Herbicide resistance in <i>Chlamydomonas reinhardtii</i> results from a mutation in the chloroplast gene for the 32-kilodalton protein of photosystem II

Article

Reference

Herbicide resistance in Chlamydomonas reinhardtii results from a mutation in the chloroplast gene for the 32-kilodalton protein of

photosystem II

ERICKSON, Jeanne Marie, et al.

Abstract

We report the isolation and characterization of a uniparental mutant of Chlamydomonas reinhardtii that is resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine). Such herbicides inhibit photosynthesis by preventing transfer of electrons in photosystem II from the primary stable electron acceptor Q to the secondary stable electron acceptor complex B, which is thought to contain a protein of 32 kDa and a bound quinone. It has been proposed that herbicide binding to the 32-kDa protein alters the B complex so that electron transfer from Q is prohibited. Both whole and broken-cell preparations of the mutant alga show a resistance to the effects of herbicide on electron transfer from Q to B, as measured by fluorescence-induction kinetics. In the absence of herbicide, mutant cells exhibit a slower rate of Q to B electron transfer than do wild-type cells. The 32-kDa protein from wild-type cells, but not mutant cells, binds azido[¹⁴C]atrazine at 0.1 μM. We have isolated psbA, the chloroplast gene for the 32-kDa protein, from both wild-type and [...]

ERICKSON, Jeanne Marie, et al. Herbicide resistance in Chlamydomonas reinhardtii results from a mutation in the chloroplast gene for the 32-kilodalton protein of photosystem II.

Proceedings of the National Academy of Sciences, 1984, vol. 81, no. 12, p. 3617-3621

DOI : 10.1073/pnas.81.12.3617

Available at:

http://archive-ouverte.unige.ch/unige:127864

Disclaimer: layout of this document may differ from the published version.

Proc.Nadl.Acad. Sci. USA Vol. 81, pp. 3617-3621, June 1984 Biochemistry

Herbicide resistance in Chlamydomonas reinhardtii results from a mutation in the chloroplast gene for the 32-kilodalton protein of photosystem II

(psbA/3-(3,4-dichlorophenyl)-1,1-dimethylurea/triazine herbicides/photosynthesis)

JEANNE M. ERICKSON*, MICHtLE RAHIRE*, PIERRE BENNOUNt, PHILIPPE DELEPELAIREt, BRUCE DINERt, ANDJEAN-DAVID ROCHAIX*

*Departments of Molecular Biology and Plant Biology, University of Geneva, CH-1211 Geneva 4,Switzerland;andtInstitut de Biologie Physico-Chimique, 13, ruePierre etManeCurie,75005Paris, France

Communicated byPierreJoliot, February IS, 1984

ABSTRACT We report the isolation and characterization of auniparental mutant ofChlamydomonas reinhardti that is resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atra- zine). Such herbicides inhibit photosynthesis by preventing transfer ofelectronsinphotosystem II from the primary stable electron acceptorQto the secondary stable electronacceptor complexB,which is thought to contain a protein of 32 kDa and abound quinone. It has been proposed that herbicide binding to the 32-kDa protein alters the B complex so that electron transfer from Q is prohibited. Both whole and broken-cell preparations of the mutant alga show a resistance to theeffects ofherbicide on electron transfer from Q to1B,as measuredby fluorescence-induction kinetics. In the absence of herbicide, mutant cells exhibit a slower rate ofQ to B electron transfer than do wild-type cells. The 32-kDa protein from wildtype cells, but not mutant cells, binds azido['4C]atrazine at 0.1 FuM. We have isolatedpsbA, the chloroplast gene for the 32- kPaprotein, from both wild-type and herbicide-resistant algae and sequenced the coding regions of the gene that are con- tainedinfive exons. The onlydifferencebetween the exonnu- cleotide sequences of thewild-typeand mutantpsbAisasingle T-AtoG-C transversion. This mutation results inapredicted amino acidchange of serine in thewild-typeproteintoalanine in the mutant. We suggest that thisalteration inthe 32-kDa protein is the molecular basisforherbicide resistance in the C.

reinhardtiimutant.

