FERTILIZATION in flowering plants begins when define the nature of this product, we have generated a

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1 Copyright 1999 y the Genetics Society of Americ A Moleculr Description of Muttions Affecting the Pollen Component of the Nicotin lt S locus J. F. Golz, 1 V. Su, A. E. Clrke nd E. Newigin Plnt Cell Biology Reserch Center, School of Botny, University of Melourne, Prkville, Victori 3052, Austrli Mnuscript received Decemer 15, 1998 Accepted for puliction Mrch 22, 1999 ABSTRACT Muttions ffecting the self-incomptiility response of Nicotin lt were generted y irrdition. Mutnts in the M 1 genertion were selected on the sis of pollen tue growth through n otherwise incomptile pistil. Twelve of the 18 M 1 plnts otined from the mutgenesis screen were self-comptile. Eleven self-comptile plnts hd muttions ffecting only the pollen function of the S locus (pollen-prt mutnts). The remining self-comptile plnt hd muttion ffecting only the style function of the S locus (style-prt mutnt). Cytologicl exmintion of the pollen-prt mutnt plnts reveled tht 8 hd n extr chromosome (2n 1) nd 3 did not. The pollen-prt muttion in 7 M 1 plnts ws followed in series of crosses. DNA lot nlysis using proes for S-RNse genes (encoding the style function of the S locus) indicted tht the pollen-prt muttion ws ssocited with n extr S llele in 4 M 1 plnts. In 3 of these plnts, the extr S llele ws locted on the dditionl chromosome. There ws no evidence of n extr S llele in the 3 remining M 1 plnts. The rekdown of self-incomptiility in plnts with n extr S llele is discussed with reference to current models of the moleculr sis of self-incomptiility. FERTILIZATION in flowering plnts egins when define the nture of this product, we hve generted pollen grin ering the mle gmetes lnds on series of pollen-prt muttions of the S locus (pollen femle stigm. Severl mechnisms enle the stigm prt mutnt, PPM). nd style to discriminte etween the different types In previous studies, muttions ffecting the pollen of pollen it my receive, the est studied eing self- component of the S locus hve een generted in N. incomptiility. If pollen grin from self-incompti- lt (Pndey 1965, 1967; vn Gstel nd de Nettnle plnt lnds on its own stigm, or on the stigm of court 1975), Petuni inflt (Brewker nd Ntr geneticlly relted plnt, the pollen either will fil to jn 1960), nd Solnum tuerosum (Olsder nd Hermgerminte or will germinte to produce pollen tue sen 1976; Hermsen 1978). Styles of PPM plnts retin tht grows poorly in the style nd does not rech the the ility to reject incomptile pollen. ovry (de Nettncourt 1977). In mny cses, this pro- Different types of lesions cn cuse muttions ffectcess is controlled y single, multillelic locus clled ing the self-incomptiility response of pollen. The mthe S locus. In solnceous plnts such s Nicotin lt jority of pollen-prt muttions in solnceous plnts re (ornmentl tocco), the S locus cts gmetophyticlly ssocited with duplictions of n S llele (Brewker nd hploid pollen grin is rejected y diploid style nd Ntrjn 1960; Pndey 1965, 1967; vn Gstel when the sme S llele is present in oth. The only nd de Nettncourt 1975). The duplicted S llele is known product of the solnceous S locus is n extrcel- frequently on short, dditionl chromosome known lulr rionuclese produced y the style (the S-RNse; s centric frgment, which segregtes independently McClure et l. 1989). S-RNses control the stylr pheno- of the S locus. The self-incomptiility phenotype of type of self-incomptile plnts ut do not control the pollen from these plnts is only ltered when the duplipollen phenotype (Lee et l. 1994; Dodds et l. 1999). cted S llele nd the llele present t the S locus re This suggests the S locus is iprtite, with different genes different (Brewker nd Ntrjn 1960). This pheencoding the pollen component (pollen-s) nd the style nomenon is clled competitive interction nd requires component (S-RNse) of the S locus. The product of two different S lleles to e present in the plnt. In the pollen-s gene is not known. As prt of strtegy to ddition, competitive interction results in progeny of ckcross nd selfed fmilies hving two different S lleles (Pndey 1967; vn Gstel nd de Nettncourt Corresponding uthor: Adrienne E. Clrke, Plnt Cell Biology Reserch 1975; see de Nettncourt 1977). Center, School of Botny, University of Melourne, Prkville, A lesion in the pollen-s gene cuses the other type of Victori 3052, Austrli. E-mil:.clrke@otny.unimel.edu.u 1 muttion ffecting the self-incomptiility response of Present ddress: Institute of Cell nd Moleculr Biology, Rutherpollen. These re true pollen-prt muttions nd cn ford Bldg., Kings Bldgs., Myfield Rd., Edinurgh, EH9 3JR, United Kingdom. e distinguished from plnts crrying duplicted S l- Genetics 152: ( July 1999)

2 1124 J. F. Golz et l. TABLE 1 Pollintion responses of plnts from the M 1 genertion Cross Phenotype Plnt Self S 3 S 6 M 1 S 2 S 2 M 1 M 1 S 2 S 2 M 1 S 3 S 3 M 1 S 6 S 6 Pollen Pistil M1-1 PPM S 3 S 6 M1-2 PPM S 3 S 6 M1-3 INC S 3 S 6 M1-4 INC S 3 S 6 M1-5 PPM S 6 S 6 M1-6 PPM S 3 S 3 M1-7 PPM S 3 S 3 M1-8 INC S 3 S 3 M1-9 PPM S 3 S 6 M1-10 PPM S 3 S 6 M1-11 PPM S 3 S 6 M1-12 PPM S 3 S 6 M1-13 c S 3 S 6 M1-14 INC S 3 S 6 M1-15 PPM S 3 S 6 M1-16 PPM S 3 S 6 M1-17 INC S 3 S 3 M1-18 INC S 3 S 3 SPM WT INC S 3 S 6, comptile pollintion;, incomptile pollintion; WT, n unmutted S 3 S 6 plnt; PPM, pollen-prt mutnt; SPM, style-prt mutnt; INC, pollen incomptiility response ws the sme s WT plnt. The femle plnt is listed first. These plnts were self-sterile ut were not chrcterized ny further. c The pollen phenotype of M1-13 could not e determined ecuse of low pollen viility. leles ecuse they my e homozygous for S lleles nd directly in pollintions or stored t 70 until needed. When- cn produce homozygous progeny following ckcross ever prcticle, ech pollen smple ws used to pollinte two flowers from n unmutted S 3 S 6 plnt (i.e., n incomptile or self-pollintions. pollintion). Following ech pollintion, 1% indole-3-cetic Becuse none of the N. lt PPM plnts generted cid in lnolin ws pplied to the se of the flower. At mtuin previous studies were ville, we generted PPM rity, the cpsules were opened nd the seeds collected. Before plnts using the sme strtegy pplied in erlier