The Plant Cell, Vol. 10, 1163–1180, July 1998, www.plantcell.org © 1998 American Society of Plant Physiologists

Stoichiometric Shifts in the Common Bean Mitochondrial Genome Leading to Male Sterility and SpontaneousReversion to Fertility

Hanna Janska,

a,1

Rodrigo Sarria,

b,1

Magdalena Woloszynska,

a

Maria Arrieta-Montiel,

b

and Sally A. Mackenzie

b,2

a

Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka, 2, 50-137 Wroclaw, Poland

b

Department of Agronomy, Lilly Hall, Purdue University, West Lafayette, Indiana 47907

The plant mitochondrial genome is characterized by a complex, multipartite structure. In cytoplasmic male-sterile(CMS) common bean, the sterility-inducing mitochondrial configuration maps as three autonomous DNA molecules,one containing the sterility-associated sequence

pvs-orf239.

We constructed a physical map of the mitochondrial ge-nome from the direct progenitors to the CMS cytoplasm and have shown that it maps as a single, circular masterconfiguration. With long-exposure autoradiography of DNA gel blots and polymerase chain reaction analysis, we dem-onstrate that the three-molecule CMS-associated configuration was present at unusually low copy number within theprogenitor genome and that the progenitor form was present substoichiometrically within the genome of the CMS line.Furthermore, upon spontaneous reversion to fertility, the progenitor genomic configuration as well as the moleculecontaining the

pvs-orf239

sterility-associated sequence were both maintained at substoichiometric levels within the re-vertant genome. In vitro mitochondrial incubation results demonstrated that the genomic shift of the

pvs-orf239

–containing molecule to substoichiometric levels upon spontaneous reversion was a reversible phenomenon. Moreover,we demonstrate that substoichiometric forms, apparently silent with regard to gene expression, are transcriptionallyand translationally active once amplified. Thus, copy number suppression may serve as an effective means of regulat-ing gene expression in plant mitochondria.

INTRODUCTION

The organizational features of the plant mitochondrial ge-nome have been the subject of numerous investigationsduring the past several years. Unlike most other well-investi-gated eukaryotic systems, the plant kingdom has demon-strated striking variability in mitochondrial genome size andstructure (reviewed in Wolstenholme and Fauron, 1995). Al-though it is unclear whether all plant species contain a mito-chondrial genome maintained in both linear and circularconformations, this appears to be the case for several spe-cies (Bendich, 1993; Oldenburg and Bendich, 1996). More-over, a high degree of recombinational activity at repeatedsequences interspersed throughout the genome produces apopulation of molecules that contain only a portion of themitochondrial genome; however, taken together, they giverise to a high degree of coding redundancy. These arereferred to as subgenomic DNA molecules, and mappingevidence suggests that although able to undergo intermo-lecular and intramolecular recombination, at least some

molecules may be replicated autonomously (Folkerts andHanson, 1991; Yamato et al., 1992; Janska and Mackenzie,1993).

The origin and mode of DNA replication in the plant mito-chondrial genome have not yet been defined. Recent evi-dence suggests that a rolling-circle model might account forobservations made using electron microscopy, pulsed-fieldgel electrophoresis, and various hybridization procedures(Backert et al., 1997). What is strikingly unusual about mostplant systems, however, is the observation that subgenomicDNA molecules can be maintained at dramatically differentrelative copy numbers within the genome. In fact, it is possi-ble to observe some genomic configurations only by usingpolymerase chain reaction (PCR) amplification or unusuallylong DNA gel blot exposure times (Small et al., 1987). Theselow-copy-number configurations, referred to as sublimons,have been suggested to account for one mechanism bywhich genetic variability is accumulated within the mito-chondrion (Small et al., 1989). In this study, we suggest thatthe suppression of copy number provides an effective meansof repressing dominant mutations within the mitochondrialgenome. Furthermore, we provide evidence to suggest that

1

The first two authors contributed equally to this work.

2

To whom correspondence should be addressed. E-mail smackenz@purdue.edu; fax 765-494-6508.

1164 The Plant Cell

this suppression of copy number is a function of nucleargenotype. This function can be relaxed by the incubation ofpurified mitochondria in vitro.

Cytoplasmic male sterility (CMS) in common bean hasbeen associated with the expression of a mitochondrial se-quence encoded within a 2.4-kb mitochondrial region desig-nated

pvs

(for

Phaseolus vulgaris

sterility sequence). Thisregion contains at least two unique open reading frames(ORFs) designated

pvs-orf98

, encoding a 98–amino acidORF, and

pvs-orf239

, encoding a 239–amino acid ORF. Wehave detected translation of only

pvs-orf239

, which pro-duces a 27-kD polypeptide that accumulates in reproductivetissues (Abad et al., 1995) but is rapidly degraded in vegeta-tive tissues (Sarria et al., 1998). Evidence suggests that theORF239 product may induce the sterility phenotype (Abadet al., 1995; He et al., 1996). Mapping of the CMS commonbean mitochondrial genome by overlapping cosmid cloneanalysis indicates that the genome is most likely organized asthree interrecombining and highly redundant mitochondrialmolecules of 394, 257, and 210 kb (Janska and Mackenzie,1993). The

pvs

mutation resides on the 210-kb form; thismolecule disappears from the genome upon spontaneouscytoplasmic reversion to fertility (Mackenzie et al., 1988;Janska and Mackenzie, 1993) or the introduction of a nucleargene designated

Fr

(Mackenzie and Chase, 1990). In thisstudy, we identified the origin of the 210-kb molecular con-figuration within the progenitors of the CMS source and in-vestigated the nature of CMS induction and its suppressionupon spontaneous reversion.

RESULTS

Confirmation of Progenitors to the CMSCommon Bean Line

The fertile progenitor of the CMS common bean line CMS-Sprite is designated accession number G08063 (Mackenzie,1991; Centro Internacional de Agricultura Tropical, Cali, Co-lombia); its mitochondrial genomic configuration is indistin-guishable from that of the CMS-Sprite line used in our studies,and it has been confirmed to contain a sterility-inducing cy-toplasm. Accession line G08063 reportedly originated with thecross POP

3

NEP-2 (maternal parent not defined; Mackenzie,1991; pedigree data obtained from Centro Internacional deAgricultura Tropical). To investigate the origin of the sterility-inducing mitochondrial configuration, it was necessary to con-firm the pedigree giving rise to the CMS source. Therefore,random amplified polymorphic DNA (RAPD)–based finger-printing was used to assess genetic distances separatingNEP-2, its progenitor San Fernando, POP, its progenitorsOpal, and the derived sister lines G08063, G08064, andG08065, using line CMS-Sprite as an outlier. In Figure 1, wepresent a phenogram based on the analysis of 68 scoredbands derived from eight primer reactions. Our results are inclear agreement with the stated pedigree of line G08063.This conclusion is based on the high degree of relatednessamong progeny, all intermediate between parental types, rel-ative to the average genetic distances observed within com-

Figure 1. Estimation of Genetic Distances between Putative Progenitors That Gave Rise to Sister Lines G08063, G08064, and G08065.

The bean phenogram demonstrates the relationships among lines based on a compiled data set derived from RAPD data. POP and NEP-2 arepostulated to be the parental lines giving rise to G08063 to G08065. San Fernando is one of the parents to NEP-2; Opal is one of the parents toPOP. CMS-Sprite contains the G08063 cytoplasm in combination with the snapbean cultivar Sprite nuclear genotype that is not expected to beclosely related to any of the selected materials; CMS-Sprite was included to represent average distance between unrelated bean lines.

Shifts in the Bean Mitochondrial Genome 1165

mon bean germplasm (Tohme et al., 1996). Therefore, linesPOP and NEP-2 were utilized for subsequent constructionof a physical map of the progenitor mitochondrial genome.