An increased understanding of herbicide susceptibility and resistance has obvious practical applications for crop pro- duction butmayalso reveal much about the basicphotosyn- theticapparatusinthechloroplasts of greenorganisms.Pho- tosyntheticelectrontransporttakesplaceinthechloroplast thylakoid membranes, mediated by the membrane-bound protein complexes ofphotosystem II (PSII) and photosys- temI(PSI). Herbicides of the s-triazine or ureaclass, such as atrazine

(2-chloro-4-ethylamino-6-isopropylamino-s-tri-

azine)orDCMU

[3-(3,4-dichlorophenyl)-1,1-dimethylurea],

disruptelectron flowatthereducingside ofPSII,thus inhib- itingphotosynthesis (1, 2).Bothalgaeandhigher plantshave developedresistancetotheseherbicides (3-5).Themolecu- larbasis for this resistance hasbeenunder intense

investiga-

tion in whichgenetic, biochemical, and

biophysical

linesof evidence are nowmerging.

The greenalgaChlamydomonasreinhardtii isparticularly well-suited for ajoint biochemical and genetic analysis of herbicide resistance. It hasasingle large chloroplast,under- goes asexual

zygotic

stage inwhich the

chloroplasts

from

eachoftwo gametes fuse, and is the only organism in which chloroplastgenerecombinationhas been observed(6). Both genetic (7, 8) and physical (9) maps of the chloroplast genome have been constructed and can be correlated by analysis of specificmutants.

Primary*

DCMU-resistant mu- tants have been isolated from C.reinhardtii(3, 4).These mu- tants exhibita uniparental mode of inheritance, suggesting thataproteinencoded in thechloroplastgenome iscentral to the herbicide mode of action. Herbicide resistance in higher plants is also maternally inherited (10). These genetic data are consistent with the observation that a chloroplast-en- codedthylakoid membrane polypeptide of 32 kDa(11) spe- cifically binds the triazine analog 2-azido-4-ethylamino-6- isopropylamino-s-triazine (azidoatrazine) (12).

The 32-kDaprotein has been extensively characterized in algaeandhigherplants (13-16). It has recently been postu- lated that this protein is part ofthe protein-plastoquinone complexB (17),whichacts asthesecondary stable electron acceptorofPSII (18). There is nodirect proofthatthequi- none itself binds to the 32-kDa protein. The quinone may bindtoanother membranepolypeptideorseveralproteins of thePSII complex mayinteract to create aquinone binding site. One model proposedfor the action of herbicidessuch as atrazineorDCMU suggests that thesecompoundsbind with high affinitytothesecondary electronacceptorsite inPSII (19),altering this sitesuch that thetransferof electronsfrom the primary electron acceptor Q is inhibited (5). Herbicide binding, presumably tothe-32 kDaprotein, mayinhibit oxi- dation ofQ- by preventingbindingofthe quinone cofactor of the B complex (20). According tothis model, herbicide resistance could be conferred tocells witha32-kDaprotein altered in such away that it no longer binds herbicide at levelsthatinterfere significantly with quinone

binding.

We report here the isolation and characterization of a DCMU- and atrazine-resistant uniparental mutant of the greenalga C. reinhardtii. We havecompared the wild-type and mutant strainswithrespect toherbicide bindingand flu- orescence induction in the presence or absence of DCMU andatrazine.Inaddition, wehaveidentified and sequenced the C. reinhardtii chloroplast genefor the 32-kDapolypep- tide. Comparison of the deduced amino acid sequence for the protein from wild-type and herbicide-resistant cells showsadirect correlation between herbicide resistanceand an altered 32-kDaprotein.

Abbreviations:atrazine,2-chloro-4-ethylamino-6-isopropylamino-s- triazine; azidoatrazine, 2-azido-4-ethylamino-6-isopropylamino-s- triazine; PSI, photosystemI; PSII, photosystemII;B, aprotein- quinone complex servingas secondarystable electron acceptorof PSII; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; kb, kilo- base(s); bp,basepair(s); Q,plastoquinone servingasprimarystable electronacceptorofPSIL.

$Primary

herbicideresistance is describedinref. 3.

Thepublicationcostsofthis articleweredefrayedinpartbypagecharge payment.This articlemusttherefore beherebymarked "advertisement"

inaccordancewith 18U.S.C. §1734solelytoindicate this fact.