studies germintion, seeds were surfce sterilized for 1 hr with hypo- (Pndey 1967; vn Gstel nd de Nettncourt chlorite solution (1% HClO in 0.1% Tween) nd rinsed thoroughly in sterile wter. Seeds were then plced on sterile 1975). Following irrdition of S 3 S 6 N. lt plnts, 18 MS gr contining 3% sucrose nd incuted t 22. When M 1 individuls were isolted nd chrcterized y polli- sufficiently strong, ech seedling ws trnsplnted into soil ntion, cytologicl exmintion of root tip cells, DNA nd grown s descried y Anderson et l. (1989). lot nlysis with S-RNse gene proes, nd protein lot Pollintion nlysis: Plnts were self-pollinted y spreding nlysis with S-RNse-specific ntiodies. Eleven plnts pollen from dehiscent nther over the stigms of four or more flowers. A pollintion ws comptile if lrge cpsule hd muttions ffecting the pollen component of the developed nd incomptile if the flower scised in the week S locus nd inheritnce of the pollen-prt muttion in following pollintion. To determine the stylr self-incomptiseven of these PPM plnts ws followed through series ility phenotype of plnt, immture florl uds were emsculted of crosses. The nture of the muttion in these plnts nd pollinted with pollen from plnt of known S is discussed with reference to current models of selfwere genotype soon fter petl opening. Four such pollintions usully done for ech plnt. Similr crosses were used incomptiility. to determine the self-incomptiility phenotype of pollen from the plnt. MATERIALS AND METHODS DNA lot nlysis: Genomic DNA ws extrcted from the leves of N. lt plnts s descried y Berntzky nd Tnksley Screen for pollen-prt mutnts: Mture N. lt plnts (genotype (1986). Lef DNA (5 g) ws digested to completion with S 3 S 6 ) received totl dose of either 8 or 10 Gy from HindIII or BmHI (Promeg, Mdison, WI). The DNA ws 60 Co source (1.4 Gy/min) housed t the CSIRO Division of frctionted on 0.8% grose gel run in 1 TBE nd trns- Plnt Industry, Cnerr, Austrli. The trget tissue ws florl ferred to nylon memrne (Amershm, Buckinghmshire, uds contining pollen mother cells (see Dodds et l. 1993). UK) s descried y Smrook et l. (1989). S-RNse cdna Of 149 uds irrdited, 108 received 8 Gy nd 41 received 10 frgments were rdioleled with rndom primers (Prim- Gy. Following irrdition, the florl uds were leled nd gene, Promeg). Hyridiztion of the rdioleled S-RNse the pollen ws collected t nthesis. This ws either used cdnas to the DNA lots ws done in 50% formmide, 5

3 Pollen-Prt Mutnts of N. lt 1125 Figure 1. DNA lot nlysis of the M 1 plnts. Ech lne contins genomic DNA (5 g) from the indicted M 1 plnt or n unmutted S 3 S 6 plnt (WT). DNA ws digested with BmHI (top) or HindIII (ottom) nd proed with the S 3 - RNse cdna nd the S 6 -RNse cdna seprtely (top) or oth the S 3 - nd S 6 -RNse cdnas (ottom). Moleculr weight stndrds (in kiloses) re shown on the left of the figure nd the identity of the S-RNse hyridizing nds is indicted t Figure 2. S-RNse ccumultion y the pistils of unmu- the right of the figure. tted N. lt plnts nd selected M 1 plnts. Buffer-solule protein ws extrcted from pistils of the indicted M 1 plnts nd pistils of unmutted S 3 S 3, S 6 S 6, nd S 3 S 6 plnts. M 1 plnts SSPE, 5 Denhrdt s solution, 0.5% SDS, 50 g/ml dentured tht hve n S 6 -RNse gene ut fil to reject S 6 pollen re herring sperm DNA t 42 for 12 hr. After hyridiztion, the indicted (SPM). Proteins were seprted y SDS-PAGE nd memrnes were wshed twice (30 min ech time) t 42 in either stined with silver (A) or lotted onto nitrocellulose 0.2 SSPE, 0.2% SDS nd exposed to film. nd incuted with n ntiserum specific to the S 6 -RNse (B). Western lot nlysis: Styles were collected nd stored t Protein nds corresponding to the S 3 - nd S 6 -RNses (A) or 70. Proteins were extrcted from plnt tissue in n extrc- the S 6 -RNse (B) re indicted. Moleculr weight stndrds tion uffer (100 mm Tris-HCl, ph 8, 50 mm EDTA, 0.1% (in kilodltons) re shown t the left of the figure. polyvinylpyrrolidone, 28 mm -mercptoethnol) to give 25% solution. Protein concentrtions were determined using colorimetric ssy (Brdford 1976) with BSA s stndrd. ges were cptured with Zeiss MC63 photogrphic unit using Stylr proteins (15 g) were frctionted on 15% SDS-poly- Tmx100 film (Kodk, Rochester, NY). crylmide gel ccording to the method of Lemmli (1970). Proteins were trnsferred to nitrocellulose memrne (Schleicher nd Schuell, Dssel, Germny) in trnsfer uffer (48 mm Tris-HCl; 39 mm glycine; % SDS; 20% methnol) using semidry electrophoretic trnsfer cell pprtus Production of the M 1 genertion: Developing N. lt RESULTS (Trnslot, Bio-Rd, Richmond, CA). A replicte gel with 5 g stylr proteins in ech lne ws stined with silver (Bio-Rd). flower uds (genotype S 3 S 6 ) were irrdited with either The memrne ws incuted with protein G-purified sheep 8or10Gyof -rys from 60 Co source. Pollen ws ntiserum for the S 6 -RNse (Dodds et l. 1993) s descried susequently collected t nthesis nd used to pollinte y Hrlow nd Lne (1988). Bound ntiodies were detected pistils of unmutted S 3 S 6 plnts. From 300 pollintions, using iotinylted nti-sheep immunogloulin nd streptvi- only three cpsules tht contined seeds were recovered. din-horserdish peroxidse (Amershm) ccording to the mnufcturer s instructions. Two cpsules, contining 7 vile seeds, were produced Cytology: Root tips were collected from hydroponiclly following pollintion with pollen irrdited with 8 Gy grown plnts. After hrvest, the root tips were plced in nd one cpsule, contining 11 seeds, ws from pollen sturted solution of -romonphthlene nd incuted for irrdited with 10 Gy. The seeds were germinted nd 2 hr t room temperture with occsionl gittion. Root tips the seedlings were grown to mturity. Plnts were numwere then wshed with wter nd fixed in ethnol:cetic cid (3:1) for 12 hr t 4. After fixtion, the root tips were plced ered M1-1 to M1-18. M1-1 to M1-4 cme from one in 70% ethnol solution nd stored t 4 for up to 1 month cpsule, nd M1-5 to M1-7 from the other cpsule efore nlysis. For cytology, fixed root tips were treted with formed y pollen irrdited with 8 Gy. The remining 0.2 n HCl solution for 10 min t 55. After cid hydrolysis, plnts cme from the cpsule formed y pollen irrdithe root tips were wshed with wter nd plced in stining ted with 10 Gy. The plnts grew normlly nd were not solution [2% synthetic orcein (Gurr) in 45% cetic cid] for 40 min. Root tips were destined in solution of 45% cetic visily different from nonmutgenized N. lt plnts. cid. Mcerted root tips were spred nd exmined under Twelve of the 18 M 1 plnts formed lrge cpsules fter phse contrst optics using Zeiss Universl microscope. Im- self-pollintion nd the remining 6 plnts were self-