Construction of a Physical Map of the Mitochondrial Genome of Progenitor Lines POP and NEP-2

To rapidly identify the differences in mitochondrial genomeorganization between our CMS line, designated CMS-Sprite(contains a Sprite nuclear genotype with the G08063 cyto-plasm), and NEP-2 and POP (G08063 progenitors), we per-formed comparative hybridizations. Total genomic DNApreparations were digested with PstI, SalI, XhoI, and EcoRI.Nineteen overlapping mitochondrial cosmid clones, span-ning the entire mitochondrial genome of CMS-Sprite, wereused as probes against blots prepared with genomic DNAfrom the male-sterile line and both progenitor lines. The ma-jority of clones revealed identical hybridization patterns forall lines, regardless of the restriction enzyme used. From thisobservation, we concluded that long stretches of the investi-gated genomes have a similar configuration. Figure 2 showsthat comparative hybridizations helped to identify four re-gions, designated A, B, C, and D, in the CMS-Sprite mito-chondrial genome. These regions were rearranged relativeto the genomes of NEP-2 and POP. The exact left and rightboundaries of these rearrangements have not yet beendefined; in Figure 2, they are presented simply as PstI frag-ments for convenience. Only EcoRI digestion allowed dis-crimination between the mitochondrial genomes of NEP-2and POP. With this enzyme, two restriction fragment lengthpolymorphisms were detected; they are outside of regions Ato D. Our preliminary investigations suggest that they resultfrom point mutations rather than sequence rearrangements(data not shown).

Once the regions distinguishing the progenitor and steril-ity-inducing mitochondrial genomes were defined, it waspossible to identify novel junction fragments within the pro-genitor genomes. Table 1 summarizes the results of com-parative hybridization experiments in regions A to D. Note thatno hybridization signals were detected on normally exposedautoradiograms for two PstI fragments: a 4.5-kb fragmentfrom region A and a 4-kb fragment from region D. Also, fourremaining PstI fragments used as probes hybridized with onlytwo fragments in the NEP-2 and POP samples. A 7.2-kbfragment of region A and a 10.2-kb fragment from region Chybridized with one 12-kb fragment in the progenitors. Like-wise, a 6.3-kb fragment in region A and a 4.8-kb fragment fromregion B hybridized with one 7-kb fragment in the progenitors.From these observations, we postulated that the 12- and 7-kbPstI fragments in the progenitor genomes represent distinctjunctions in the mitochondrial genomes of NEP-2 and POP.

To test this hypothesis, we used long-range PCR condi-tions incorporating primers localized to each side of the pu-tative junctions. In the case of the 12-kb fragment, thesequence of one primer was based on the cytochrome

b

(

cob

) gene sequence located within the cross-hybridizingCMS-Sprite 7.2-kb fragment. The second primer was de-rived from the gene encoding the

a

subunit of ATPase (

atpA

)known to be present on the cross-hybridizing CMS-Sprite10.2-kb fragment. The positions of the primers are shown inFigure 3. Based on primer locations, we expected to obtaina PCR amplification product of

z

10 kb. The results are illus-trated in Figure 4A. As predicted, a 10-kb fragment was am-plified only when DNAs isolated from NEP-2 and POP wereused as templates. In accordance with our hypothesis, theamplification was unsuccessful for CMS-Sprite becausecopies of the

cob

and

atpA

genes in this genome are sepa-rated by

z

200 kb. However, the particular long-range PCRconditions used for this experiment were not sufficientlysensitive to amplify substoichiometric forms. Only thoseforms present in the predominant genome could be ampli-fied (data not shown).

A similar PCR-mediated approach was used to confirmthe presence of a second junction specific to the NEP-2 andPOP mitochondrial genomes. We designed two primers thatallowed amplification of the 7-kb PstI fragment together witha short flanking sequence. One primer was derived from thecross-hybridizing CMS-Sprite 4.8-kb PstI fragment and theother from the 18S/5S rRNA (

rrn18

/

rrn5

) gene sequenceknown to reside close to the cross-hybridizing 6.3-kb CMS-Sprite PstI fragment. The results of the PCR amplificationsagain were consistent with those from DNA gel blot analysis(Figure 4B). The predicted fragment was amplified only inthe progenitor lines but not in samples from CMS-Sprite.These results indicate that in the progenitor genomes ofNEP-2 and POP, the left flanking sequence to region A isconnected by the 12-kb junction to the right flanking se-quence of region C. Likewise, the right flanking sequence ofregion B is linked by the 7-kb junction to the right flankingsequence of region A (see Figure 2).

Two families of recombinationally active repeats, R1 andR2, were identified previously in the mitochondrial genomeof the male-sterile line CMS-Sprite (Janska and Mackenzie,1993). Figure 5A presents the schematic diagram of thebranch points around these two repeats. Both repeats havethree flanking sequences designated a, b, and c at one endof the repeat and two designated d and e at the other.Flanking sequences b and c share a segment of homologyforming an additional branch point. As is shown in Figure 2,the genomic regions designated B and C are located nearbyto copies of repeats R2 and R1, respectively. Thus, we as-sumed that at least two genomic environments surroundingrepeats R1 and R2 were rearranged in the mitochondrial ge-nomes of NEP-2 and POP relative to the sterile line.

To examine environments surrounding the R1 and R2 re-peats in the progenitors, we hybridized probes homologousto the R1 and R2 repeats, with DNA preparations from CMS-Sprite, NEP-2, and POP digested with four different restrictionenzymes. No novel genomic environments were detected inthe progenitors, but the loss of b–d and b–e environmentsaround R1 could be observed, as demonstrated in Figure

1166 The Plant Cell

5C. Hybridization with the repeat R2 resulted in the detec-tion of like-sized fragments in both male-sterile and fertileprogenitor lines, but the relative intensity of bands differed;the observed changes in intensity are consistent with theloss of R2-surrounding environments b–d and b–e (Figure5D). We were forced to base this assumption on band inten-sity because a restriction enzyme distinguishing these con-

figurations was not identified. The lack of two genomicenvironments surrounding each repeat (b–d and b–e for R1and b–d and b–e for R2) in the progenitor genomes impliesthat R1 and R2 repeats are present in only four configura-tions (Figure 5B) rather than the six identified in CMS-Sprite.

Results of comparative hybridizations, identification ofdistinctive junctions, and assessment of genomic environ-

Figure 2. A Physical Map of the CMS-Sprite Mitochondrial Genome Indicating Regions Rearranged Relative to Progenitors NEP-2 and POP.

The CMS-Sprite map previously was constructed with restriction enzymes PstI (P), SalI (S), and XhoI (X) (Janska and Mackenzie, 1993). The re-gions rearranged relative to POP and NEP-2 are indicated (A to D) and were localized based on comparative hybridizations. The small boxesabove the map indicate the locations of recombination repeats R1 and R2, with arrows indicating their orientation. The vertical dashed lines des-ignate the junction points between the genome and the pvs sequence. The region encompassing this junction is shown at the bottom. Genomicenvironments a–e surrounding repeats are indicated.

Shifts in the Bean Mitochondrial Genome 1167

ments surrounding the recombinational repeats within theprogenitor genomes were used to construct a physical mapof the mitochondrial genome of lines NEP-2 and POP. Thismap is presented in Figure 3. We present only one map be-cause as previously mentioned, no differences were detectedbetween the NEP-2 and POP genomes in hybridization pat-terns with enzymes PstI, SalI, and XhoI. The approximate po-sitions of the two aforementioned polymorphisms revealed byEcoRI digestion are indicated on the map by asterisks. Thetwo families of repeats, R1 and R2, are indistinguishable be-tween progenitor and male-sterile lines, although their sur-rounding environments are simplified in the progenitors.Based on the linear representations of the physical maps,the mitochondrial genomes of the male-sterile line and itsprogenitors are 420 and 393 kb, respectively. This differenceresults from the reduced copy number of the recombinationrepeats and the absence of particular segments from re-gions A to D within the progenitor genomes.