3618 Biochemistry: Erickson et al.

MATERIALS AND METHODS

Strains. C. reinhardtii wild-type strain 137c was usedfor mutagenesis. Algal strains were maintained in continuous dim light on solid Tris/acetate/phosphate (TAP) medium (21). Escherichia coli strains BHB2690 [N205 recA- (Ximm434cIl'sb2 red3Daml5 Sam7)/X] andBHB2688 [N205 recA-(Ximm434cl'sb2red3 Eaml5 Sam7)/X] were used to makeX packaging extracts (22). E. coliNM539 [hsdR supF trpRmet (P2 cox3)] was obtainedfrom N. Murray and used to selectforrecombinant Xphage (23). E. coliC600(rk-mk- suIf)was the host forpropagation ofXphageandplasmids.

MutagenesisandSelection.Cells of C.reinhardtii wild-type strain 137c were grown in liquid TAP medium and treated with 1mM5-fluorodeoxyuridine (FdUrd) asdescribed (24).

Enrichmentwasachievedbygrowingcells inliquidminimal medium (25)containing 1

AM

^DCMU.Cellswere thenplated on solid minimal medium containing 1 ,uMDCMU.

Genetic Analysis. Reciprocal crosses between DCMU-4 and 137c were performed. Twenty-five complete tetrads from each cross were testedfor invivoresistance tovarying levels of DCMU andatrazine.

Fluorescence Induction. Fluorescence-induction kinetics (26) were carried out by using dark-adapted whole cells or broken-cell preparations (27).

BindingStudies. Cells at 2 x 106 cellspermlwereconcen- trated bycentrifugationandresuspendedin TAPmediumto afinaldensityof2.5 x 107cells per ml(50

Ag

ofchlorophyll perml).Azido[ 4C]atrazine(49.4mCi/mmol;1Ci =37GBq) wasadded at a finalconcentration of0.1 or10 ,M to 1.0-ml aliquots of the cellsuspension in asmallbeaker. Cells were then irradiated with UV light from an unfiltered General Electric 4 Wgermicidal lampplaced 8 cmfrom the sample.

After irradiation, 250-sul aliquots were removed andcentri- fugedand the cellpelletwasresuspended inabuffercontain- ing2% NaDodSO4,30mMsodiumcarbonate, 30mMdithio- threitol, and 12% sucrose. These samples were heated at 100'C and electrophoresed on a 12-18% gradient polyacryl- amide

§el

^containing 8 Murea (28). Gels were analyzed for

azido[l

Clatrazine by x-ray fluorography at -70'C using EN3HANCE from New England Nuclear.

Cloningand Sequencing. C. reinhardtii chloroplast DNA (29), plasmid DNA (30), and X DNA (31) were prepared as described. psbA, the gene for the 32-kDa protein, was isolat- edfromDCMU-4bycloning chloroplast DNAdigested with BamHI into the X vector XEMBL4(32). Ligated DNA was packaged in vitro (22) and phage were plated on E. coli NM539, which preventsgrowth of the parentalX. Recombi- nantphagewere screenedby the method ofBentonand Da- vis (33). Nick-translation, hybridization, and autoradiogra- phywere performedasdescribed (34). Phage containingthe DCMU-4 psbA were identified after hybridization with a

32P-labeled

fragment of the C. reinhardtii wild-type psbA contained onchloroplastEcoRI fragmentR14 (9). Thewild- typegene had been previously isolated (35) by heterologous hybridization with the cloned spinach psbA (36). The C.

reinhardtii psbA is located entirely within thechloroplast in- verted repeat (35) and hence present in two copies per genome. Digestion ofchloroplast DNA withBamHI allows fortheunambiguous separation ofthese two copies, one of which is located on the restriction fragment Bal2 and the other on Bal4 (9). The mutant gene located on Bal2 was subcloned from X into plasmid vectors as follows. The 6.0- kilobase (kb) EcoRI fragment (R14) was cloned into the EcoRI site of pBR328 (37). The adjacent 3.6-kb EcoRI- BamHI fragment was cloned in pBR322 (38). DNA frag- ments labeled at the 5' or 3' end were sequenced by the method of Maxam and Gilbert (39).

Herbicides.

Azido[14C]atrazine

was obtained from Path- findersLaboratories (St. Louis), DCMUwasfrom DuPont,

andatrazinewasfrom

Ciba-Geigy.