4 1126 J. F. Golz et l. TABLE 2 Summry of the pollintion, DNA lot, stylr protein, nd cytology nlyses of plnts from the M 1 genertion S phenotype S-RNse S-RNse Chromo- Type of Plnt Pollen Pistil genes proteins some no. muttion M1-1 PPM S 3 S 6 S 3 S 6 S 3,S 6 2n 1 PPM M1-2 PPM S 3 S 6 S 3 S 6 S 3,S 6 2n 1 PPM M1-5 PPM S 6 S 6 S 6 S 6 2n PPM M1-6 PPM S 3 S 3 S 3 S 6 S 3,S 6 (trce) 2n 1 PPM/SPM M1-7 PPM S 3 S 3 S 3 S 6 S 3,S 6 (trce) 2n PPM/SPM M1-8 INC S 3 S 3 S 3 S 3 2n 1 REV M1-9 PPM S 3 S 6 S 3 S 6 S 3,S 6 2n 1 PPM M1-10 PPM S 3 S 6 S 3 S 6 S 3,S 6 2n PPM M1-11 PPM S 3 S 6 S 3 S 6 S 3,S 6 2n 1 PPM M1-12 PPM S 3 S 6 S 3 S 6 S 3,S 6 2n 1 PPM M1-13 S 3 S 6 S 3 S 6 S 3,S 6 3n 1 Polyploid M1-14 INC S 3 S 6 S 3 S 6 S 3,S 6 2n 1 REV M1-15 PPM S 3 S 6 S 3 S 6 S 3,S 6 2n 1 PPM M1-16 PPM S 3 S 6 S 3 S 6 S 3,S 6 2n 1 c PPM M1-17 INC S 3 S 3 S 3 S 3 2n 1 REV M1-18 INC S 3 S 6 SPM S 3 S 6 S 3,S 6 (trce) 2n SPM WT INC S 3 S 6 S 3 S 6 S 3,S 6 2n REV, revertnt plnt (see text); other revitions re defined in Tle 1. 2n 18 chromosomes; 2n 1 18 chromosomes plus centric frgment; 3n 1 28 chromosomes. The pollen phenotype of M1-13 could not e determined ecuse of low pollen viility (see Tle 1). c Cytology of plnt from the ckcross fmily of M1-16 (see text). sterile (see Tle 1; dt re incomplete for M1-3 nd tues to grow through n incomptile style reverted M1-4, which were self-sterile ut were not chrcterized to n unmutted stte fter fertiliztion. The self-incomfurther). Crosses to N. lt plnts of known S genotypes ptile M 1 plnts re therefore descried s revertnts were used to chrcterize the pollen nd pistil selfincomptiility (REVs). M1-13 did not produce vile pollen s no cp- phenotype of ech M 1 plnt (Tle 1). sules formed fter pollintion of n S 2 S 2 plnt. Ten plnts hd pistils tht rejected pollen from S 3 S 3 nd DNA lot nlysis of M 1 plnts: The S genotype of 16 S 6 S 6 plnts, which indicted their pistil phenotype ws M 1 plnts ws determined y DNA lot nlysis using S 3 S 6 ; five plnts hd pistils tht rejected S 3 pollen ut the S 3 - nd S 6 -RNse cdnas s proes (Figure 1). The ccepted S 6 pollen nd therefore hd S 3 S 3 s their pistil S 3 -RNse gene ws present in ll plnts except M1-5 phenotype; nd one plnt rejected S 6 pollen ut ccepted (S 6 S 6 ). Similrly, the S 6 -RNse gene ws present in ll S 3 pollen, which indicted its pistil phenotype plnts except M1-8 nd M1-17 (oth S 3 S 3 ). Thus, with ws S 6 S 6. the exception of M1-6 nd M1-7 (oth S 3 S 3 ) nd M1-18 Cpsules formed following the pollintion of S 3 S 6 pis- (S 3 S 3 SPM), the S genotype of the M 1 plnts determined tils with pollen from 11 of the 12 self-fertile M 1 plnts. y DNA lot nlysis mtched the pistil phenotype. This showed these plnts crried muttions ffecting the Detection of S-RNses in the pistils of M 1 plnts: To self-incomptiility phenotype of their pollen. Cpsules understnd the discrepncy etween the S phenotype did not form following similr pollintions using pollen nd S genotype in plnts M1-6, M1-7, nd M1-18, the from the self-fertile plnt M1-18, indicting this plnt ccumultion of S-RNses y the styles of these plnts crried muttion ffecting the self-incomptiility phe- ws ssessed y SDS-PAGE nd Western lot nlysis. notype of its styles ( style-prt mutnt, SPM). M1-18 Buffer-solule proteins were extrcted from the styles nd 3 of the 4 self-sterile M 1 plnts (M1-8, M1-14, nd (including stigms) of unmutted plnts (S 3 S 3, S 6 S 6, nd M1-17), produced vile pollen (cpsules formed fter S 3 S 6 ), nd from the indicted M 1 plnts (Figure 2). Stylr pollintion of comptile S 2 S 2 pistil). Interestingly, protein ws seprted y SDS-PAGE nd either stined these plnts did not hve muttions ffecting the selfincomptiility with silver (Figure 2A) or trnsferred to nitrocellulose phenotype of their pollen, even though memrne nd incuted with n ntiserum specific they were ll grown from seed formed fter n incomptile for the S 6 -RNse (Figure 2B). pollintion. Presumly the norml self-incompti- In Figure 2A, the S 3 -RNse ppered s nd of M r ility response of M1-8, M1-14, M1-17, nd M1-18 pollen 35 kd nd the S 6 -RNse s nd of M r 33 kd. In Figure rose ecuse the muttion tht hd llowed the pollen 2B, the S 6 -RNse ppered s mjor nd of 33 kd

5 Pollen-Prt Mutnts of N. lt 1127 Figure 3. Microgrphs of metphse chromosomes in the root tip cells of n unmutted S 3 S 6 plnt (A) nd representtive M 1 plnts (B F). The unmutted S 3 S 6 plnt (A) nd plnt M1-5(B) oth contin 18 chromosomes. Nineteen chromosomes were seen in M1-1 (C), M1-6 (D), nd M1-14 (E). The dditionl chromosome in M1-1 nd M1-6 (C nd D) ws smller thn the rest (rrow). All chromosomes in M1-14 (E) were similr in size. Cells from M1-13 contined 28 chromo- somes (F). Br in A, 5 m. will e referred to s S 6 spm. Plnt M1-18 hd this muttion lone; plnts M1-6 nd M1-7 hd pollen-prt muttions s well. The results of protein nd Western nlyses of ll M 1 plnts re summrized in Tle 2. Cytology: Mitotic chromosomes in the root tips of n unmutted S 3 S 6 plnt nd ll M 1 plnts were stined with orcein nd exmined y phse-contrst microscopy. Typiclly, four root tips were exmined from ech plnt nd the numer of chromosomes in t lest four cells from ech root tip ws counted. Figure 3 shows representtive exmples of these cells. The results for ech M 1 plnt re summrized in Tle 2. The cells of n unmutted N. lt plnt nd four M 1 plnts contined 18 chromosomes (Figure 3, A nd B), which is the expected numer of chromosomes in this species. The morphology of individul chromosomes lso mtched n erlier description of N. lt chromosomes mde y Crluccio et l. (1974). Eleven of the M 1 plnts contined 19 chromosomes (Tle 2) nd one M 1 plnt (M1-13) contined 28 chromosomes (Figure 3F). In plnts with 19 chromosomes, the dditionl chromosome ws generlly smller thn the other chromosomes nd vried in length from 1 m (for exmple, M1-1; Figure 3C) to 1.7 m (for exmple, M1-6; Figure 3D). In plnt M1-14, none