Unlike the construction of the CMS-Sprite mitochondrialmap, the ends of a linear representation of the physical mapof NEP-2 and POP could be directly connected to produce asingle master circular form; this is because the same R1 re-peated sequence is located at both map ends. Thus, the en-tire configuration of the fertile progenitor mitochondrialgenome is contained in a circular map of 393 kb, as dia-grammed in Figure 6. In contrast, a model involving a singlemaster chromosome in the CMS-Sprite genome would re-quire the triplication of a segment of

.

200 kb. Thus, the cor-responding model for the structure of the CMS-Spritegenome consists of three autonomous chromosomes (394,257, and 210 kb) differing only in short, unique regions. Figure6 presents the two proposed models, with the correspondingregions being designated. The genomic environments flank-ing the repeats R1 and R2 were chosen arbitrarily. To date,we have identified no sequences in the POP or NEP-2 ge-nomes that were not also present in the CMS-Sprite genome;however, sequences unique to the CMS-Sprite genome are

indicated. The sequence of particular interest to our study isthe sterility-associated sequence designated

pvs.

The G08063 Sterility-Inducing Cytoplasm Arose in Part as a Consequence of a Mitochondrial Genomic Shift

With the identification of regions A to D unique to the sterility-inducing cytoplasm, it was possible to determine whetherthe G08063 genomic configuration was present at substo-ichiometric levels within the progenitor lines. To test thispossibility, gel-blotted POP and NEP-2 total DNA prepara-tions were hybridized with clones containing regions A to D,and autoradiograms were developed after extended expo-sure times. As shown in Figures 7A to 7D, low- to medium-intensity bands of expected sizes reflecting all four regionscould be detected in POP, but only region B was detectablein line NEP-2 (Figure 7B). In Figure 7D, a

pvs

probe wasused to investigate the presence of the sterility-associatedregion within progenitor lines. The lower band observed inCMS-Sprite and POP represents an EcoRI fragment of 3.1kb encompassing the

pvs

sequence. These results stronglysuggest that mitochondrial DNA regions specific to the ste-rility-inducing cytoplasm preexist in the progenitor POP mi-tochondrial genome at substoichiometric levels. This resultalso implies that POP was likely the maternal parent in thecross that gives rise to G08063. We are investigating thispossibility further.

A similar approach was used to determine whether the se-quences distinguishing the progenitor POP genome aremaintained at substoichiometric levels within the G08063cytoplasm. In Figures 8A and 8B, autoradiography was usedto demonstrate that the configurations characteristic of thefertile progenitor genome, represented as 7- and 12-kb PstIfragments described earlier, are present in CMS-Sprite. Asexpected, cross-hybridization of the probes to other previ-ously defined regions was also observed. Thus, we con-clude from these observations that the transition fromprogenitor POP mitochondrial configuration to that of thesterility-inducing configuration involved the amplification ofthe G08063 tripartite form concomitant with the suppressionof the POP form.

Evidence That Spontaneous Reversion to FertilityOccurs as a Consequence of a DistinctMitochondrial Genomic Shift

One unusual feature of the G08063-derived mitochondrialconfiguration is its propensity to undergo spontaneous cyto-plasmic reversion to fertility. These reversion events, influ-enced in frequency by nuclear genotype (Mackenzie et al.,1988), are associated with the disappearance of the

pvs

-containing mitochondrial molecule from the genome (Janskaand Mackenzie, 1993). We examined the possibility that thereversion phenomenon also could be due to mitochondrial

Table 1.

Sizes of NEP-2 and POP Mitochondrial DNAFragments Hybridizing to Regions A to D of the CMS-Sprite Mitochondrial Genome

RegionCMS-Sprite PstI FragmentsUsed as a Probe (kb)

NEP-2/POP PstI Cross-Hybridizing Fragments (kb)

A 7.2 12.04.5

2

a

6.3 7.0B 4.8 7.0C 10.2 12.0D 4.0 (

pvs

)

2

a

The (

2

) indicates that no hybridization signal was detected. Hybrid-izing fragments that were common to both progenitor and CMS-Sprite genomes are not included in this table.

1168 The Plant Cell

genome shifts. To study this reversion event, we used long-distance PCR amplification, using as primers a sequence lo-cated at the 3

9

end of the

atpA

gene (residing 5

9

to

pvs

) anda sequence derived from the 3

9

end of the

pvs-orf239

gene.These primers produce a fragment of 4437 bp unique to the

pvs

configuration. PCR-amplified fragments from revertantline WPR-3 were almost invisible when stained with ethid-ium bromide after gel electrophoresis (data not shown), buttheir identity and size were confirmed by subsequent DNAgel blot hybridization, as demonstrated in Figure 9A. Theseresults indicate that the

pvs

sequence is not lost from the

mitochondrial genome of the CMS line upon reversion but ismaintained substoichiometrically.

These observations led to the prediction that the POP pro-genitor genome configuration also should be present substo-ichiometrically in the WPR-3 revertant line. Figure 9B showsthe result of DNA gel blot hybridization to a mitochondrialDNA preparation of revertant WPR-3, using as a probe thePCR-amplified 7-kb PstI fragment specific to the POP ge-nome. Although we were able to observe evidence of thepresence of the progenitor configuration within WPR-3, ourresults suggest that the progenitor form was present in lower

Figure 3. A Physical Map of the Mitochondrial Genome of Progenitor Lines NEP-2 and POP.

The map was constructed with restriction enzymes PstI (P), SalI (S), and XhoI (X) and is colinear relative to the CMS-Sprite mitochondrial map forall regions indicated, except for two prominent junction points designated by boldface rectangles. Short double arrows indicate the positions ofprimers used to identify and PCR amplify the junctions. Boxes designate R1 and R2 repeats, with arrows indicating relative orientation. The ap-proximate positions of two polymorphisms that distinguish POP and NEP genomes, revealed by EcoRI digestion, are indicated by asterisks.

Shifts in the Bean Mitochondrial Genome 1169

amounts within the revertant genome than in the male-ster-ile genome, although it was not feasible to quantify this dif-ference accurately.

The Genomic Shift Associated with Reversion MayArise from Active Copy Number Suppression of the

pvs

-Containing Mitochondrial Molecule

Because the

pvs

-containing mitochondrial DNA moleculeexpresses a novel sequence, namely,

pvs-orf239

, it is possi-ble to monitor its presence not only by DNA analysis but byexpressional analysis as well. During this investigation, weposed the hypothesis that nuclear-directed reduction in rel-ative copy number of a mitochondrial molecule should bereversible and might then provide one means of suppressinggene expression. To examine this possibility further, we pu-rified mitochondria from lines G08063, CMS-Sprite, and twoindependently derived revertants, WPR-3 and 83-1, by frac-

tionation over Percoll gradients. An aliquot of these mito-chondria was lysed and tested by PCR and reversetranscription (RT)–PCR amplification for the presence of the

pvs-orf239

sequence and its transcript. ELISA and immuno-precipitation with anti-ORF239 polyclonal antibodies wereused to test for the presence of the ORF239 protein. The re-maining intact, purified mitochondria were incubated in thepresence of

35

S-cysteine and an energy source to allow forin organello translations. Previous studies have indicatedthat the

orf239

gene product is highly susceptible to pro-teolysis (Sarria et al., 1998), so mitochondrial incubationswere in the presence of the protease inhibitor vanadate toallow for the accumulation of ORF239 protein. Subsequentto 90-min incubations, mitochondrial preparations were ly-sed and tested for the presence of the

pvs-orf239

sequenceby using PCR amplification. RT-PCR was used to test forpresence of its transcript. Additional aliquots were used formitochondrial RNA preparations as well as for the immuno-precipitation of ORF239 protein. The 83-1 revertant samplewas not included for all treatments.

Figure 4. PCR Amplification Results Demonstrating Two DistinctJunctions Present within the Progenitor Mitochondrial Genomes ofNEP-2 and POP.