Unlabeledazidoatrazine was a gift from K. Pfister. Stock solutions of DCMU and atrazine weremade inethanol and methanol, respectively, and stored at -20'C. The concentration of alcohol in the growth mediumneverexceeded 1%.

RESULTS

Isolation of Mutants and Genetic Analysis. Ten cultures containing109cells each were independentlytreated with 1 mMFdUrd and DCMU-resistant cellswererecovered from oneof these. After a secondtreatment with 1 mM FdUrd, one clone, DCMU-4, was selected for the studies reported here. DCMU-4 grows inthe light on solid minimal medium containing10

gM

DCMU.Growth ofwild-typecells isinhib- ited by 1 ,uM DCMU. Though selected for resistance to DCMU, this strain grows on solid medium containing 0.1 mMatrazine. Growth of wild-typecells isinhibitedby1

ILM

atrazine. Reciprocal crosses between DCMU-4 and wild- type 137c show that both DCMU resistance and atrazine resistance are inherited uniparentally.

Membrane Properties. Fluorescence-induction kinetics of dark-adapted broken-cell preparations, in which both the cellular membrane andthe chloroplastenvelopearedisrupt- ed, demonstrate that the in vivo resistance ofDCMU-4 to herbicide is also reflected in vitro. Fig. 1 gives the fluores- cence-induction curves of wild-type and DCMU-4 broken cells in the presence of atrazine or DCMU. A similar result was obtained by using azidoatrazine (data not shown). The wild-type preparations show a rapid rise in thefluorescence yield during illumination. This rise corresponds tothe accu- mulation of Q-, since reoxidation ofQ- is blocked bythe herbicide (5). Inthe mutant preparations at thesame concen- tration of herbicide, Q- is reoxidized by the secondary ac- ceptor. The much slower rise in fluorescence kinetics of DCMU-4 reflects a gradual filling up ofthe plastoquinone pool.

Azido[14C]atrazine

photoaffinity-labeling ofpolypeptides from wild-type and DCMU-4 mutant cells is shown in the fluoro ram of Fig. 2. Wild-type cells treated with 0.1 ,uM azido[ 4C]atrazine show one labeled polypeptide, whereas DCMU-4 mutant cells under the same conditions show no binding of azidoatrazine. By comparison with a fluorogram of thylakoid membrane polypeptides synthesized inside the chloroplast, the peptide covalently binding azidoatrazine is identified as the32-kDaproteinthatcomigrates with D1(40).

At 10 ,M azidoatrazine, both wild-type and mutant cells showthis same protein labeledwith14C. Similarresults were obtained when isolated thylakoid membrane polypeptides from wild-type and mutant cells were photoaffinity labeled in vitro with azido[14C]atrazine (data not shown).

Fluorescence Induction inWhole Cells. Fig. 3comparesthe fluorescence-induction kinetics of dark-adapted DCMU-4 and wild-type cells in the absenceofinhibitor. Asignificant increase in the rapid phase of the fluorescent rise under strongillumination is observed inmutantcells and indicates slowing of electron transferfrom Q to B. This phenomenon

wC.) zw Cl,Q cclw

0' -JIL

WT

I P~~CMU4

I

T IME FIG.1. Fluorescence-induction kinetics ofdark-adaptedbroken- cellpreparationsofwild-type (WT) andDCMU-4mutantC. rein- hardtiiin the presence of4 AuM atrazine (Left) or 1

AtM

^DCMU

(Right).Total sweep, 1 sec.

Proc. NatLAcad Sci. USA 81

(1984)

Proc. NatL Acad. Sci. USA81 (1984) 3619

D1- ^Q

I~

Dl- 4

-

as

LU

uiz

0LLJ -JU-

.a.m

.i.d

4 _

2 3

FIG. 2. Fluorography of"4C-labeledproteins electrophoresedon a12-18% gradient polyacrylamide gel containing 8 Murea.Lanes 1 and 4, thylakoid membrane polypeptides synthesized inside the chloroplast, labeled in vivo with [14C]acetate in the^presenceof ani- somycin (15). The diffuse band marked D1includes the 32-kDapro-

tein of PSII (15). Lanes 2 and 3, labeled proteins from wild-type (WT)and DCMU-4 mutantcells,respectively, after 10min ofUV treatmentinthepresenceof 0.1 FM azido[14C]atrazine. Lanes⁵and 6,asdescribed for lanes 2 and 3, with cells irradiated in thepresence

of 10

,AM

azido[14C]atrazine. Insomegels, the protein labeled with

azido[14C]atrazine

isresolvedas alabeled doublet band.