of the chromosomes ws noticely shorter thn the others, mking it difficult to sy which ws dditionl (Figure 3E). When identifile, the dditionl chromosome hd constriction indictive of centromere. In keeping with the nomenclture used y erlier reserchers, the dditionl chromosome will e referred to s centric frgment. Breeding nlysis of four PPM M 1 plnts tht hd centric frgment: One hypothesis to ccount for the pollen-prt muttion in the four PPM M 1 plnts with centric frgment is tht the centric frgment in these plnts crries duplicted S llele. This hypothesis ws tested y correlting the presence of centric frgment with the S phenotype nd S genotype of plnts produced y ckcrossing n M 1 plnt to n unmutted S 3 S 6 plnt or outcrossing it to n unmutted S 2 S 2 plnt. In one cse, fmily produced y self-pollinting n M 1 plnt ws used insted of ckcrossed fmily. S genotypes were determined y DNA lot nlysis using S-RNse cdnas s proes. Tle 3 summrizes the results of this nlysis for plnts M1-1, M1-2, M1-6, nd M1-11. All plnts in the ckcross fmily of M1-1 hd S 3 S 6 s their pistil phenotype nd, with one exception, ll were PPMs. The S genotype of five PPM plnts from the ckcross fmily ws determined y DNA gel lot nlysis nd, s expected, oth the S 3 - nd S 6 -RNse genes were present. Cytologicl nlysis of root tip cells from two PPM plnts showed oth plnts contined 19 chromosomes, with the dditionl chromosome pprently identicl in size to the protein seen in Figure 2A. A second nd of M r 31.5 kd nd third, less undnt nd of M r 30 kd were lso seen. The mount of S 3 - nd S 6 -RNse extrcted from styles of M1-11 nd the other nine S 3 S 6 M 1 plnts (dt not shown) ws similr to tht in the style of n unmutted S 3 S 6 plnt. Likewise, the mount of S 3 -RNse in the style of M1-8 nd M1-17 (oth S 3 S 3 ) nd the mount of S 6 -RNse in M1-5 styles (S 6 S 6 ) ws similr to tht in unmutted S 3 S 3 nd S 6 S 6 styles, respectively (Figure 2A nd dt not shown). However, lthough M1-6, M1-7, nd M1-18 hd the S 6 - RNse gene, little or no S 6 -RNse could e detected in their styles. It is likely tht the level of S 6 -RNse in these plnts ws less thn tht required to reject S 6 pollen identicl to the centric frgment in plnt M1-1 (Figure 3C). (Tle 1). All three plnts re therefore SPMs with lesion ffecting the style-prt of the S 6 llele. This llele Two types of plnts were present in the outcross fmily

6 1128 J. F. Golz et l. TABLE 3 Pollintion, DNA lot, nd cytology dt for progeny derived from four M 1 PPM plnts crrying centric frgment S phenotype of progeny No. of S-RNse Centric Cross Pollen Pistil progeny genes frgment c S 3 S 6 M1-1 INC S 3 S 6 1 ND ND PPM S 3 S 6 12 S 3 S 6 (5) 2 (2) S 2 S 2 M1-1 INC S 2 S 6 5 S 2 S 6 (5) 0 (2) PPM S 2 S 3 S 6 6 S 2 S 3 S 6 (5) 2 (2) S 3 S 6 M1-2 PPM S 3 S 6 8 S 3 S 6 (5) 1 (1) PPM S 6 S 6 10 S 6 (5) 1 (1) S 2 S 2 M1-2 INC S 2 S 3 5 S 2 S 3 (5) 1 (2) INC S 2 S 6 3 S 2 S 6 (3) 0 (1) PPM S 2 S 6 1 S 2 S 6 (1) 1 (1) S 3 S 6 M1-6 PPM S 3 S 3 SPM 11 S 3 S 6 (5) 1 (1) PPM S 3 S 6 9 S 3 S 6 (5) 1 (1) S 2 S 2 M1-6 INC S 2 S 3 5 S 2 S 3 (5) 0 (2) INC S 2 S 2 SPM 4 S 2 S 6 (4) 0 (2) PPM S 2 S 3 SPM 1 S 2 S 3 S 6 (1) 1 (1) M1-11 self PPM S 3 S 3 SPM 5 S 3 S 6 (5) 1 (1) PPM S 3 S 6 10 S 3 S 6 (10) 1 (1) S 2 S 2 M1-11 INC S 2 S 6 26 S 2 S 6 (26) 0 (2) INC S 2 S 2 SPM 10 S 2 S 6 (10) 0 (2) PPM S 2 S 3 S 6 2 S 2 S 3 S 6 (2) 1 (1) PPM S 2 S 3 SPM 3 S 2 S 3 S 6 (3) 3 (3) ND, not determined; other revitions re defined in Tle 1. The femle plnt is listed first. The numer of progeny exmined y DNA lot hyridiztion is indicted in prentheses. c The numer of progeny with centric frgment (numer of plnts exmined) is indicted. of M1-1; self-incomptile S 2 S 6 plnts nd PPM plnts ments. Similr types of plnts were lso found in fmily with n S 2 S 3 S 6 (trillelic) pistil phenotype. DNA lot produced y crossing M1-2 to n S 1 S 1 plnt (dt not nlysis of five plnts from ech clss confirmed the shown). presence of the S 2 - nd S 6 -RNse genes in the self-incomptile S 3 S 3 or S 3 S 6 PPM plnts were found in the ckcrossed plnts nd the S 2 -, S 3 -, nd S 6 -RNse genes in the fmily of M1-6 nd the selfed fmily of M1-11 (Tle trillelic PPM plnts (Figure 4A). Cytologicl nlysis 3). The S 3 - nd S 6 -RNse genes were detected in repre- found centric frgment in the root tip cells of two senttive S 3 S 6 nd S 3 S 3 plnts from oth fmilies (Figure trillelic PPM plnts ut not in two self-incomptile 4, D nd E). This indicted some plnts inherited n S 2 S 6 plnts. Similr clsses of plnts were lso found in S 6 spm llele. As mentioned ove, plnt M1-6 hd muttion fmilies produced y outcrossing M1-1 to S 1 S 1 nd S 7 S 7 ffecting expression of the S 6 -RNse gene nd the plnts (dt not shown). S 3 S 3 PPM plnts in the ckcross fmily presumly inherited PPM plnts with either S 3 S 6 or S 6 S 6 s their pistil phenotype this muttion. SDS-PAGE nd Western nlyses were found in the ckcross fmily of M1-2 (T- of plnts from the selfed fmily of M1-11 found trce le 3). DNA gel lot nlysis detected oth the S 3 - nd levels of S 6 -RNse in the pistils of S 3 S 3 PPM plnts (dt S 6 -RNse genes in representtive S 3 S 6 plnts ut only the not shown). This showed M1-11 hd the S6 spm llele nd S 6 -RNse gene in representtive S 6 S 6 plnts (Figure 4B). ws oth pollen nd style-prt mutnt (SPM/PPM). Both types of PPM plnt inherited the centric frgment Both S 3 S 3 SPM/PPM nd S 3 S 6 PPM plnts inherited centric present in plnt M1-2. frgments from M1-6 nd M1-11 (Tle 3). Three types of plnts were found in the outcross fm- Three types of plnts were found in the outcrossed ily of M1-2: self-incomptile plnts, either S 2 S 3 or S 2 S 6, fmily of plnt M1-6: self-incomptile S 2 S 3 plnts, S 2 S 2 nd n S 2 S 6 PPM plnt. The expected S-RNse genes SPM plnts, nd S 2 S 3 PPM plnt (Tle 3). DNA lot were detected y DNA lot nlysis in representtives nlysis found the S 2 - nd S 6 -RNse genes in the S 2 S 2 of ech type of plnt (Figure 4C). The S 2 S 6 PPM plnt SPM plnts nd S 2 -, S 3 -, nd S 6 -RNse genes in the S 2 S 3 nd t lest one of the S 2 S 3 self-incomptile plnts inherited PPM plnt. Both types of plnts hd presumly inher- the centric frgment from M1-2 (Tle 3). ited the S6 spm llele from M1-6. Cytologicl exmintion Other self-incomptile plnts lcked centric frg- found centric frgment in the PPM plnt.

