increasedfluorescence intensity, low-temperatture absorptionlspectra -and filuorescence-emission spectra of themutants (not shown here) failed to revea.l abnor-malities when com,pared with spectra of normalleavets.

The enzymes of the photosynthetic carbon cycleare present in the mutants and have abouit normalaotivities. The very slow formation of radioactivesugtar phosphates from 14CO2 by the mutants (15)can be accounted for, at 'least in part, by photo-system-1 activity. The ATP content of mutanttissue increased tupon illumination (14), apparenitlydue to svstem-i mediated photophosphorylation.Photooxidation of ascorbate by system 1, or perhapstransport of reducing equivalents from the cyto-plasm, may lead to the slow formation of someNADPH.

The reduiced ch'lorophyll. content of the leavesmay be considered as a secondary consequence ofthe mutation. A block in the electron transportchain, which prevents 'light energy from beingeffectiveily channeled into photosynthesi!s, may beexpected to cause photooxida'tive pigment destruc-tion as observed in the mutants (14). Likewise,starvation phenomena stuch as increased .appearaniceof free amino acids may also be considered assecondary events (15).

A Mutant Hazving Imp/'aired Photosvstent-r Ac-tizvitv. Mutant ITa wN-as different from all]l theother mutants investigated in that it had impaire(lfunctioning of system 1. In this multant even ahigh intensity far-recl light was insufficient to causeappreciahle oxidation of cyto'ch'rome f. A pro-nouinced oxidatilon coulld only be observed 'afteraddition of DCMU. Apparentliy there was suffi-cient excitation of photosystem 2 even in far-redlight to couniteract the accuimuflation of cvtochromef by system 1. In mu1tant Ha 'the rate of cvto-chrome oxidati'on in far-red light after bilockingelectron flow from sysitem 2 wvith DCM\U was about12 times 'less onI a uinit chlorophyll basis than in thenormal or in mutant IIy.

The 518-mpt change of muitant Ila liad the fast,transient system-1 component as wel;l as a distinctsecond, slow phase, sensitive to DC\IIU, character-istic of tihat produced tupon excitation of sxstem 2.Photosystem 2, therefore, is operative in thfis miltant.However, it does not appear to be comipletely uin-changed in its activity. While the half times ofthe rise in fluorescence in DCMU-treaited leavesindicate even faster redtuction of QH in mutantIHa than in normall leaves, the nuimber of 'trappinacenters of system 2 appears to be .somewhat redtuced,

610

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FORK AND HEBER-PHOTOSYNTHESIS IN PLASTOMIE MUTANTS OF OENOTHERA

perhaps as a secondary consequence of the muta-tion. As discussed above, it is cjlear ithat photo-oxidative reactions induced by a block in system Ishould secondarily influence photosystem II. StilIl,the activity of photosystem 2 is much higher th-anthat of photosystem 1 and the liatter is clearlylimiting photosynthesis. Mutant IIa has an ap-

preciable residual photosynthetic activity and canalmost compensate respiration. As wiith the ot'hermutants, it conntains all the enzymes of the photo-synithetic carbon cycle in about normal activities.

Genetic-Physiologicail Aspects. There is an

overwheilming body of evidence to ilink point changesof the genome caused by mutations to specificalterations of individual proteins. One of the aimsof the present investigation was to determinewhether it was possible to link, in a similar way,the changes caused by mutation of the plastome tospecific events in the biochemical machinery ofchloroplasts. Spontaneously-occurring plastome mu-

tants are particularly well suited to prove tha-t them-utated genetic material is associated closely withchloroplasts. The main criteria (cf. 18) are quan-titative differences in the extent of variegation inhybrids from reciprocal crosses between normal and

defective types. The nature of the plastome is stillunknown. DNA Ihas 'been 'identified in recent yearsas a constituent of chloroplasts and evidence for a

relationship between chloroplast DNA and chloro-plast development has been presented (21). Ftur-thermore, chloroplast DNA appears to be engagedin the formation of chloropl-ast ribosomes (22).However, in all mutants deficient in photosynthesis,listed in a recent survey by Kirk (16), that have

been investigated biochemically and genetically, themuitations appear to be centered in the nucleus. Theresults of the present work indicate that the plas-tome exercises control over specific chloroplastreactions. Since none of the photosynthetically-deficient mutanits investigated had a 1block in itscarbon metabolism, and all were affected in theirelectron-transport chain, it appears that the plas-tome control steps of electron transport which occur

in the lamellar struictture of chloroplasts.

Acknowledgment

It is a pleasure to thank Prof. Dr. XV. Stubbe whokiindly supplied the mutants used in this work and whoprovided valutable criticism of certain parts of thismanuiscript.

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