is notseenunderconditions of weak illumination. The fluo- rescence-induction kinetics of both wild-type and mutant dark-adapted cells in thepresence orabsence ofDCMU is shown in Fig. 4. The typical increase in the initial fluores-

cence yield of wild-type cells in the presence of DCMU is duetothetransfer ofanelectron fromthe reduced second- ary acceptor B- to the primary acceptor Q (18). This in- creased initial fluorescenceyield induced by DCMU is sig- nificantly diminishedin theDCMU-4mutantcells. Fluores- cence-induction curves ofwild-type and DCMU-4 cells at varying concentrations of azidoatrazine (datanotshown) in- dicate that blockage of Q- reoxidationoccurswithIC50val-

uesof 50 nM and 5 uM, respectively.

Sequence Analysis ofthe DCMU4 32-kDa Protein Gene.

psbA, the^geneforthe 32-kDaprotein, has beenpreviously identified andsequencedinthewild-typeC. reinhardtii 137c (35). This algalgenecontainsfourintrons andcovers6.7 kb.

psbA located ^on restriction fragment 13a12 was initially cloned from DCMU-4 into a X vector and subcloned into plasmidvectors. DNAfragmentsweresequenced according tothestrategyillustratedinFig.5.Thus,thecompletenucle- otide sequencefor each of the five ^{exons was}determined.

IIJ

0

WW

0

U_

Co

-J

TIME

FIG. 3. Fluorescence-inductionkinetics of dark-adapted cells of wild-type (WT) and DCMU4mutantC.reinhardtii. Totalsweep,2 sec.

WT

T IME FIG.4. Fluorescence-induction kinetics ofdark-adapted cells of wild-type (WT) and DCMU-4 mutant C. reinhardtiiin the absence (Left) orpresence (Right) of 40

AuM

^DCMU.Total sweep, 0.1 sec.

Both strands were sequenced in every exon except for the first42 base pairs (bp) of exon 3. In this case, sequencing from the 5'-labeled Pvu II site was repeated several times.

There isonlyonedifferencebetween theexon sequences of the mutant and the wild-type psbA. Fig. 6 shows a single basepairchange ofT-A toG-C, which results in the conver- sion ofaserineresiduein the wild type toanalanine residue in the herbicide-resistantmutant. No otherdifferences were observed between the wild-type and DCMU-4 exon se- quences.

Fig. 7 shows the position of the serine residue in the 32- kDaprotein that is affected by the psbA mutation.This ser- ine,atposition 264, is the twelfth aminoacid residue in the fifth exon. It is in a region of 121 predicted amino acid resi- dues that aretotally conserved between S. oleracea (36),N.

debneyi (36), A. hybridus (41), soybean (42), and C. rein- hardtii (35).

DISCUSSION

Sequence analysis of C. reinhardtii psbA, the chloroplast genefor the 32-kDa protein, has revealedasingleT-Ato G-C transversionintheDCMU-4mutantthat results inadeduced amino acid change of serine toalanine. This change corre- lates with the observation that

azido[14C]atrazine

at0.1 ,uM bindsto the 32-kDaprotein from wild-type, butnotmutant, cells. An atrazine-resistantbiotype of the pigweedA. hybri- dus showsasimilar,reducedaffinity of the herbicide analog for the 32-kDaprotein (12). Theexactsite of herbicide bind- ing to the wild-type protein has not yet been determined.

Prediction of the hydrophobic helical structure ofthe spin- ach32-kDaprotein places this serine residue in, butnearthe edge of,ahydrophobic helix that spans the membrane(43).

The serine residue that is altered in the mutant may be an amino acid residuedirectlyinvolved in thebindingofherbi- cideormayhaveaconformational effectonthebindingsite.

The deduced amino acid substitution of alanineinthemu- tantprotein for serinein the wildtype occursinaregion of one-third of the protein that is totally conserved between spinach, tobacco, pigweed, soybean, andthe alga(Fig. 7).