(A) Primers P2 and P8757 (locations designated in Figure 3) ampli-fied a 10-kb fragment when mitochondrial DNA templates fromNEP-2 and POP were used. Under the same conditions, no amplifi-cation product was detected with CMS-Sprite. A PstI-digested l

size standard (MW) is included for size estimation as well as the re-sults of a reaction run without added template DNA (Control).(B) Primers P18/5r and P4.8 (locations designated in Figure 3) am-plified a 7-kb fragment when samples of mitochondrial DNA fromNEP-2 and POP were used as templates. When CMS-Sprite mito-chondrial DNA was used as a template, nonspecific products weredetected; these products did not hybridize with the amplified 7-kbfragment used as probe in DNA gel blot experiments (data notshown). A size marker of HindIII-digested l is included (MW) as wellas a control reaction minus template (Control).Numbers at right indicate fragment lengths in kilobases.

Figure 5. Genomic Environments Surrounding RecombinationallyActive Repeats R1 and R2 in the CMS-Sprite (G08063 Cytoplasm)Genome versus Progenitor Lines POP and NEP-2.

(A) and (B) Schematic diagrams show the branch points surround-ing repeats R1 and R2 within the mitochondrial genomes of theCMS-Sprite (A) and progenitor (B) lines. Lowercase letters indicatedifferent flanking sequences.(C) DNA gel blot hybridization with blotted mitochondrial DNA di-gested with EcoRI and probed with repeat R1. Lowercase letters in-dicate the respective combinations of the flanking sequences.(D) DNA preparations were digested with SalI and probed with re-peat R2.

1170 The Plant Cell

From these experiments, we obtained the results that areillustrated in Figures 10 and 11. PCR amplification experi-ments demonstrated a marked increase in the concentrationof the

pvs

-

orf239

sequence after 90-min incubations in alllines, suggesting that DNA replication occurs in vitro underthe conditions used in our experiment. In the case of rever-tant WPR-3, the PCR product was undetectable at time zerowith both ethidium bromide staining of the agarose gel afterelectrophoresis and DNA gel blot analysis. After 90-min in-cubations, the

pvs-orf239

sequence was difficult to detectin WPR-3 samples by using ethidium bromide staining ofagarose gels, but it was readily detected by subsequentDNA gel blot hybridization, as indicated in Figures 10A to10F. These results suggest that the

pvs-orf239

sequence isnot only present in substoichiometric amounts within theWPR-3 line, as demonstrated, but that it is replicatively ac-tive. The difficulty encountered in detecting the sequencewas presumably due to the relatively low concentration ofmitochondria in our incubation sample as well as the lack ofadded exogenous nucleotides to our reaction, but the in-crease over time was dramatic and highly reproduciblenonetheless. Of additional interest was our observation withprimers specific for the

cob

gene. In the revertant samples,amplification with these primers also demonstrated an in-crease in intensity after incubation; however, incubation in

the G08063 and CMS-Sprite samples demonstrated a de-tectable and reproducible decrease in amplification of the

cob

sequence. These results imply that incubation condi-tions led not only to amplification of some genomic regionsbut also to a possible decline in copy number of others.

To confirm that these results reflect de novo DNA synthe-sis, we performed similar incubations in the presence of alabeled nucleotide with and without the addition of ddT TPas a mitochondrial DNA replication inhibitor. Figures 10Gand 10H demonstrate good incorporation of label into mito-chondrial DNA fractions, markedly lowered with the additionof ddTTP to the incubation. As expected, the addition of thenuclear DNA replication inhibitor aphidicolin showed no ef-fect. Our analysis of counts-per-minute values indicated thatafter the 90-min in organello incubation, 600 cpm was incor-porated into 200 ng of mitochondrial DNA in the treatmentswith no inhibitor or added aphidicolin, and 250 cpm was in-corporated into the ddTTP treatment.

Mitochondrial RNA preparations derived from purifiedCMS-Sprite and WPR-3 were used for RT-PCR, in vitrotranslation, and immunoprecipitation experiments to test forthe presence and translational competence of the

pvs-orf239

transcript in the revertant line after a 90-min incu-bation. Figure 11A shows the results of these experiments.RT-PCR amplification experiments, using primers specific for

Figure 6. Comparison of the Mitochondrial Genome Organization of CMS-Sprite (G08063 Cytoplasm) and Its Fertile Progenitors NEP-2and POP.

Regions showing the same restriction pattern are indicated by the same color. Sequences of the CMS-Sprite genome rearranged relative to thegenomes of NEP-2 and POP are designated in red, with the letters A to D corresponding to Figure 2. Two distinctive junctions exist within thePOP and NEP-2 genomes and are shown as rectangles marked J-7 and J-12. The positions of recombinationally active repeats are representedby boxes with arrows to indicate relative orientations. The filled box shows the location of the pvs sequence.

Shifts in the Bean Mitochondrial Genome 1171

the

pvs-orf239

transcript, demonstrated that the transcriptwas undetectable in mitochondrial RNA preparations fromthe revertant before incubation but readily detectable afterincubation. Although, admittedly, these experiments were notquantitative, they allow us to conclude that a marked increasein concentration of the

pvs-orf239

transcript occurred overthe 90-min incubation period. Similar in organello transcrip-tion activity in the absence of added exogenous ribonu-cleotides was reported previously in rat mitochondria byEnriquez et al. (1993). Immunoprecipitation of the in vitrotranslation product with anti-ORF239 antibodies confirmedthat the

pvs-orf239

transcript was present and translation-ally competent in both the CMS-Sprite and revertant WPR-3samples after incubation, although significantly less productwas obtained from the WPR-3 preparation.

These results suggest that the

pvs-orf239

sequence istranscriptionally active within the revertant once DNA copynumber is increased. This is important, because no

pvs-orf239

transcript is detectable in mitochondria purified fromwhole plant tissues of a revertant line (Chase, 1994), sug-gesting that the substoichiometric form of

pvs-orf239

ispresent in sufficiently low copy numbers to render it effec-tively silent. It is not possible, based on these experiments,to state that the

pvs-orf239

sequence is fully silent in the re-vertant, because it is possible that transcripts are producedat levels below detection.

In organello translation experiments using mitochondriapurified from CMS-Sprite and revertant WPR-3 likewisedemonstrated an accumulation of the ORF239 protein afterincubation (Figure 11B). Again, lower levels were detected in

Figure 7. Overexposed Autoradiograms Reveal G08063-Specific Mitochondrial Sequences at Substoichiometric Levels in the Progenitors POPand NEP-2.

Total genomic DNAs prepared from CMS-Sprite, NEP-2, and POP were blotted and hybridized with probes representing regions A to D from theCMS-Sprite mitochondrial genome (see Figure 2). For (A) to (D), the CMS-Sprite sample was underloaded intentionally by approximately one-fourth relative to the progenitor samples, and numbers denote molecular weights of fragments indicated by arrowheads.(A) DNA samples were digested with EcoRI and hybridized with a cosmid clone that overlaps region A and its right flanking sequence. Arrow-heads indicate the EcoRI fragments derived from the region.(B) Samples were digested with PstI and hybridized with a cloned 4.8-kb PstI fragment that itself represents region B. Other bands evident areknown to correspond to homologous regions within the genomes.(C) Samples were digested with EcoRI and hybridized with a cosmid clone that overlaps region C and its flanking sequences. The 2.8-kb frag-ment represents the junction between region C and pvs.(D) Samples were digested with EcoRI and hybridized with a PCR-amplified pvs sequence; the band indicated by an arrowhead represents themitochondrial fragment encompassing pvs. The upper, more intensely hybridizing band represents a chloroplast fragment that hybridizes by vir-tue of a small region of chloroplast tRNAAla intron homology contained within the pvs region (Chase and Ortega, 1992; Johns et al., 1992). All mi-tochondrial DNA fragments present in the CMS-Sprite genome were detected in the genome of fertile progenitor POP.In (A) to (D), numbers at right indicate fragment lengths in kilobases.