7 Pollen-Prt Mutnts of N. lt 1129 Figure 4. DNA lot nlysis of PPM plnts with centric frgment nd representtive progeny. Genomic DNA ws isolted from the indicted plnt, digested with HindIII nd frctionted on n grose gel. After trnsfer onto nylon memrne, the lots were proed with 32 P-leled S- RNse cdna proes. (A) M1-1 nd representtives of the phenotypic clsses (self-incomptile S 2 S 6 nd S 2 S 3 S 6 PPMs) identified in the S 2 S 2 outcross fmily (see Tle 3). DNA from unmutted S 2 S 2 nd S 3 S 6 plnts is lso shown. (B) M1-2 nd representtives of the phenotypic clsses identified in the S 3 S 6 ckcross fmily (S 3 S 6 PPM nd S 6 S 6 PPM plnts). (C) M1-2 nd representtives of the phenotypic clsses identified in the S 2 S 2 outcross fmily (selfincomptile S 2 S 3 nd S 2 S 6 plnts nd S 2 S 6 PPM plnt). (D) M1-6 nd representtives of the phenotypic clsses identified in the S 3 S 6 ckcross fmily (S 3 S 3 SPM/PPM nd S 3 S 6 PPM plnts). (E) M1-11 nd representtives of the phenotypic clsses identified in the S 3 S 6 ckcross fmily (S 3 S 3 SPM/PPM nd S 3 S 6 PPM plnts). Moleculr weight stndrds (in k) re shown on the left of the figure nd the S- RNse hyridizing nds re indicted t the right of the figure. A similr rnge of pollintion phenotypes ws lso S genotype ws determined y DNA lot nlysis using present in the outcross fmily of plnt M1-11, except S-RNse cdnas s proes. Tle 4 summrizes reeding tht this fmily included self-incomptile S 2 S 6 plnts dt for plnts M1-5, M1-7, nd M1-10. nd S 2 S 3 S 6 PPM plnts (Tle 3). DNA lot nlysis PPM plnts with S 3 S 3,S 3 S 6, nd S 6 S 6 pistil phenotypes found the S 2 - nd S 6 -RNse genes in the S 2 S 6 self-incom- were found in the ckcross fmily of M1-5 (Tle 4). ptile nd the S 2 S 2 SPM plnts nd the S 2 -, S 3 -, nd S 6 - DNA gel lot nlysis detected the S 3 - nd S 6 -RNse RNse genes in the S 2 S 3 nd S 2 S 3 S 6 PPM plnts. The genes in S 3 S 3 nd S 3 S 6 PPM plnts, ut only the S 6 -RNse S6 spm llele from plnt M1-11 hd therefore een inherited gene ws present in S 6 S 6 PPM plnts (Figure 5A). SDScl y the S 2 S 2 SPM nd S 2 S 3 SPM/PPM plnts. Cytologi- PAGE nd Western nlyses found only trce mounts nlysis found centric frgment in the root tip of the S 6 -RNse in the pistils of S 3 S 3 PPM plnts, showing cells of four PPM plnts, ut not in either the self- these plnts hd inherited n S6 spm llele previously undetected incomptile or SPM plnts. in M1-5 (dt not shown). Breeding nlysis of three PPM M 1 plnts tht lcked Self-incomptile S 2 S 6 plnts nd PPM plnts with either centric frgment: One wy to ccount for the pollenprt S 2 S 2 or S 2 S 6 s their pistil phenotype were found in muttion in the three PPM plnts tht lck centric the outcross fmily of M1-5 (Tle 4). The S 2 - nd S 6 - frgment is to ssume the muttion is cused y lesion RNse genes were found in ll plnts tested (Figure in the pollen-s gene. This hypothesis ws tested y exm- 5B). The S 2 S 2 PPM plnt ws presumly lso n SPM ining the S phenotype nd S genotype of plnts produced nd hd inherited the S6 spm llele from M1-5. y ckcrossing n M 1 plnt to n unmutted PPM plnts with either S 3 S 3 or S 3 S 6 s their pistil phe- S 3 S 6 plnt or outcrossing it to n unmutted S 2 S 2 plnt. notype were found in the ckcross fmily of plnt

8 1130 J. F. Golz et l. TABLE 4 Pollintion nd DNA lot dt for progeny derived from three PPM plnts lcking centric frgment S phenotype of progeny Cross Pollen Pistil No. of progeny S-RNse genes S 3 S 6 M1-5 PPM S 3 S 3 SPM 3 S 3 S 6 (3) PPM S 3 S 6 4 S 3 S 6 (4) PPM S 6 S 6 13 S 6 (5) S 2 S 2 M1-5 INC S 2 S 6 10 S 2 S 6 (5) PPM S 2 S 2 SPM 1 S 2 S 6 (1) PPM S 2 S 6 11 S 2 S 6 (5) S 3 S 6 M1-7 PPM S 3 S 3 SPM 8 S 3 S 6 (5) PPM S 3 S 6 12 S 3 S 6 (7) S 2 S 2 M1-7 INC S 2 S 3 19 S 2 S 3 (5) INC S 2 S 2 SPM 2 S 2 S 6 (2) S 3 S 6 M1-10 PPM S 3 S 6 8 S 3 S 6 (8) PPM S 6 S 6 4 S 6 (2) S 2 S 2 M1-10 INC S 2 S 3 9 S 2 S 3 (5) PPM S 2 S 6 7 S 2 S 6 (5) The femle plnt is listed first. The numer of progeny exmined y DNA lot hyridiztion is indicted in prentheses. M1-7. As plnts from oth clsses contined the S 3 - nd specific ntiodies to identify S-RNse products of the S 6 -RNse genes, the S 3 S 3 PPM plnts presumly inherited S locus. This gives us n independent mens of identi- the S6 spm llele present in plnt M1-7 (see ove). fying S lleles nd n opportunity to get more precise Two types of plnts were found in the outcrossed fmily description of the nture of the muttions. of plnt M1-7: self-incomptile S 2 S 3 plnts nd S 2 S 2 SPM Ionizing rdition cuses chromosoml ltertions plnts (Tle 3). No PPM plnts were mong the 21 such s inversions nd deletions nd hs frequently een plnts exmined. The S 2 S 2 SPM plnts in this fmily used to induce pollen-prt muttions (de Nettnpresumly inherited the S6 spm llele nd, consistent with court 1977). The clsses of plnts produced y mutthis, DNA lot nlysis found the S 2 - nd S 6 -RNse genes genesis in this study were similr to those descried in these plnts. erlier (Pndey 1967; vn Gstel nd de Nettn- S 3 S 6 nd S 6 S 6 PPM plnts were found in the ckcross court 1975). Self-comptiility ws minly due to mutfmily of M1-10 (Tle 4). DNA gel lot nlysis de- tions ffecting pollen phenotype (PPMs). The mjority tected the S 3 - nd S 6 -RNse genes in the S 3 S 6 PPM plnts, of PPMs expressed two S lleles (S 3 nd S 6 ) in their pistil ut only the S 6 -RNse gene in the S 6 S 6 PPM plnts (dt ut few expressed single S llele (either S 3 or S 6 ). not shown). Self-incomptile S 2 