Such conservation mayreflectstrongfunctionalconstraints ontheprotein. Recently, psbAfromwild-typeandatrazine- resistant biotypes of the pigweedA. hybridus havebeen se- quenced by Hirschberg and McIntosh (41). Deduced from the nucleotidesequence,aserineresiduein thewild-type32- kDaprotein of pigweedischangedto aglycineresidue in the herbicide-resistant biotype. This is the same serine residue that wefind altered to alaninein the C. reinhardtiimutant.

However, thewild-typeserine codons differat thisposition (Fig. 6). There is no way that a single point mutation can convertthealgalTCTserine codontoglycineor, converse-

ly, thepigweedAGT serine codonto alanine.

It has been proposed that the triazine

binding

domain shares residues in common with the plastoquinone

binding

site(20). Thus, onemightexpect thatastructuralchangein the 32-kDa protein that alters atrazine binding would also

Biochemistry:

^{Erickson etaL}

3620 Biochemistry: Erickson et al.

0

2 3 4 5 6 7 8 9 10kb

i --i

R H H_IH

I

_~^H_I

₂

^X

₃

^{X RH}_I ^{H KK B}

- I

4

^I

,1, 1 3 4 5'-_

H Hf

1-I ^{D x}

^SP

^x DHf R

^H

f

^Hf

I

^'

200 bp

FIG. 5. Strategy for Maxam- Gilbert sequencing (39) of the psbA exon regions (_)from the DCMU-4 mutant C. reinhardtii.

Fragmentsrecovered from acryl- amidegels werelabeledatthe 5'

(.->)

or3'(o->) end. The restric- tion mapfor thechloroplastEcoRl fragmentR14(9)and theadjacent

EcoRI-BamHI fragment

^{from R24}

is given,indicating thepositionsof thefive exons.Restriction enzyme sites for BamHI (13), Dde I (D),

EcoRI (R), HindI1 (H), Hinfl

(Hf), Kpn I (K), Pvu

¹¹

(P), Sau3A

(S),and Xba I (X)areindicated.

cause achangeinthe rateofelectron transfer in PSII in the absence of herbicide. Fluorescence-induction kinetics of DCMU-4 show thatthis is, in fact, thecase. In theabsence of DCMUoratrazine, thesecondaryacceptor site ofPSI is impaired in the mutantcells ascomparedto wild type(Fig.

3). Asimilar change in PSII electron transferwaspreviously reported for the atrazine-resistant biotype of pigweed(44). A change inplastoquinonebinding in DCMU-4 is also reflected by the loweramountof stableB-in themutantascompared to thewild-type cells (Fig. 4). Gallowayand Mets have re- ported four classes of uniparental herbicide-resistant mu- tantsofC.reinhardtii (45). TheseareresistanttoDCMU,to atrazine, to DCMU and bromacil, or toDCMU, atrazine, andbromacil. Only the lattermutant showsanalteration in PSIIelectrontransportin the absence ofherbicide.This mu- tantis 84-foldmoreresistanttoatrazine than wild-typecells, compared to 15-fold for the mutant resistant to atrazine alone. Sequenceanalysis ofpsbA from thesemutantsshould reveal much about the residues affecting binding of various herbicides and the plastoquinone.

Anipvertedrepeat, containing theDNAencoding rRNA, isfound inmostof the chloroplast genomes studiedto date (46). The C.reinhardtiipsbA, unlike that of higher plants, is located withintheinvertedrepeatandhencepresentattwo copies per

genpMe

(35). Recentevidence obtained withmu- tants deleted for aportion ofthe inverted repeat suggests that gene conversion occurs between the two segments of

therepeat (47). Both copies ofthe C. reinhardtii wild-type psbA haveaserinecodonataminoacid residue264(Fig. 6).

The mutant sequencewe reportis fromone copy of psbA.

Sequence analysis of both copies of the mutant gene isre- quired beforeanyconclusions can bereached regardingthe dominantorrecessive nature ofthepsbA mutation andthe possible occurrenceofgene conversion atthis locus.

Inadditiontoits

implications

for themechanism ofherbi- cide resistance, the DCMU-4mutant provides anexcellent chloroplast marker. Suchamutation is important in relating the genetic and physical maps of the chloroplast genome.