1172 The Plant Cell

the WPR-3 line, but the product is clearly visible. This obser-vation suggests that expression of the

pvs-orf239

sequencecan be detected upon amplification of the

pvs-orf239

se-quence, even if it is undetectable before incubation (notshown) and in mitochondria prepared from whole plant tis-sues (Abad et al., 1995). Taken together, these results sup-port our hypothesis that mitochondrial genome shifts arereversible events and that molecules maintained at substoi-chiometric levels are suppressed in their expression buttranscriptionally and translationally competent once ampli-fied to above-threshold levels.

DISCUSSION

Based on the observations presented here, we propose thefollowing model to account for the mitochondrial genomechanges observed between progenitor line POP, derived ac-cession line G08063, and the fertile revertants WPR-3 and83-1. Our model is outlined schematically in Figure 12. Wepropose that the present-day mitochondrial genome of pro-genitor line POP contains not only the predominant form,represented as a single master chromosome, but also thesterility-associated tripartite configuration characteristic ofderived line G08063 at substoichiometric levels. Generation

of the G08063-associated configuration then would involvethe amplification of the tripartite form and the suppression ofthe POP progenitor form to substoichiometric levels. Uponspontaneous reversion, a second suppression event is pos-tulated, now involving only the

pvs

-containing mitochondrialmolecule to substoichiometric concentrations.

Only with the availability of progenitors to accession lineG08063 has investigation into the origin of the CMS-associ-ated mitochondrial configuration become feasible. Althoughconstruction of a physical map of the progenitor genome inlines POP and NEP-2 produced a model predicting a singlecircular master chromosome, the greatest proportion of thegenome is similar to that found in the male-sterile line. Directcomparison of the progenitor and G08063 tripartite mitochon-drial configurations does not allow us to deduce the series ofevents that mediated the formation of one from another overevolutionary time; most likely, the two forms have evolvedindependently for some period. From mapping data, wepostulate that several sequential events have occurred inthe derivation of the tripartite form, with one important eventinvolving the creation of the

pvs

region. This would be con-sistent with the means of generating mitochondrial genome

Figure 8. Overexposed Autoradiograms Demonstrating the Pres-ence of Progenitor (NEP-2 and POP)-Specific DNA Fragments inCMS-Sprite Mitochondrial DNA Preparations.

Mitochondrial DNA preparations were digested with PstI and hybrid-ized with probes derived from amplified junction fragments uniqueto the NEP-2 and POP genomes. Probes were PCR amplified fromthe POP mitochondrial genome. Other bands correspond to homol-ogous regions identified in the genomes.(A) Probed with the 7-kb junction fragment.(B) Probed with the 12-kb junction fragment.

Figure 9. Evidence for the Presence of the pvs Sequence and thePOP and NEP-2 Progenitor Genome Forms within Revertant LineWPR-3.

(A) PCR amplification of the pvs region (4.4 kb) from the mitochon-drial genome of revertant line WPR-3 by using long-distance PCRamplification with primers ATP-A5 and orf239-39 (see Methods). ThePCR product from revertant samples is barely visible by ethidiumbromide staining of an agarose gel (data not shown). The identity ofthe PCR product is confirmed by DNA gel blot hybridization, using acloned orf239 sequence as a probe.(B) Overexposed autoradiogram demonstrating the presence of the7-kb junction fragment specific to NEP-2 and POP progenitor ge-nomes within the genome of fertile revertant WPR-3.The mitochondrial DNA samples were digested with PstI, blotted,and hybridized with the PCR probe encompassing the 7-kb junctionfragment. The position of this fragment is indicated.

Shifts in the Bean Mitochondrial Genome 1173

diversity suggested by Leaver et al. (1988). Alternatively, thepossibility exists that the single circular genome form foundin the POP and NEP-2 lines is derived from the tripartiteconfiguration, with the deletion of the pvs sequence as onestep in the transition. Ongoing experiments to investigatethe origin of the pvs sequence may provide a clearer modelfor this process.

Our discovery that the sterility-associated genome config-uration is maintained within at least one progenitor line,POP, at substoichiometric levels provided the first evidencethat the mitochondrial genome alterations giving rise to theG08063 cytoplasm involved genomic shifts in relative copynumber of existing molecules. What induced these shifts isnot yet clear. The crossing of POP 3 NEP-2 gave rise toseveral progeny, of which accessions G08063, G08064, andG08065 represent sister lines. Although DNA fingerprintingresults support the close genetic relationship of these threelines, as demonstrated in Figure 1, the mitochondrial ge-nome shift occurred only in G08063, with mitochondrial ge-nomes in G08064 and G08065 appearing identical to thePOP/NEP-2 parental types (data not shown). Thus, althoughit is conceivable that the combining of the POP and NEP-2nuclear genotypes was sufficient to induce the mitochon-drial alteration of G08063, it is clear that such changes donot occur at high frequency or demonstrate the predictedMendelian behavior expected if a nuclear genetic factor iscausing the transition.

Likewise, we have demonstrated the presence of both theprogenitor and sterility-inducing mitochondrial genome formsat substoichiometric levels within the genome of the sponta-neous revertants WPR-3 and 83-1. Reversion is an unusualphenomenon not common to most other CMS systems. Inbean, reversion appears to consist of the suppression of thepvs-containing mitochondrial molecule, with retention of the re-maining two subgenomic molecules at apparently normal copynumber. The reversion event is spontaneous, and its frequencyvaries with changes in genetic background (Mackenzie et al.,1988). Those factors, environmental or otherwise, that areinvolved in the induction of a genomic shift upon reversionare not yet defined.

It is important to note that changes in genome organiza-tion during the transition from progenitor to G08063 (malesterility–inducing) mitochondrial forms include not only anincrease in complexity of environments surrounding recom-bination repeats but also DNA sequence differences in fourregions, A to D, distinguishing progenitor and G08063 ge-nomes. We have investigated only the implications of thesedifferences with regard to modulation of pvs-orf239 expres-sion contained within region D. It is possible that otherchanges in gene expression occurred in the transition fromprogenitor to G08063. However, it is not likely that thesecontribute to a phenotype that is pronounced like that of theORF239 product, because upon reversion to fertility, onlyregion D is suppressed; regions A to C remain intact (Janskaand Mackenzie, 1993), and the phenotype of the revertantappears normal.

The observed amplification and expression of the pvs-containing mitochondrial molecule in vitro in mitochondriapurified from the revertant lines suggest that the reversionphenomenon is reversible and that the male-fertile pheno-type of the revertant is a direct consequence of reduced pvscopy number. Moreover, our results imply that the genomicshift occurring upon reversion involves differential replica-tion as opposed to mitochondrial population shifts or inter-cellular competition, as have also been postulated by others(Albert et al., 1996). This conclusion is based on the fact thatwe were able, with a 90-min incubation of a defined popula-tion of mitochondria isolated from revertant seedling tissues,to observe a reversal of the reversion phenomenon in theform of amplification and expression of the pvs-orf239 se-quence. Because we have, in previous studies, associatedexpression of the pvs-orf239 sequence with the sterility phe-notype (Abad et al., 1995; He et al., 1996), we assume thatthe changes observed in vitro would have led to the reestab-lishment of the male-sterile phenotype in vivo. We are cur-rently conducting experiments to test for the reversibility invivo of the fertile revertant to the male-sterile phenotype.