S 3 nd S 2 S 6 plnts nd The proportion of homozygous to heterozygous PPMs PPM plnts with n S 2 S 6 pistil phenotype were found in seen in this study ws lso similr to tht reported erlier. the outcross fmily of M1-10 (Tle 4). As oserved in erlier studies, mny plnts hd centric Breeding nlysis of two revertnt M 1 plnts with frgment. centric frgment: The revertnt plnts, M1-8 nd M1- Style nd pollen-prt muttions re independent: We 17, were crossed s the mle prent to self-incomptile identified style-prt muttions (SPMs) in three M 1 plnts S 2 S 2 plnts. All progeny of these crosses were self-incom- (M1-6, M1-7, nd M1-18). Ech plnt hd n S6 spm llele ptile with n S 2 S 3 pistil phenotype (Tle 5). DNA nd two hd pollen-prt muttion s well (Tle 2). gel lot nlysis detected the S 2 - nd S 3 -RNse genes in Moleculr nlyses of plnts in the M 2 genertion lso ll the plnts. The centric frgment ws found in two identified S6 spm lleles in plnts M1-5 nd M1-11. Funcof four plnts exmined from ech fmily. tionl S 6 lleles msked the style-prt muttions in these M 1 plnts, which indictes the S6 spm llele is recessive. In DISCUSSION plnts with n S6 spm llele nd no functionl S 6 llele, low levels of stylr S 6 -RNse were detected y Western lotting. Our study of pollen-prt muttions is prt of roder The lesion in the S6 spm llele is unknown ut ppers study imed t identifying the pollen-s gene. Erlier to ct in cis nd ffects the level of expression of the S 6 - exmintions of pollen-prt muttions relied on pollinindictes RNse gene. However, our nlysis of the M 2 fmilies tion ehvior to identify S lleles in individul plnts. the S6 spm llele my not e completely penetrnt A significnt dvnce of this study ws the vilility nd cn in some instnces ecome functionl S 6 llele. of cdna proes to identify prticulr S llele nd Breeding experiments showed tht the pollen function

9 Pollen-Prt Mutnts of N. lt 1131 of the S6 spm llele is norml nd the muttion is indepen- The inheritnce of the pollen-prt muttion in seven dent of the pollen-prt muttion. For exmple, 10 of M 1 plnts ws studied. Four plnts (M1-1, M1-6, M1-7, the outcross progeny of M1-11 inherited the S6 spm llele nd M1-11) hd duplicted S 3 llele s judged y DNA ut not the pollen-prt muttion (Tle 3). Style-prt lot nlysis with S-RNse proes (Tle 6) nd the muttions ffecting the expression of the S 6 llele hve three remining plnts (M1-2, M1-5, nd M1-10) did lso een noted in N. lt plnts recovered from tissue not (Tle 7). We will discuss the pollen-prt muttions culture (H. Du, A. E. Clrke nd T. Bcic, personl in these two groups seprtely. communiction). The S6 spm llele in these plnts rose The outcross fmilies of M1-1, M1-6, nd M1-11 con- in the sence of irrdition. It my well e tht the tined trillelic progeny, which indictes some of the S6 spm llele descried here lso rose spontneously in pollen produced y these plnts contined oth the S 3 our stock lines nd ws not produced y irrdition. nd S 6 lleles. For M1-1 nd M1-11, the lck of S 2 S 3 This muttion is not discussed further. progeny in the outcross fmilies showed tht the S 3 llele Evidence of duplicted S llele in four M 1 plnts: ws not t the S locus. The duplicted S 3 llele (ds 3 )is therefore proly on the centric frgment s centric frgments were found in ll trillelic progeny. On this sis, the S genotypes of M1-1 nd M1-11 re S 6 S 6 duplicted S 3 (S 6 S 6 ds 3 ) nd S 6 S6 spm ds 3, respectively. The outcross progeny of M1-6 indicte tht this plnt hs oth the S 3 nd S6 spm lleles t its S locus. The duplicted llele in M1-6 is therefore either S 3 or S 6 spm nd is ssocited with centric frgment. The reltive intensities of the S 3 - nd S 6 -RNse hyridizing nds on lots of M1-6 DNA indicte S 3 S6 spm ds 3 s the most likely S genotype of M1-6 (Figure 4D). The lck of PPM plnts in the outcross fmily of M1-7 limited interprettion of the nture of the muttion in this plnt. Like M1-6, DNA lot nlysis indicted tht ll ckcross progeny hd oth S 3 nd S 6 lleles. Lck of segregtion in the ckcross fmily is indictive of duplicted S llele (see elow). Presumly the duplicted S llele is poorly trnsmitted through pollen unless selection is pplied for the pollen-prt muttion. The S genotype of M1-7 is the sme s tht of M1-6 s judged y S-RNse nd intensities (dt not shown). As M1-7 does not hve centric frgment, the duplicted S 3 llele must hve een trnslocted to nother chromosome. In four M 1 plnts pollen-prt muttions rise through competitive interction of S lleles: According to their S genotypes, M1-1 nd M1-11 cn produce either true hploid pollen contining the S 6 llele or S 6 ds 3 -contining pollen. An S 3 S 6 pistil will reject S 6 pollen. If competitive interction occurs, S 6 ds 3 -contining pollen will e comptile on n S 3 S 6 pistil nd ll the progeny of Figure 5. DNA lot nlysis of M1-5 nd representtive progeny. Genomic DNA ws isolted from the indicted plnt, digested with HindIII, nd frctionted on n grose gel. After trnsfer onto nylon memrne, the lots were proed with 32 P-leled S-RNse cdna proes. (A) M1-5 nd representtives of the phenotypic clsses identified in the S 3 S 6 ckcross fmily (S 3 S 3 SPM/PPM, S 3 S 6 PPM, nd S 6 S 6 PPM plnts). (B) M1-5 nd representtives of the phenotypic clsses identified in the S 2 S 2 outcross fmily (self-incomptile S 2 S 6 plnts, S 2 S 2 SPM/PPM, nd S 2 S 6 PPM plnts). Moleculr weight stndrds (in k) re shown on the left of the figure nd the S-RNse hyridizing nds re indicted to the right of the figure.