Onecorrelation sitewas recentlyestablished foramutation in rbcL, the gene forthe large subunit ofribulose bisphos- phate carboxylase/oxygenase (48). Genetic mapping ofthe psbA mutationrelativeto other chloroplast markers willpro- videanadditional siteof reference betweenthegenetic and physicalchloroplastmaps. AnotherDCMU-resistant unipa- rentalmutantofC.reinhardtii mapped withrespect toeryth- romycin and streptomycin markers has establisheda

prelilmi-

nary genetic location for that mutation (49). The DCMU-4 mutantwillmost likely beatthesamelocus.

Themutant genefor the 32-kDa protein has the potential to producea selectable phenotype in the transformation of chloroplasts. Since C. reinhardtii has a single large chloro- plast, it ismoreamenabletochloroplast transformationthan arehigher plants. Suchatransformation system,using psbA from the herbicide-resistant mutant, would provide an in-

G A+G A G CJT

T X _ __

A

A mi

A ^...

G

A X_

Ga_ *

G _

T~W

tyr WT IsTAC

DCMU-4-TAC S.o.N~d. -TAT

tyr

GC-

TA

^A

T AGC AT A T

AT -A

G C

T-*G

AT G CC G G

C

TAA T.-

A+G A

vkA--:.-

G C T+C

.._ _

-~~~~~~~0 ^-~a-sn

meN

ss^{.;.> - -} -~~~~~~~~~~~~~~~~~i

DCMU-4

264

ala ser phe asn

GCT

TCT

^{TTC AAC}

ala

GCT AGT TTC AAC

ala ser phe asn

FIG. 6. Comparison of the nucleotide se-

quencing

gels for the C. reinhardtiiwild-type (WT)andDCMU-4psbA showingthe T-Ato G-C transversion. Theposition of the muta- tion is marked with a dark circle. Thewild- type and mutantamino acid codons for this region (mutation affects amino acid residue 264) are given and compared to those from Spinaciaoleracea (S.o.)(36), Nicotianadeb- neyi (N.d.) (36), and Amaranthus hybridus (A.h.) (41).

Proc. NatLAcad Sci. USA 81

(1984)

0 >

IE oo---->

4E 4 " >

\

Aba.,-I.

/ .*.,!:",.W".

V..

Proc. NatL Acad. Sci USA 81 (1984) 3621

111 11 II

100 200

ala DCMUR

ser

t

WT

W 1 11

300

JIMCOOH

amino acid 352 residues

[ EXON 1 IL EXON 2 ][EXON 3][ EXON 4

][

^{EXON 5 ]}

FIG. 7. Comparison oftheaminoacidsequenceof the 32-kDa protein of C. reinhardtii(35)and S. oleracea (36). The positionsof the amino acidresiduesthatdifferareindicated with verticalbars.Thesinglemutation site in DCMU-4 is indicated withanasterisk. The mutationresults inanaminoacid residuechangebetweenthewild-type(WT)andmutant(DCMUR)strains^asindicated.Exonboundaries inthe C. reinhardtii geneareindicated by brackets.

valuable tool for studying the function and regulation of chloroplast^genes and their products.

Note Added in Proof. BothcopiesofpsbA from DCMU-4 havethe

samemutantcodonataminoacid residue264.

WearegratefultoL. McIntosh and L. Mets for communicating unpublished results andH.Bohnertforsendingusthespinach psbA

geneto use as aprobe. We thank M. Goldschmidt-Clermont and R.J. Spreitzer for helpful suggestions and critical reading of the manuscript, ^0.Jenni fordrawingsandphotography, andN. Murray for supplying NM 539 and ^X EMBL4. We also thank M. Gold- schmidt-Clermont for providing in vitro packaging extracts. This workwassupported by SwissNational Foundation Grant3.258.0.82 toJ.D.R. and EECGrantESF017toP.B.

1. Moreland,D. E.(1980)Annu.Rev.Plant Physiol.31,597-638.

2. Trebst, A. (1980) MethodsEnzymol.69,675-715.

3. Galloway, R. E. & Mets, L. (1982) Plant Physiol. 70, 1673- 1677.

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111L 11

NH2

6

Biochemistry:

EricksonetaL