The in organello experiments to test for reversibility ofpvs-orf239 suppression were intentionally conducted forDNA amplification, RNA amplification, and protein expres-sion simultaneously, using identical incubation conditionsoptimized for in organello protein synthesis. This is becauseour observation of in organello ORF239 protein accumulationin mitochondria from revertant lines over a 90-min incubationdirectly implied concomitant DNA and RNA amplification un-der these conditions. However, it can be assumed that theaddition of exogenous nucleotides to the incubation mixturewould have enhanced dramatically the rate of DNA and RNAsynthesis in vitro. Enriquez et al. (1994) showed incorpora-tion of 32P-dCTP in the absence of added dTTP, dATP, anddGTP in in organello incubations of rat mitochondria, using 1mg of protein per incubation, and then compared these re-sults with those obtained after adding the four 32P-labeleddeoxynucleotide triphosphates. They obtained up to 100,000cpm over a 10-hr in organello incubation period, when allfour labeled nucleotides were added, and a 75% reductionin this value in the absence of the added nucleotides; how-ever, it was not clear how much mitochondrial DNA wasused to determine these values. In the same study, it wasshown that a concentration of mitochondria equivalent to 2mg/mL of protein represents an optimal amount to achievegood incorporation. In our study, the amount of mitochon-dria included per incubation was equivalent to 0.2 mg ofprotein, representing 20% of the amount used by Enriquezet al. (1994), with the incubation lasting only 90 min. Be-cause of the striking differences in reaction conditions aswell as the organisms from which mitochondria were pre-pared, it is difficult to compare directly the efficiency of ourreaction with that reported previously. However, the resultsreported by Enriquez et al. do support our conclusion thatmitochondrial DNA replication occurs under the conditionsused in our experiment.

1174 The Plant Cell

Figure 10. Evidence of Amplification of the pvs-orf239 DNA Sequence within the Mitochondrial Genome of Revertant Lines after in Organello In-cubation.

Mitochondrial samples (200 mg of protein) taken from Percoll gradient–purified mitochondria were used for isolation of DNA template to test forchanges in stoichiometry within the mitochondrial genome after a 90-min in organello incubation (two time points: 0 and 90 min). A 5-ng sampleof mitochondrial DNA was used for the PCR amplification reactions, and amplified samples subsequently were subjected to electrophoresis in a1% agarose gel.(A) PCR amplification results, using the orf59 and orf39 primers, are shown on an ethidium bromide–stained gel. Numbers at left indicate frag-ment size in kilobases.

Shifts in the Bean Mitochondrial Genome 1175

It becomes increasingly clear that substoichiometric formsare prevalent within the mitochondrial genomes of many plantspecies (Small et al., 1987; Bonhomme et al., 1992; Pla etal., 1995; Yesodi et al., 1995; Suzuki et al., 1996; Gutierres et al.,1997) and that changes in their relative stoichiometry can bereversed (Kanazawa et al., 1994). Although the tracking ofgenomic shifts over generations has not previously been re-ported to the extent that we describe here, it would not besurprising to find that these phenomena occur in severalplant lineages. In fact, such spontaneous genomic shifts arepredicted to account for much of the heterogeneity found inplant mitochondrial populations (Lonsdale et al., 1988). Whatis relevant about our observations in common bean is thatthey demonstrate that these shifts can occur in a very shorttime span within a single plant lineage under natural growthconditions.

Evidence suggests that some genomic shifts are underthe control of specific nuclear loci. For instance, introductionby crossing of the fertility restorer Fr in common beanproduces a phenomenon indistinguishable from reversion(Mackenzie and Chase, 1990), and in fact, the pvs-orf239 se-quence is present substoichiometrically in fertile Fr-restoredlines (W. Van Houten and S.A. Mackenzie, unpublished data).It is conceivable that our in vitro mitochondrial incubationsto amplify the pvs molecule simply have allowed the relax-ation of control by the Fr locus in the experiments reportedhere. Moreover, in Arabidopsis, mutation at the chloroplastmutator (CHM) locus results in the selective amplification ofspecific substoichiometric forms, depending on the cytoplasmcontained in the line to which the chm mutation is intro-duced (Sakamoto et al., 1996). These examples provide themost compelling evidence to date that nuclear genotype di-

rectly influences the stoichiometric relationships among mi-tochondrial molecules.

One of the distinguishing features of plants with regard tomitochondrial biology is the subdividing of their genome intoa complex, recombinationally active organization. The evo-lutionary rationale for such a complex structure has notbeen immediately obvious. However, our observations inthis study suggest that such a structure may provide onemeans of modulating expression of particular regions of thegenome. In plant mitochondria, where the roles of transcrip-tional, post-transcriptional, and translational mechanisms ingene inactivation are not altogether clear and where domi-nant mitochondrial mutations are prevalent (Hanson, 1991),the ability to dramatically and reversibly suppress copynumber of a subsegment of the genome most likely providesan important mechanism for maintaining proper mitochon-drial function while retaining tremendous adaptive geneticdiversity.

METHODS

Plant Materials

Common bean (Phaseolus vulgaris) accession lines G04459 (NEP-2)and G14067 (POP), G08063, G08064, and G08065, and line SanFernando were obtained from the Centro Internacional de AgriculturaTropical Germplasm Collection Center (Cali, Colombia). The cultivarOpal was provided by Rogers Brothers Seed Co. (Twin Falls, ID).CMS-Sprite was derived from recurrent backcrossing (20 genera-tions) of the G08063 cytoplasm to a cultivar Sprite nuclear genotype.Spontaneous reversions were selected as single seed-bearing pods

Figure 10. (continued).

(B) Primers orf59 and cob39 were used for PCR amplification, and the products are shown on an ethidium bromide–stained gel.(C) An autoradiogram of the gel shown in (A) hybridized with the orf239 probe.(D) The corresponding autoradiogram from the gel shown in (B), using the orf239 sequence as probe.(E) Primers cob59 and cob39 were used for PCR amplifications. These primers were selected as controls to monitor an unrelated region of themitochondrial genome. Revertant lines consistently showed an increase in amplification product after the in organello incubation, whereas CMS-Sprite and G08063 showed a slight decrease in amplification product. We assume that changes in concentration of amplified product are mostlikely reflective of the changes in the relative amount of template after the in organello incubations because equal amounts of mitochondrial DNAtemplate were included in each reaction. Numbers at left indicate fragment size in kilobases.(F) Diagram depicting the relative location of the primers used in the regions containing the pvs-orf239 and cob genes, respectively.(G) and (H) Results of 32P-dCTP incorporation experiments to demonstrate de novo DNA synthesis by purified bean mitochondria in the absenceof added deoxynucleotides during in organello incubations. In organello incubations (200 mg of mitochondrial protein) were performed in thepresence of 32P-dCTP, with or without the addition of the replication inhibitors ddTTP or aphidicolin. Samples for three time points (0, 30, and 90min) in each of the treatments described above were taken during the in organello incubations to test for 32P-dCTP incorporation into newly syn-thesized mitochondrial DNA. DNA samples of 200 ng each were subjected to electrophoresis in 0.8% agarose, DNA gel blotted, counted, andexposed to x-ray film. Because of the lack of identified specific mitochondrial g DNA polymerase inhibitors, the nonspecific replication inhibitorddTTP (20 mM) was used to demonstrate that the observed incorporation of 32P-dCTP was dependent on mitochondrial DNA replicative activity.The count-per-minute values obtained for each of the bands show that a 45% inhibition of 32P-dCTP incorporation occurs in the presence ofddTTP when compared with the aphidicolin and no-inhibitor treatments after 90 min. Subsequent hybridization of the resulting DNA gel blot witha chloroplast DNA probe (ATP synthase subunit 1) indicated that chloroplast DNA contamination of the prepared mitochondrial DNA sample wasundetectable (data not shown).

1176 The Plant Cell

on otherwise male-sterile CMS-Sprite plants. They were confirmedto be cytoplasmic events, as described by Mackenzie et al. (1988).

DNA Isolation, Gel Blotting, and Hybridization Procedures

Mitochondrial DNA was purified from 7-day-old etiolated seedlings,according to the procedure described by Mackenzie et al. (1988). To-tal genomic DNA was extracted from leaves of greenhouse-grownplants, as described by He et al. (1995). Cosmid or plasmid DNAswere prepared as minipreparations by the method described bySambrook et al. (1989).