10 1132 J. F. Golz et l. TABLE 5 Pollintion, DNA lot, nd cytology dt for progeny derived from two REV plnts S phenotype of progeny Centric Cross Pollen Pistil No. of progeny S-RNse genes frgment c S 2 S 2 M1-8 INC S 2 S 3 13 S 2 S 3 (13) 2 (4) S 2 S 2 M1-17 INC S 2 S 3 10 S 2 S 3 (10) 2 (4) The femle plnt is listed first. The numer of progeny exmined y DNA lot hyridiztion is indicted in prentheses. c The numer of progeny with centric frgment (numer of plnts exmined) is indicted. ckcross or self-pollintion will e heterozygous. This of the ds 3 llele is to complement the lethl muttion. ws found experimentlly. However the S 2 S 6 spm progeny in the outcross fmily of M1-6 nd M1-7 cn produce true hploid pollen con- M1-6 nd M1-7 mkes the presence of lethl muttion tining either S 3 or S6 spm or pollen contining two S lleles, ner the S6 spm llele unlikely. either S 3 ds 3 or S6 spm ds 3. An S 3 S 6 pistil will reject ll hploid Self-incomptiility models nd competitive interction: pollen from M1-6 nd M1-7. If competitive interction As our nlysis of four M 1 plnts led us to conclude occurs, only S6 spm ds 3 pollen will e comptile on n S 3 S 6 tht self-incomptiility reks down in pollen grins pistil nd ll the ckcross progeny will hve oth S 3 - contining ds 3 nd either S 6 or S6 spm, we sought to explin nd S 6 -RNse genes. As this ws oserved, ds 3 must e competitive interction using current models of the moleculr le to interct with S6 spm nd not S 3 to produce PPM sis of self-incomptiility in the Solncee. phenotype. It is formlly possile, s suggested y Pn- The two current models of self-incomptiility re dey (1967), tht the S6 spm llele hs true pollen-prt the receptor model nd the inhiitor model (Figure 6; muttion nd second muttion linked to the S 6 spm llele McClure et l. 1989; Thompson nd Kirch 1992). The tht is lethl in pollen. According to this model, the role receptor model proposes tht the pollen product of the TABLE 6 Summry of the genetics of four PPM plnts crrying duplicted S 3 llele Expected progeny c Oserved progeny: Plnt S phenotype S genotype Cross S phenotype S genotype S phenotype M1-1 S 3 S 6 PPM S 6 S 6 ds 3 S 3 S 6 S 3 S 6 PPM S 3 S 6 ds 3, S 6 S 6 ds 3 S 3 S 6 PPM S 2 S 2 S 2 S 6 S 2 S 6 S 2 S 6 S 2 S 3 S 6 PPM S 2 S 6 ds 3 S 2 S 3 S 6 PPM M1-6 S 3 S 3 SPM/PPM S 3 S spm 6 ds 3 S 3 S 6 S 3 S 3 SPM/PPM S 3 S spm 6 ds 3 S 3 S 3 SPM/PPM S S PPM S S spm ds S 3 S 6 PPM S 2 S 3 S 2 S 3 S 2 S 3 S 2 S 3 S 2 S 2 SPM S 2 S spm 6 S 2 S 2 SPM S 2 S 3 PPM S 2 S 3 ds 3 Not found S 2 S 3 SPM/PPM S 2 S6 spm ds 3 S 2 S 3 SPM/PPM 6 ds 3 S 3 S 3 SPM/PPM S S PPM S S spm ds S 3 S 6 PPM S 2 S 2 S 2 S 3 S 2 S 3 S 2 S 3 S 2 S 2 SPM S 2 S6 spm S 2 S 2 SPM S 2 S 3 PPM S 2 S 3 ds 3 Not found S 2 S 3 SPM/PPM S 2 S6 spm ds 3 Not found 6 ds 3 S 3 S 3 SPM/PPM S S PPM S S ds, S 6 S6 spm ds S 3 S 6 PPM S 2 S 2 S 2 S 6 S 2 S 6 S 2 S 6 S 2 S 2 SPM S 2 S6 spm S 2 S 2 SPM S 2 S 3 SPM/PPM S 2 S spm 6 ds 3 S 2 S 3 SPM/PPM S 2 S 3 S 6 PPM S 2 S 6 ds 3 S 2 S 3 S 6 PPM M1-7 S 3 S 3 SPM/PPM S 3 S spm 6 ds 3 S 3 S 6 S 3 S 3 SPM/PPM S 3 S spm M1-11 S 3 S 6 PPM S 6 S spm 6 ds 3 Self S 3 S 3 SPM/PPM S spm 6 S spm ds 3 denotes n dditionl S 3 llele. In M1-1, M1-6, nd M1-11, ds 3 is on centric frgment. In ech cse the M 1 plnt ws the stminte prent in cross to the indicted pistillte prent. c Expecttions re sed on the competitive interction model (see text).

11 Pollen-Prt Mutnts of N. lt 1133 TABLE 7 Summry of the genetics of three PPM plnts ssuming the pollen-prt of the S 3 llele hs een duplicted Expected progeny c Oserved progeny: Plnt S phenotype S genotype Cross S phenotype S genotype S phenotype M1-2 S 3 S 6 PPM S 3 S 6 ds3 p S 3 S 6 S 3 S 6 PPM S 3 S 6 ds3 p S 3 S 6 PPM S 6 S 6 PPM S 6 S 6 ds3 p S 6 S 6 PPM S 2 S 2 S 2 S 3 S 2 S 3 S 2 S 3 S 2 S 6 S 2 S 6 S 2 S 6 S 2 S 3 PPM S 2 S 3 ds3 p S 2 S 3 S 2 S 6 PPM S 2 S 6 ds3 p S 2 S 6 PPM M1-5 S 6 S 6 PPM S 6 S6 spm ds3 p S 3 S 6 S 3 S 3 SPM/PPM S 3 S6 spm ds3 p S 3 S 3 SPM/PPM S 3 S 6 PPM S 3 S 6 p S 3 S 6 PPM S 6 S 6 PPM S 6 S 6 ds3,s p 6 S6 spm ds3 p S 6 S 6 PPM S 2 S 2 S 2 S 6 S 2 S 6 S 2 S 6 S 2 S 2 SPM S 2 S6 spm Not found S 2 S 2 SPM/PPM S 2 S6 spm ds3 p S 2 S 2 SPM/PPM S 2 S 6 PPM S 2 S 6 ds3 p S 2 S 6 SPM M1-10 S 3 S 6 PPM S 3 S 6 ds3 p S 3 S 6 S 3 S 6 PPM S 3 S 6 ds3 p S 3 S 6 PPM S 6 S 6 PPM S 6 S 6 ds3 p S 6 S 6 PPM S 2 S 2 S 2 S 3 S 2 S 3 S 2 S 3 S 2 S 6 S 2 S 6 Not found S 2 S 3 PPM S 2 S 3 ds3 p Not found S 2 S 6 PPM S 2 S 6 ds3 p S 2 S 6 PPM ds p 3 denotes n dditionl pollen-prt of the S 3 llele. In M1-2, ds p 3 is on centric frgment. In ech cse the M 1 plnt ws the stminte prent in cross to the indicted pistillte prent. c Expecttions re sed on the competitive interction (see text). S locus (pollen-s) is receptor tht llows extrcellulr S-RNses to enter the pollen tue in n llele-specific mnner (Figure 6A). Specific uptke of n ctive rionuclese y the pollen tue cuses n incresed rte of RNA rekdown nd n inility to synthesize protein. This leds to the drmticlly slowed growth rte chrcteristic of incomptile pollen tues (Lush nd Clrke 1997). The ehvior of pollen-prt muttions resulting from duplicted S llele cn e ccommodted y the recep- tor model if it is ssumed tht the self-incomptiility response of pollen is criticlly dependent on the numer of functionl receptors. A pollen tue with two different S lleles will hve fewer functionl receptors thn pollen tue with single S llele if the receptor (pollen-s) is multimer nd only homomeric forms of pollen-s re functionl. However, heteromeric forms of pollen-s will occur only if the monomers encoded y different S lleles ssort rndomly. The second model, the inhiitor model, proposes Figure 6. Two models of events involved in inhiiting the growth of n S 1 pollen tue in n S 1 S 2 style. (A) The receptor model; (B) the inhiitor model. See text for detils.