DNAs were digested with restriction enzymes according to themanufacturers’ instructions (PstI and EcoRI from Gibco BRL; XhoIand SalI from Promega). Agarose gel electrophoresis, DNA transferto Hybond-N nylon membrane (0.45 mm; Amersham), extraction ofDNA fragments, labeling of DNA, and filter hybridizations were con-ducted according to the procedures described by Janska andMackenzie (1993). Autoradiography was performed using an Instant-imager (Packard, Meriden, CT) and exposure to x-ray film (Fuji,Stamford, CT) with intensifying screens at 2708C. Short exposuretimes were generally overnight, with long exposure times to visualizesubstoichiometric forms extended to z1 week.

DNA Fingerprint Analysis

Oligonucleotide primers (10mers) were purchased from OperonTechnologies (Alameda, CA). The polymerase chain reaction (PCR)conditions were optimized for this study. Reaction mixtures (20 mLtotal volume) consisted of 10 mM Tris-HCl, pH 9.0, at 258C, 50 mMKCl, 2.25 mM MgCl2, nucleotides dATP, dTTP, dCTP, and dGTP(125 mM each), 0.2 mM primer, 60 ng of template DNA, and 1.25 unitsof Taq DNA polymerase (Promega). The amplifications were per-formed in a thermal cycler (model PTC100; MJ Research, Water-town, CT) programmed for 1 min at 948C followed by 35 cycles of 35sec at 948C, 50 sec at 368C, 50 sec at 728C, and ending with 5 min at728C. The primers were used individually.

Rapid amplified polymorphic DNA (RAPD) profiles were scored vi-sually. Variation among samples was evaluated from pairwise com-parisons of the proportion of shared fragments among samples, thatis, two times the number of shared fragments divided by the totalnumber in the profiles being compared. This method was equivalentto calculating Nei and Li’s index (1979) for genetic similarity (Sxy) forrestriction fragment length polymorphism comparisons. Relation-ships among samples were evaluated with a phenetic cluster analy-sis, using the unweighted pair group method for arithmetic averages,and plotted in a phenogram. All computations and statistical analy-ses were performed using SAS (version 6; Cary, NC) programs.

Figure 11. Presence of the ORF239 Transcript and Peptide in Re-vertant WPR-3 Mitochondria after in Organello Incubations.

(A) Results of an RT-PCR experiment show that the amount oforf239 transcript increased after 90-min in organello incubations ofmitochondria prepared from revertant line 83-1 and CMS-Sprite.Control reactions labeled RNA (DNase I) represent DNase I–treatedRNA samples that were subjected to PCR amplification without priorreverse transcriptase reaction to confirm elimination of DNA con-tamination. In the revertant line 83-1 sample, the orf239 transcriptwas detected only after the 90-min in organello incubation. In thecase of the male-sterile line CMS-Sprite, transcripts were evidentbefore and after incubation. A 90-min incubation of the CMS-Spritemitochondria resulted in a detectable increase in the orf239 tran-script amount.(B) Effect of a 90-min in organello incubation on accumulation of theORF239 peptide in mitochondria prepared from the revertant WPR-3line. The product shown is the product of immunoprecipitation, usinganti-ORF239 polyclonal antibodies. Lanes labeled Anti-ORF239 andPreim show the results of experiments with anti-ORF239 polyclonalantibody and preimmune serum, respectively, used to immunopre-cipitate the in vitro–transcribed and –translated (IVT) ORF239 pep-tide. In all remaining lanes, the products were immunoprecipitatedusing the anti-ORF239 polyclonal antibody. The product of the pre-dicted size was obtained (30 kD). Longer exposure of IVT samplesrevealed the presence of two higher molecular weight products (notshown; also presented in Sarria et al., 1998) of identical size to thoseshown in the fifth and sixth lanes. These higher molecular weightproducts are thought to represent aggregation or multimeric formsof ORF239 because they were present in both immunoprecipitatedIVT and in organello samples when ORF239 antibodies (purified IgGfraction only) were used. These larger forms never have been ob-served at time zero of an in organello incubation or when ORF239

was absent (e.g., in the absence of added protease inhibitor). Lanesdesignated CMS (CMS-Sprite) and WPR-3 IVT-mtRNA show the re-covery of ORF239 peptide by immunoprecipitation with anti-ORF239antibodies after in vitro translation of mitochondrial RNA (1 mg perreaction) isolated after a 90-min in organello incubation. Lanes des-ignated in organello show recovery by immunoprecipitation of theORF239 protein product after in organello translation reactions withpurified CMS-Sprite and WPR-3 mitochondria in the presence of theprotease inhibitor vanadate (4 mM).

Shifts in the Bean Mitochondrial Genome 1177

Mitochondrial DNA Probes and DNA Cloning

Cosmid clones used in comparative DNA gel blot hybridizations werederived from previously developed mitochondrial DNA libraries ofCMS-Sprite (Mackenzie and Chase, 1990) and WPR-3 (Janska andMackenzie, 1993). Other mitochondrial probes were derived by re-striction endonuclease digestion of cosmid DNA and fragment purifi-cation from agarose, as described by Janska and Mackenzie (1993).Probes used to identify regions unique to the progenitor NEP-2 andPOP genomes were derived by PCR amplification, using total DNAas a template.

The 4.8-kb PstI restriction fragment derived from cosmid clone12G4 (WPR-3 library) was purified and ligated to vector pBluescript IIKS1 (Stratagene, La Jolla, CA) for transformation to Escherichia colistrain XL-1 Blue MRF9 (Stratagene). The orientation was deduced by

restriction pattern, and the appropriate fragment end was sequenced(Sequenase 2.0; Amersham) to identify a suitable primer site for sub-sequent PCR amplification experiments.

Long-Distance PCR

To amplify two distinctive junctions present in the NEP-2 and POPmitochondrial genomes, we performed reactions in a ThermocyclerPoly-Chain II (PolyGen, Kiev, Ukraine). Amplification between atpAand cob utilized the following primers: primer P2 (59-ATAGCAGGT-CTAATTCCGCG-39) is homologous to the atpA gene from commonbean at positions 1084 to 1103 (Chase and Ortega, 1992), and primerP8757 (59-AGCTATGCATTACACACCTC-39) was derived from themaize cob gene at positions 168 to 187 (Dawson et al., 1984). These

Figure 12. A Proposed Model for Mitochondrial Genomic Shifts Predicted to Occur upon Transition from the Progenitor Configuration (POP andNEP-2) to a Sterility-Inducing Configuration (G08063 and CMS-Sprite) to a Fertility-Conferring Configuration of Revertants WPR-3 and 83-1.

Molecules are diagrammed to correspond to those presented in Figure 6. In the POP progenitor genome, the tripartite configuration characteris-tic of G08063 is present at substoichiometric levels (dashed lines). Transition to the male-sterile G08063/CMS-Sprite form involves the suppres-sion of the POP progenitor form (dashed lines) and an amplification of the tripartite configuration. Spontaneous reversion involves suppressionof the pvs-containing 210-kb molecule, leaving a bipartite mitochondrial organization and restoring the fertility phenotype by suppression of pvs-orf239 expression.

1178 The Plant Cell

primers allowed amplification of the 10-kb fragment. The secondspecific junction, z7 kb in length, utilized primers P4.8 (59-GTG-CTATAGTGGCCTTGGAC-39) derived from sequencing the 4.8-kbPstI fragment at the junction site and P18/5r (5 9-CATGCTCCA-CCGCTTGT-39) derived from the soybean rrn18 gene sequence atpositions 929 to 945 (Grabau, 1985). The locations of all primers areshown in Figure 3.

PCR amplifications involved a 50-mL reaction mixture of 50 mMTris-HCl, pH 9.0, 10 mM (NH4)2SO4, 0.1% Triton X-100, 2 mM MgCl2,400 mM of each deoxynucleotide triphosphate, 800 nM of eachprimer, and 100 ng of template mitochondrial DNA. The sampleswere overlaid with 50 mL of mineral oil. After an initial denaturationstep of 2 min at 928C, the reaction was initiated by adding 2.5 units ofPrimeZyme DNA polymerase (Biometra, Gottingen, Germany). Am-plification of the 10-kb fragment was performed for 30 cycles withdenaturation at 928C for 10 sec, annealing at 628C for 30 sec, and ex-tension at 688C for 12 min. During the last 20 cycles, 100 sec wasadded to the extension time after each fifth cycle. The reaction wasfollowed by a 25-min incubation at 688C. To amplify the 7-kb frag-ment, we lowered the annealing temperature to 588C, and the exten-sion times were as follows: 10 cycles of 7 min in duration, with thefollowing 20 cycles lengthened each fifth cycle by 60 sec. The finalelongation was 15 min. In the CMS-Sprite sample, nonspecific bandsof various sizes amplified using these conditions; none of these am-plification products demonstrated hybridization with the 4.8-kb PstIfragment. PCR products were agarose gel fractionated.

For long-distance PCR amplification of the pvs region in revertantWPR-3 and 83-1 mitochondrial DNA preparations, primers ATP-A5(59-GGTGGACTAACTAGCGAA-39) from the 39 end of the atpA gene(Chase and Ortega, 1992) and orf23939 (59-CCATTAGCGGGGATG-CTT-39) derived from the 39 end of the pvs-orf239 sequence wereused. The Expand long-template PCR system was used (BoehringerMannheim). Reaction conditions involved a 30-mL final volume with1.75 mM MgCl2, 500 mM of each deoxynucleotide triphosphate, 66mM primers, 1.55 units of Taq DNA polymerase and Pwo DNA poly-merase, and 60 ng of template DNA per reaction. Amplification con-ditions were 928C for 2 min followed by 10 cycles of 928C for 15 sec,588C for 40 sec, and 688C for 4 min. This was followed by 20 cyclesat 928C for 15 sec, 588C for 40 sec, and 688C for 4 min, adding 15 secper cycle. Final elongation was 688C for 7 min. DNA gel blot hybrid-izations to confirm the identity of the 4.4-kb amplification productwere conducted using standard hybridization conditions indicatedabove, with a PCR-amplified orf239 sequence as probe.

Mitochondrial Preparation for in Organello Incubations

Dark-grown 10-day-old seedlings of common bean lines CMS-Spriteand WPR-3 were used for mitochondria isolation, according to Fordeet al. (1978), and purified over a 26% Percoll gradient. Purified mito-chondria were resuspended in 10 times the final pellet volume, whichgives an approximate protein concentration of 40 mg/mL.

In Organello Incubations and Nucleic Acid andProtein Isolations

Mitochondrial in organello incubations were conducted withoutmodifications, according to the procedure of Forde et al. (1978). Aftera 90-min incubation, samples were used for mitochondrial RNA,DNA, or protein isolation. In organello reactions to demonstrate de

novo DNA synthesis in the absence of added deoxynucleotide tri-phosphates were conducted in the presence of 4 mL of Redivue-32P-dCTP (Amersham), with a specific activity of 3000 Ci/mmol (0.05 mM)with and without 20 mM ddTTP (Sigma) and 20 mg/mL aphidicolin(Fluka, Milwaukee, WI). For RNA and DNA isolation, mitochondriawere lysed in lysis buffer (150 mM NaCl, 100 mM EDTA, 10 mM Tris-HCl, pH 8.0, and 2% SDS). Samples were incubated at 658C for 10min, after which potassium acetate (0.5 M) was added. Sampleswere then centrifuged for 5 min at 14,000 rpm and precipitated over-night with 2 volumes of absolute ethanol. Preparations were resus-pended in TE buffer (10 mM Tris-HCl and 1 mM EDTA). For thepurification of RNA, samples were treated with RNase-free DNase,and for the isolation of DNA, with DNase-free RNase. For in organellotranslations, 35S-cysteine and the protease inhibitor vanadate (4 mM)were included to recover labeled ORF239 product. For protein isola-tion, mitochondria were treated as described by Ragan et al. (1987),without fractionation. Counts per minute in 32P-dCTP incorporationexperiments were determined using a Packard Instantimager.

Reverse Transcription–PCR Reactions

Reverse transcription (RT) and RT-PCR reactions for detection ofpvs-orf239 were performed in the presence of 1 mL of avian myelo-blastosis virus reverse transcriptase (25 units/mL; Promega), 4 mL of5 3 avian myeloblastosis virus reverse transcriptase buffer, 2 mL ofdeoxynucleotide triphosphates (10 mM each), 1 mL of Rnasin (40units/mL), 1 mL of orf39 primer (12.5 mM), and 400 ng of template mi-tochondrial RNA in a total volume of 20 mL. The cDNA first-strandsynthesis reaction was incubated at 558C for 1 hr. Samples subse-quently were incubated at 658C for 15 min to stop the reverse tran-scription reaction. For subsequent PCR reactions, 5 mL of cDNAfirst-strand synthesis was used. PCR amplification of pvs-orf239 wasas described below.

In Vitro Translation and Immunoprecipitation

The clone or f239 (1 mg) was transcribed and translated using theTNT-coupled transcription/translation reticulocyte lysate system(Promega). Transcription was performed using T7 RNA polymerase.Reactions were incubated for 90 min. The rabbit reticulocyte system(Promega) was used to translate purified mitochondrial RNA sam-ples. Reactions were incubated for 90 min. Immunoprecipitations ofboth in organello translations as well as in vitro translations were per-formed according to Sarria et al. (1998). Anti-ORF239 polyclonal an-tibodies used were those described by Abad et al. (1995). Proteinsamples were run in an SDS-PAGE discontinuous system, as de-scribed by Laemmli (1970).

PCRs and DNA Gel Blotting from in OrganelloIncubation Samples

Primers orf59 (59-ATGTTCCTCCCATTCAACCC-39), orf39 (59-TAA-CGGTTGCCCCATTAGCGG-39), cob59 (59-ATGACTATAAGGAAC-CAA-39), and cob39 (59-TGGAATTCCTCTTCCAAC-39) were used forthe PCR reactions. Primers or f59 and orf39 amplify a 720-bp frag-ment that comprises the pvs-orf239 gene. Primers orf59 and cob39

generate a 783-bp fragment that contains the pvs-or f239 gene andthe flanking 83 bp that have 100% homology with the 39 end of thecob gene. Primers cob59 and cob39 generate a 1148-bp fragment

Shifts in the Bean Mitochondrial Genome 1179

that corresponds to most of the coding region of the cob gene. A5-ng sample of mitochondrial DNA obtained before and after in or-ganello incubations was used per PCR reaction. PCR cycling condi-tions were an initial denaturation step of 948C for 1 min, 35 cycles of928C for 30 sec, 568C for 1 min, and 728C for 1 min and 30 sec, witha final elongation step of 728C for 5 min. DNA gel blots were pre-pared and hybridized as described by Sambrook et al. (1989).

ACKNOWLEDGMENTS

We thank the Biological Illustrations Department at Purdue Universityfor their assistance in the preparation of illustrations. This work wassupported by Grant No. 9630252 MCB from the National ScienceFoundation to S.A.M. and Grant No. 6P20302307 from the PolishState Committee for Scientific Research (KBN) to H.J.

Received January 20, 1998; accepted April 28, 1998.

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DOI 10.1105/tpc.10.7.1163 1998;10;1163-1180Plant Cell

Hanna Janska, Rodrigo Sarria, Magdalena Woloszynska, Maria Arrieta-Montiel and Sally A. MackenzieSpontaneous Reversion to Fertility

Stoichiometric Shifts in the Common Bean Mitochondrial Genome Leading to Male Sterility and

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