12 1134 J. F. Golz et l. tht S-RNses enter pollen tues nonspecificlly (Figure dupliction of only prt of the S locus, or y muttion 6B). Once inside, S-RNses encounter pollen-s, which in n llele-specific modifier locus unlinked to the S is n inhiitor tht cn inctivte ny S-RNses except locus. Accordingly, it is possile tht duplicted pollenprt of the S 3 llele (ds3), p ut not the style-prt of the those encoded y mtching S llele. According to this model, the inility of pollen tue to detoxify S 3 llele (the S 3 -RNse gene), cn ccount for the pollenprt muttions in M1-2, M1-5, nd M1-10 (see Tle 7). mtching S-RNses leds to incresed rtes of RNA degrdtion nd consequently slowed growth. To explin In M1-2, ds p 3 is presumly on the centric frgment. In why pollen tue expressing two different S lleles cn M1-5 nd M1-10, ds p 3 my e linked to n S 6 llele. grow through n incomptile pistil, it is necessry to Whether the lesion in M1-2, M1-5, nd M1-10 is cused ssume tht the presence of two types of pollen-s inhiitor cn inctivte ll S-RNses, regrdless of their llelic dupliction of the pollen-prt of the S 3 llele is currently y true muttion in the pollen-prt of the S 6 llele or origin. eing investigted y DNA lot nlysis using moleculr Although either model cn explin competitive inter- mrkers linked to the S locus. ction, they mke different predictions out the mut- We thnk Bruce McGinness for his ssistnce in the glsshouse nd ility of the pollen-s gene. According to the receptor Drs. Peter Chndler nd Jim Pecock from the CSIRO Division of Plnt model, pollen-prt muttions could rise from deletions Industry, Cnerr, Austrli, for help with the irrdition experiment s well s duplictions of the pollen-s gene. A pollen nd ccess to the 60 Co source. We thnk Dr. Mrilyn Anderson of LTroe University, Melourne, Austrli for her dvice t vrious tue lcking the pollen-s gene would e unle to llow stges of this project. J.F.G. ws supported y n Overses Postgrdute S-RNses to enter nd thus would e le to grow Reserch Scholrship from the Austrlin Government. Reserch t through n incomptile style. the Plnt Cell Biology Center is funded y Specil Reserch Center The inhiitor model, on the other hnd, predicts tht grnt from the Austrlin Reserch Council. PPMs cn rise only y dupliction of n S llele s pollen tue lcking the pollen-s gene is unle to detoxify ny S-RNse. This mkes muttions of the pollen-s LITERATURE CITED gene lethl, s pollen tues crrying these muttions Anderson, M. A., G. I. McFdden, R. Berntzky, A. Atkinson, T. re rejected y styles expressing S-RNses. Orpin et l., 1989 Sequence vriility of three lleles of the Using PPM plnts to test the models of self-incompti- self-incomptiility gene of Nicotin lt. Plnt Cell 1: ility: Identifying PPM plnts tht pper to lck duplisequences in tomto. Theor. Appl. Genet. 72: Berntzky, R., nd S. D. Tnksley, 1986 Genetics of ctin-relted cted S llele is one wy to test the two self-incomptiil- Brdford, M. M., 1976 A rpid sensitive method for the quntifiction of microgrm quntities of protein using the principle of ity models. In our study, three plnts fll into this ctegory: M1-2, M1-5, nd M1-10. M1-5 is homozygous protein-dye inding. Anl. Biochem. 72: Brewker, J. L., nd A. T. Ntrjn, 1960 Centric frgments t the S locus, nd the ckcross fmilies of M1-2 nd nd pollen prt muttions of incomptiility lleles in Petuni. M1-10 include oth homozygous nd heterozygous Genetics 45: PPMs. There re no trillelic progeny in the outcross Crluccio, F., D. de Nettncourt nd A. J. G. vn Gstel, 1974 On possile involvement of chromosome 3 in the formtion fmilies of M1-2, M1-5, nd M1-10. We might conclude of self-comptiility muttions in Nicotin lt Link nd Otto, tht these plnts hve true pollen-prt muttions nd pp in Polyploidy nd Induced Muttions in Plnt Breeding. therefore support the receptor model. Certinly the IAEA, Vienn. de Nettncourt, D., 1977 Incomptiility in Angiosperms. Springerpresence of S 3 S 6 nd S 6 S 6 plnts in the ckcross fmily Verlg, Berlin. of M1-2 suggests true pollen-prt muttion in the S 6 Dodds, P. N., I. Bonig, H. Du, J. Rodin, M. A. Anderson et l., 1993 llele. However, there re some uncertinties, in prticu- S-RNse gene of Nicotin lt is expressed in developing pollen. Plnt Cell 5: lr the ssocition etween centric frgment nd the Dodds, P. N., C. Ferguson, A. E. Clrke nd E. Newigin, 1999 PPM phenotype. Pollen-expressed S-RNses re not involved in self-incomptiility M1-5 nd M1-10 hve no dditionl chromosome or in Lycopersicon peruvinum. Sex. Plnt Reprod. (in press). Hrlow, E., nd D. Lne, 1988 Antiodies: A Lortory Mnul. other evidence of dupliction. The inheritnce of the Cold Spring Hror Lortory Press, Cold Spring Hror, NY. pollen-prt muttion cn e explined y ssuming the Hermsen, J. G. T., 1978 Genetics of self-comptiility in dihploids muttion in these plnts is linked to n S of Solnum tuerosum L. 2. Detection nd identifiction of ll 6 llele. It is possile incomptiility nd comptiility genotypes in six F 1 s possile M1-5 nd M1-10 crry true muttions in the from interdihploid crosses. Euphytic 27: pollen-prt of the S 6 llele. Lemmli, U. K., 1970 Clevge of structurl proteins during the To dte, the only ttempt to understnd PPMs t ssemly of the hed of cteriophge T4. Nture 227: Lee, H. S., S. Hung nd T. H. Ko, 1994 S proteins control rejection moleculr level hs een y Thompson et l. (1991). of incomptile pollen in Petuni inflt. Nture 367: Working with self-comptile dihploid lines of S. tuerogrowth in Nicotin lt nd their implictions for the mech- Lush, W. M., nd A. E. Clrke, 1997 Oservtions of pollen tue sum, ering wht ppered to e trnsloction of n nism of self-incomptiility. Sex. Plnt Reprod. 10: S llele, Thompson et l. (1991) used S-RNse gene McClure, B. A., V. Hring, P. R. Eert, M. A. Anderson, R. J. proes nd gels of stylr proteins to serch for evidence Simpson et l., 1989 Style self-incomptiility gene products of of duplicted S locus, ut found none. On the sis of Nicotin lt re rionucleses. Nture 342: Olsder, J., nd J. G. T. Hermsen, 1976 Genetics of self-comptiility this lck of evidence, Thompson et l. (1991) concluded in dihploids of Solnum tuerosum L. 1. Breeding ehvior of tht the pollen-prt muttion ws cused either y the two self-comptile dihploids. Euphytic 25:

13 Pollen-Prt Mutnts of N. lt 1135 Pndey, K. K., 1965 Centric chromosomes frgments nd pollen- Thompson, R. D., H. Uhrig, J. G. T. Hermsen, F. Slmini nd H. prt muttions of the incomptiility gene in Nicotin lt. Kufmnn, 1991 Investigtion of self-comptile muttion Nture 206: in Solnum tuerosum clones inhiiting S-llele ctivity in pollen Pndey, K. K., 1967 Elements of the S-gene complex. II. Muttions differentilly. Mol. Gen. Genet. 226: nd complementtion t the S I locus in Nicotin lt. Heredity vn Gstel, A. J. G., nd D. de Nettncourt, 1975 The effects 22: of different mutgens on self-incomptiility in Nicotin lt Smrook, J., E. F. Fritsch nd T. Mnitis, 1989 Moleculr Clon- Link nd Otto II. Acute irrdition with X-rys nd fst neutrons. ing: A Lortory Mnul. Cold Spring Hror Lortory Press, Heredity 34: Cold Spring Hror, NY. Thompson, R. D., nd H. H. Kirch, 1992 The S locus of flowering Communicting editor: J. Chory plnts: when self-rejection is self-interest. Trends Genet. 8: