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Studies on wheat mitochondrial DNA organization. Comparison of mitochondrial DNA from normal and cytoplasmic male sterile varieties of wheat
Plant Science, 43 (1986) 141--149 Elsevier Scientific Publishers Ireland Ltd.
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S T U D I E S ON W H E A T M I T O C H O N D R I A L D N A O R G A N I Z A T I O N . C O M P A R I S O N O F MITOCHONDRIAL DNA FROM NORMAL AND CYTOPLASMIC MALE STERILE V A R I E T I E S OF WHEAT
BERt~NICE RICARD *'a, BERNARD LEJEUNEb and ALEJANDRO ARAYA**~ Institut de Biochimie Cellulaire et Neurochimie du CNRS, 1 Rue Camille Saint Saens, 33077 Bordeaux Cedex and bLaboratoire de Biologie Moldculaire Vdgdtale, Universitd de Paris-Sud, Batiment 430, 91405 Orsay Cedex (France)
(Received July 29th, 1985) (Revision received December 5th, 1985) (Accepted December 5th, 1985) The mitochondrial genome of fertile, male-sterile and restored cytoplasm lines of wheat has been studied by means of recombinant DNA and hybridization techniques. Using cloned fragments of mitochondrial DNA (mtDNA) from fertile wheat cytoplasms as probes, about 40% of the genome is shown to have a differential hybridization pattern. The use of wheat rRNA and corn eytochrome oxidase subunit II probes indicates that duplication and rearrangement of genes or parts of genes may account for the differences observed. DNA synthesis in isolated mitochondria showed neither preferential labeling of part of the mtDNA nor the presence of extrachromosomal elements. Key words : wheat; mitochondriai DNA ; cytoplasmic male sterility
Introduction T h e m t D N A of higher plants is unusually variable in size, ranging b e t w e e n 2 0 0 kilo basepairs ( k b p ) and 2 5 0 0 k b p [ 1 , 2 ] . O n e striking feature is the h e t e r o g e n e o u s population of molecules isolated f r o m p l a n t mitoc h o n d r i a , as indicated by t h e c o m p l i c a t e d restriction endonuclease patterns obtained [3]. Studies o f t h e m o l e c u l a r o r g a n i z a t i o n of B r a s s i c a c a m p e s t r i s [4] and Z e a m a y s [5[ m t D N A have led to the idea t h a t m o s t m i t o c h o n d r i a l genes reside in a ' m a s t e r ' c h r o m o some and t h a t a subset o f m t D N A m o l e c u l e s arises f r o m r e c o m b i n a t i o n events. The pres* Present address: Laboratoire de physiologie v6g6tale. I.N.R.A. 33 Pont de la Maye. France. **To whom correspondence should be addressed. Abbreviations: CMS, cytoplasmic male sterility; COII, subunit II of cytochrome c oxidase; kbp, kilo base pairs; PVP, polyvinylpyrolidine; SDS, sodium dodecyl sulfate.
ence o f r e p e a t e d sequences in plant m t D N A s u p p o r t s this h y p o t h e s i s . T h a t t h e r R N A genes are f o u n d on f o u r restriction f r a g m e n t s in w h e a t m t D N A indicates t h a t such a m o d e l m a y also be applicable to w h e a t [6]. I n t r a m o l e c u l a r r e c o m b i n a t i o n as a source o f m t D N A h e t e r o m o r p h i s m has been described in f u n g u s [ 7 ] . A n o t h e r source o f heteromorphism c o u l d be a differential replication process, such as the a m p l i f i c a t i o n o f m i n i c h r o m o s o m e s in P o d o s p o r a a n s e r i n a [8]. T o u n d e r s t a n d the f u n c t i o n i n g o f plant m t D N A , an interesting a p p r o a c h c o u l d be t h e s t u d y o f m t D N A in a h e t e r o l o g o u s c o n t e x t . In w h e a t , c y t o p l a s m i c male sterility (CMS) results f r o m r e p e a t e d interspecific back-crossing, leading t o t h e association o f t h e nucleus o f t h e h e x a p l o i d , T r i t i c u m a e s t i v u m , with t h e c y t o p l a s m o f the tetraploid, T. t i m o p h e e v i . In a variety o f plants in a d d i t i o n t o corn, evidence is a c c u m u l a t i n g t h a t t h e genetic
0168-9452/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland
142 determinants that control CMS are carried by the mitochondrion and not the chloroplast [9]. CMS has been associated with differences in restriction enzyme patterns of mtDNA and identical patterns of cpDNA in wheat [3,10]. In male sterile cytoplasms of Vicia faba, sorghum, sugarbeet, Brassica and corn, episomal DNA can be isolated from mitochondria [11--15], although the available evidence does not suggest that episomes are correlated with CMS. In wheat, the mitochondria are not the sole determinants of CMS since the cytoplasm of T. timopheevi is male fertile in association with the nucleus of T. timopheevi but male sterile in association with the nuclei of T. aestivum. We have searched for episomal-like DNA as well as a modification of mtDNA polymerase which we speculated might be correlated with such DNA in mitochondria isolated from male sterile cytoplasms of wheat. In the second part of our study, we show that the mtDNA from the male sterile cytoplasms is homologous to that in normal cytoplasm of wheat, but organized differently. Such differences in organization may explain in part the incompatibility of T. timopheevi mitochondria with T. aestivum nuclei. Materials and methods
Wheat genetic stocks Seeds of alloplasmic fertile and male sterile lines of wheat (62.2.3 and 62.2.3.T) were kindly provided by P. Auriau (Station d'Am61ioration des Plantes de Versailles, France and P. B6rard (Station d'Am61ioration des Plantes de Clermont-Ferrand, France, who also supplied the fertile hybrid F1 (62.2.3.T × 149 Trf). The fertile hybrid or male-fertile (restored) has the cytoplasm T of T. timopheevi Zhuk of the male-sterile seed parent and the restorer genes of 149 Trf, the pollen parent. One hundred forty-nine Trf combines genes for the restoration of fertility from three different sources T. timopheevi and two T. aestivum varieties.
These restorer genes are present in all three wheat genomes.
Growth and harvesting conditions For etiolated seedlings, seeds were surfacesterilized for 20 min in i.5% Mucocit, washed and germinated for 7--10 days in the dark at 22°C in humid boxes. Isolation o f mitochondria and purification of mitochondrial DNA DNA was isolated from mitochondria prepared as described previously [16] according to the technique of Qu6tier and V6del [3] from etiolated wheat seedlings. Preparation o f mitochondrial extracts Preparation of mitochondrial lysates and synthesis of DNA in whole mitochondria from wheat embryos has been described previously [ 16]. Endonuclease digestion and agarose gel electrophoresis Mitochondrial DNA (0.4 pg) was digested with 4 units of the restriction enzyme Sal I (Genofit) in a 20-ul reaction mixture containing 10 mM Tris--HC1 (pH 7.5), 6 mM MgC12, 0.2 mM EDTA, 150 mM NaC1 for 4 h at 37°C. The reaction was stopped by adding 5 ul of stop buffer (50% glycerol, 0.1% bromphenol blue, 1.25% sodium dodecyl sulfate (SDS), 30 mM EDTA). DNA samples were then loaded on 0.4% agarose gels in 89 mM Tris--borate, 2 mM EDTA buffer containing 0.5 ug/ml of ethidium bromide. Electrophoresis was performed at 40 volts for 20 h. The gel was dried and autoradiography was performed or the DNA was transferred to nitrocellulose filters by the procedure of Southern [ 171. Cloning o f wheat mtDNA Partial or complete Sal I digests of fertile wheat mtDNA were cloned in pHC79 or pBR322 as described [18]. Cytochrome oxidase subunit II gene (mox I) was a generous gift of C.J. Leaver (Edinburgh).
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Labeling and hybridization of cloned fragme n ts Approximately 1 pg of cloned m t D N A was added to a nick translation mixture which contained 50 mM Tris--HC1 (pH 7.2), 10 mM MgSO4, 0.1 mM dithiothreitol, 100 uM dATP, dGTP, dCTP and 20 uCi [a_32p]. TTP (Amersham France, 3000 Ci/mmol). Five units of DNA polymerase I were added and the reaction was allowed to proceed for 1 h at 15°C. Reaction was stopped by the addition of EDTA to 20 mM. After purification by gel filtration on Sephadex {G-50 medium), the DNA was heated at 100°C for 5 min, quick-cooled and added to hybridization buffer at a radioactive concentration of 106 cpm/ml. Hybridizations were carried out at 37°C for 40 h in 50% formamide, 3 × SSC (1 × SSC = 0.15 M NaC1, 0.015 M sodium citrate), 0.02% polyvinylpyrolidone (PVP)--Ficoll, 0.1% SDS, 1 mM EDTA, 50 mM sodium phosphate buffer (pH 6.5), 50 pg/ml denatured, sonicated salmon sperm DNA. After annealing, filters were washed at 37°C once in 3 × SSC, 0.02% PVP--Ficoll, 0.1% SDS, 1 mM EDTA, 50 mM sodium phosphate buffer (pH 6.5), twice in 50% formamide, 3 × SSC, 0.1% SDS, 1 mM EDTA, twice in 3 ×SSC, 0.1% SDS and twice in 0.1 ×SSC, 0.1% SDS. Filters were dried and autoradiography was performed. Reverse transcription of total mtRNA Total RNA was prepared by two successive phenol/chloroform (1 : 1) extractions of mitochondria lyzed 30 min at r o o m temperature with sodium N-lauryl sarcosine (0.2%, w/v) and 200 ug/ml of proteinase K. Ten micrograms of total RNA was incubated for 1 h at 37°C in a 50-~1 reaction mixture containing 25 mM Tris--HC1 (pH 8.3), 6 mM MgC12, 8 mM dithiothreitol, 200 t~M dATP, dGTP, TTP, 75 uCi [a-32P]dCTP (3000 Ci/mmol), 500 t~g/ml calf thymus random primer DNA, 20 ug/ml actinomycin D, 17 units of AMV reverse transcriptase {Life Sciences). The reaction was stopped with 5 ~l of 0.1 M
EDTA and 3 ul of 5% SDS. RNA templates were removed by adding NaOH to 0.3 M and incubating overnight at 37°C. After neutralization, the labeled cDNA was separated from unincorporated nucle~tides by chromatography through a Sephadex G-100 column equilibrated with 10 mM Tris--HC1 (pH 9), 100 mM NaC1, 1 mM EDTA. Hybridization conditions were the same as described above for DNA labeled by nick translation. Results arid discussion
Replication of DNA in mitochondria from male sterile and fertile cytoplasms Mitochondria from both male sterile and fertile cytoplasms are capable of incorporating TTP into the entire mitochondrial genome. A comparison of Fig. 1B and C shows that the Sal I restriction enzyme profile of DNA labeled in organello {Fig. 1B) is indistinguishable from that of unlabeled mtDNA (Fig. 1C). In particular, the variable intensities of restriction fragments are reproduced, indicating that the entire genome is involved in DNA synthesis. No preferential synthesis of particular fragments is evident, as has been shown to occur with episomal elements in corn {V. Walbot, pers. commun.). This is supported by the autoradiograph of undigested mtDNA synthesized in organello (Fig. 1A): only chromosomal DNA is labeled. Likewise, no episomal elements were found, either by analysis of unlabeled mtDNA or by labeling of mtDNA in organello. The mitochondrial DNA polymerases from male sterile and fertile cytoplasms of alloplasmic lines were isolated as described previously [16]. No differences were evident in the template specificities, cation activation profiles, and inhibitor sensitivities. The specific activities correlated with the percentage of embryos capable of germination and not with type of cytoplasm (male sterile or fertile) {data not shown). These results are not surprising if we assume a nuclear
144
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A
C
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i
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i
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Fig. 1. A, B; Autoradiogram of 32P-labeled DNA synthesized in mitochondria isolated from malesterile (lanes 1, 3) and fertile (lanes 2, 4) cytoplasms of wheat without digestion (A) and following digestion (B) with restriction enzyme Sal I. The lettering of fragments in fertile m t D N A of wheat is that of Lejeune and Qu~tier (unpublished data). (C) restriction enzyme analysis of mtDNA isolated from fertile
cytoplasms of T. aestivurn (lane 5), male-sterile T. aestivum (cytoplasm T. tirnopheevi) (lane 6) and restored male-fertile T. aestivum (cytoplasm T. timopheevi) (lane 7). origin of mtDNA polymerases, as has been shown in the case of yeast [19]. Organization o f m t D N A from normal and male sterile cytoplasms o f wheat When Sal I restriction enzyme fragments
of mtDNA from normal T. aestivum cytoplasm are fractionated by 0.4% agarose gel electrophoresis, the pattern obtained is complex but characteristic. It is easily distinguished from that of mtDNA from male sterile cytoplasms, even though the majority of the fragments have similar mobilities (Fig. 1C). Approximately 33 Sal I fragments can be visualized by EtBr staining. Twentynine appear to be shared by the two types of mtDNA. T h e Sal I restriction profiles of male sterile cytoplasm is indistinguishable whether in association with T. timopheevi or T. aestivum nuclei, in the presence or absence of the genes for the restoration of fertility (Ref. 10, Fig. 1). These results suggest that no apparent alterations at the mtDNA level occur as a consequence of the introduction of a T. aestivum nucleus to a T. timopheevi cytoplasm. In T. timopheevi induced male sterility, mtDNA alterations due to cytoplasmic mutations cannot be invoked to explain male sterility. It seems more probable that the defect lies at the level of nucleo-cytoplasmic interactions. These interactions may be affected by a change in the molecular organization of certain genes, perhaps at the level of leader sequences involved in transport, initiation/termination sites, precursor/ product relationships of RNA transcripts or time sequence of transcription. It therefore seemed interesting to us to analyze the region of the mitochondrial genome which differs in both cytoplasms. It is at these regions that potential defects in nucleo-cytoplasmic interactions m a y originate. We sought first to evaluate the degree of similarity between the two mt genomes and second, to identify that part of the mt genome which was different. This was done using cloned fragments of mtDNA from fertile wheat cytoplasms. The cloned fragments ranged in size from 0.85 to 29 kbp and summed to a total of 170 kbp. This corresponds to 39% of the estimated total molecular weight of the wheat mt genome {unit genome size estimated to be 430 kbp, B. Lejeune and F. Qu~tier, pers.
145
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Cloned fragments with identical hybridization patterns in mtDNA from fertile and male sterile cytoplasms. Eleven cloned fragments, summing to 71 kbp, hybridized with a single band of similar mobility in m t D N A from fertile and male sterile cytoplasms {Fig. 2). This result shows that no major homology differences exist in these mitochondrial fragments and that they have been conserved in the two cytoplasms. In the case of clone H2 (Fig. 2A), hybridization appears less intense with the homologous normal mtDNA of fertile wheat than with the mtDNA of male sterile cytoplasms. This seems at first surprising since the restriction profiles indicate that band H is of comparable intensities in the three mt genomes. According to Qu~tier et al., however (pers. commun.), band H is composed of two fragments of identical size (clone H1 and H2) in fertile wheat mtDNA, so that it is possible that H2 is present in different amounts in the m t D N A from male sterile and fertile cytoplasms. Cloned fragments hybridizing differently in mtDNA from fertile and male-sterile cytoplasms. Six cloned fragments, summing to 84 kbp, hybridized with a band of similar mobility b u t also to different bands in m t D N A from fertile and male sterile cytoplasms (Fig. 3A). This result shows redundancy of at least a part of the cloned fragment. One of the fragments is conserved in the two cytoplasms, while the other may be located in a different chromosomal environment. Three cloned fragments, summing to 21 kbp, hybridized with a single band of different mobilities in m t D N A from fertile and male-sterile cytoplasms (Fig. 3C). In each case, hybridization with the m t D N A from male-sterile cytoplasms was weak, suggesting that these fragments are not present in equimolar amounts. One cloned fragment, of 22.7 kbp, hybridized with its band of origin in fertile wheat m t D N A and with t w o bands of different mobilities in malesterile wheat mtDNA. It is difficult to explain I
1
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Fig. 2. Hybridization of cloned and labeled Sal I fragments H2 (A) and X1, Y, AB (C) to a Southern transfer (B) of Sal I-digested mtDNA male-sterile (lane 4), fertile (lane 5), and male-fertile (restored) (lane 6) wheat cytoplasms.
commun.). Every clone hybridized with its fragment of origin in fertile wheat mtDNA and with either the same or a different fragment in male sterile mtDNA. None of the clones studied hybridized differently to male fertile (restored) m t D N A as compared to male sterile mtDNA. Thus, our studies so far do not permit us to distinguish any differences in the m t D N A of male fertile (restored) and male sterile cytoplasms.
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Recent sequence data for different plant mtDNA genes has shown that the coding regions are highly conserved [21,22]. It is interesting to speculate that the differences observed in wheat mtDNA may occur in non-coding or control regions of the mitochondrial genome.
W
O
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Fig. 3. Hybridization of cloned ai~d labeled Sal I fragments O (A) and K2 (C) to a Southern transfer (B) o f DNA from mitochondria o f male-sterile (lane 4), fertile (lane 5), and male-fertile (restored) (lane 6) wheat cytoplasms.
these differences as involving a sequence change in a single Sal I cleavage site. To explain the complex patterns, one could evoke multiple mutational events that create and/or abolish several restriction recognition sites. Alternatively, these patterns could be explained by sequence rearrangements. It has been shown that most plant mitochondrial genomes contain repeated sequences. The precise role of these potential recombination regions, that are different in different species, is n o t clear [20]. A summary of the hybridization results is presented in Table I. Of 169 kbp of mtDNA analyzed (the sum of the molecular weights of the cloned fragments), nearly 100 kbp showed an identical pattern of hybridization in the two cytoplasms, while 69 kbp displayed a different pattern.
Organization o f r R N A genes and the cytochrome oxidase H gone in m t D N A from normal and male sterile cytoplasms Falconet et al. [6] have shown that in normal wheat mtDNA, the 18--5 S rRNA genes are located on four Sal I fragments: F2, G2, R1, T (corresponding to fragments 5a, 5b, 19, 21 in their notation). Sal I fragm e n t T, 5.5 kbp, contains the 18--5 S rRNA genes in a structural unit at least 4 kbp long. This probe was used to detect the rRNA genes on mtDNA from male sterile cytoplasms. Figure 4, lane 5, confirms Falconet's results: fragment T hybridizes to three bands in the Sal I digest of fertile mtDNA (FG, R, and T). On our gels, bands F and G are not resolved. Fragment T also hybridizes to bands R and T in male sterile mtDNA but, in addition, to three other bands (Fig. 4, lanes 4 and 6). Likewise fragm e n t M, used to detect the 26 S rRNA gone on Sal I fragments o f mtDNA from malesterile cytoplasms, co-hybridizes to band M in both cytoplasms but also to different bands. In order to confirm that the multiple hybridization is indeed due to the presence of multiple rRNA genes, we synthesized labeled cDNA from total RNA by reverse transcription, as described in Materials and methods. Ribosomal RNA comprises the bulk of total RNA so that the probe prepared detects essentially ribosomal RNA genes. Figure 4, lane 8, shows that the rRNA probe hybridizes to the same five bands as fragments M and T in the Sal I digest of fertile wheat mtDNA. In male sterile wheat mtDNA seven Sal I bands were homologous to the rRNA probe; these were the same as those detected by fragments M and T.
147 Table I.
Summary of hybridization experiments.
A. Cloned fragments with identical hybridization patterns in Sal I restricted m t D N A from male-sterile and fertile cytoplasms o f wheat
Cloned fragment used as probe
kbp
Hybridization in Sal I digest of fertile wheat mtDNA to band :
Hybridization to co-migrating band in Sal I digest of male sterile wheat mtDNA
C3 H: L~ U: W X~ Y AA AB AC AE/AF
24 16.8 9.8 4.6 3.6 3.2 3.1 1.8 1.7 1.5 0.85
C H L U W X Y AA AB AC AE/AF
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
B. Cloned fragments with different hybridization patterns in Sal I restricted m t D N A from male sterile and fertile cytoplasms o f wheat
Cloned fragment used as probe
kbp
Hybridization in Sal I digest of fertile wheat mtDNA to band:
Hybridization to comigrating band in Sal I digest of male sterile wheat mtDNA: number of non-co-migrating bands
kbp of comigrating band(s)
A D, K~ K: O R: T X2
29 22.7 11.7 11.7 7.6 6.2 5.5 3.2
A + C D K ~ K O + R F +G X
Yes (Q):2 No : 2 Yes (M}:I No :1 Yes ( Z ) : I No :1 Yes(R + T ) : 3 No :1
6.4
~ Q M Z + R + T
The mox I clone corresponding to subunit II o f c y t o c h r o m e c o x i d a s e ( C O I I ) f r o m c o r n mitochondria [ 2 2 ] was u s e d t o i d e n t i f y homologous s e q u e n c e s in m t D N A from male-sterile a n d -fertile cytoplasms. Figure 5 shows that mox I hybridizes to an identical b a n d o f 3.9 k b p i n b o t h c y t o p l a s m s b u t also t o d i f f e r e n t b a n d s . R e c e n t l y B o n e n e t al. [21] have published the complete sequence o f t h e w h e a t c y t o c h r o m e o x i d a s e s u b u n i t II g e n e . T h e g e n e c o n t a i n s a Sal I site i n t h e i n t r o n v e r y n e a r t h e 3' e x o n . B o n e n e t al.
8.6 2.3 6.2 4- 5.5
[211 also d e m o n s t r a t e d t h e p r e s e n c e o f a repeated sequence corresponding to an i n t e r n a l p o r t i o n o f t h e 5' e x o n . T h e r e s u l t s s h o w n i n Fig. 5 m a y b e i n t e r p r e t e d i n t h e l i g h t o f t h e s e r e s u l t s . T h e m o x I p r o b e hybridizes to two b a n d s of m t D N A f r o m n o r m a l c y t o p l a s m s ( 7 . 6 a n d 3.9 k b p ) ; v e r y f a i n t h y b r i d i z a t i o n can be d e t e c t e d on the original a u t o r a d i o g r a m ( b u t n o t in t h e f i g u r e ) t o a t h i r d b a n d o f 13 k b p . T h e t w o b a n d s o f 19 a n d 3.9 k b p s h o win g the most in te n s e hybridization probably correspond to the cyto-
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Fig. 4. Identification of restriction fragments of mtDNA from male-sterile cytoplasms containing rRNA gene sequences. Labeled Sal I fragment T (B) was hybridized to a Southern blot (A) of Sal Idigested mtDNA from male-sterile (lanes 1,4), fertile (lanes 2,5), and male-fertile (restored) (lanes 3,6) wheat cytoplasms. Labeled mtRNA (C) was hybridized to a Southern blot of Sal I-digested mtDNA from male-sterile (lane 7) and fertile (lane 8) wheat cytoplasms.
c h r o m e o x i d a s e II gene separated on 2 fragm e n t s by t h e Sal I site. This site is a p p a r e n t l y c o n s e r v e d in t h e c y t o c h r o m e oxidase gene in male-sterile w h e a t m t D N A . T h e 5' e x o n a p p e a r s t o be l o c a t e d o n a Sal I f r a g m e n t of identical size in t h e t w o c y t o p l a s m s . The 3' end o f t h e gene is o n a f r a g m e n t o f larger size in male sterile w h e a t m t D N A . T h e b a n d with v e r y w e a k h y b r i d i z a t i o n (11 k b p ) c o u l d r e p r e s e n t the r e p e a t e d sequence.
1
2
3
Fig. 5. Identification of restriction fragments of mtDNA from male-sterile cytoplasms containing sequences homologous to the maize cytochrome oxidase II gene (pZmE1 clone). Labeled 2.4 kbp Eco RI fragment from pZmE1 recombinant plasmid was hybridized to a Southern blot of Sal I-digested mtDNA from restored (lane 1), male-sterile (lane 2) and fertile (lane 3) wheat cytoplasm.
Conclusion M i t o c h o n d r i a l D N A p o l y m e r a s e f r o m normal a n d male-sterile w h e a t c y t o p l a s m s are capable o f replicating t h e entire m i t o c h o n drial g e n o m e . N o episomes or preferential synthesis o f p a r t o f the g e n o m e or o f extrac h r o m o s o m a l e l e m e n t s c o u l d be detected.
149 M i t o c h o n d r i a l D N A s f r o m n o r m a l and male sterile w h e a t c y t o p l a s m s are largely h o m o logous and conserve the same sequence o r g a n i z a t i o n over nearly 60% o f the g e n o m e . H y b r i d i z a t i o n studies revealed differential h y b r i d i z a t i o n p a t t e r n s for a b o u t 40% o f the g e n o m e . These differences are unlikely t o be t h e result o f single m u t a t i o n a l events creating or abolishing a single restriction e n z y m e site. Detailed m a p p i n g studies o f r R N A genes f r o m fertile w h e a t m t D N A [6] as well as o u r h y b r i d i z a t i o n studies using w h e a t r R N A and c o r n c y t o c h r o m e oxidase II p r o b e s indicate t h a t d u p l i c a t i o n and r e a r r a n g e m e n t events are likely to a c c o u n t for m u c h o f the h y b r i d i z a t i o n differences d e t e c t e d . R e c e n t l y Isaac et al. [23] have s h o w n t h a t in mitoc h o n d r i a l D N A f r o m the male sterile c y t o plasm o f maize, the c y t o c h r o m e oxidase s u b u n i t ! gene is present o n several restriction f r a g m e n t s p r o b a b l y as t h e result o f rearrangem e n t s u p s t r e a m o f t h e c o d i n g region. App a r e n t l y m o s t o f these events do n o t lead t o f u n c t i o n a l differences in the m i t o c h o n d r i a . B o u t r y et al. [24] have studied the p r o t e i n s s y n t h e s i z e d in m i t o c h o n d r i a f r o m n o r m a l fertile lines o f T. aestivum and male-sterile lines c o n t a i n i n g T. timopheevi c y t o p l a s m . Many o f the p o l y p e p t i d e s were identical b u t one or t w o a d d i t i o n a l p o l y p e p t i d e s were f o u n d to be s y n t h e s i z e d in male-sterile c y t o p l a s m s . We have r e p e a t e d these experim e n t s and o b t a i n e d similar results. We are c o n t i n u i n g o u r studies of the s e q u e n c e o r g a n i z a t i o n of identified m t genes with the h o p e of correlating differential e~/pression with varying s e q u e n c e organizations. Acknowledgements The a u t h o r s are e x t r e m e l y grateful t o Drs. F. Quetier (Orsay) and S. Litvak ( B o r d e a u x ) for their c o n s t a n t s u p p o r t a n d
e n c o u r a g e m e n t . This w o r k was s u p p o r t e d b y t h e C N R S (ATP Biologic Mol~culaire V~g~tale 4 5 9 5 t o A.A. and S. Litvak) a n d t h e University o f B o r d e a u x II. References
1 D.C. Wallace, Mierobiol. Rev., 46 (1982) 208. 2 D.M. Lonsdale, T.P. Hodge and C.M.-R. Fauron, Plant Mol. Biol., 3 (1984) 201. 3 F. Qu~tier and F. V~del, Nature, 268 (1977) 365. 4 J.D. Palmer and C.R. Shields, Nature, 307 (1983) 437. 5 D.M. Lonsdale, Nucl. Acid Res., 12 (1984) 9249. 6 D. Falconet, B. Lejeune, F. Querier and M.W. Gray, EMBO J., 3 (1984) 297. 7 S.R. Gross, T. Hsieh and P.H. Levine, Cell, 38 (1984) 233. 8 R.M. Wright, M.A. Horrum and D.J. Cummings, Cell, 29 (1982) 505. 9 C.J. Leaver and M.W. Gray, Annu. Rev. Plant Physiol., 33 (1982) 373. 10 F. Vedel, F. Qu~tier, F. Dosba and G. Doussinault, Plant Sci. Lett., 13 (1978) 97. 11 M. Boutry and M. Briquet, Eur. J. Biochem., 127 (1982) 129. 12 L.K. Dixon and C.J. Leaver, Plant Mol. Biol., 1 (1982) 89. 13 A. Powling, Mol. Gem Genet., 183 (1981) 82. 14 J.D. Palmer, C.R. Shields, D.B. Cohen and T.J. Orton, Nature, 301 (1983) 725. 15 C.S. Levings, III, B.D. Kim, D.R. Pring, M.F. Conde, R.J. Mans, J.R. Laughnan and S.G. Gabay-Laughnan, Science, 209 (1980) 1021. 16 B. Ricard, M. Echeverria, L. Christophe and S. Litvak, Plant Mol. Biol., 2 (1983) 167. 17 E.M. Southern, J. Mol. Biol., 98 (1975) 503. 18 S. Delrome, DSc Thesis, No. 3540, University of Paris-Sud, Orsay, France, 1983. 19 U. Wintersberger and E. Wintersberger, Eur. J. Biochem., 13 (1970) 20." 20 D.B. Stern and J.D. Palmer, Nucl. Acids Res., 12 (1984) 6141. 21 L. Bonen, P.H. Boer and M.W. Gray, EMBO J., 3 (1984) 2531. 22 T.D. Fox and C.J. Leaver, Cell, 26 (1981) 315. 23 P.G. Isaac, V.P. Jones and C.J. Leaver, EMBO. J., 3 (1985) 1617. 24 M. Boutry, A. Faber, M. Charbonnier and M. Briquet, Plant Mol. Biol., 3 (1984) 445.
141
S T U D I E S ON W H E A T M I T O C H O N D R I A L D N A O R G A N I Z A T I O N . C O M P A R I S O N O F MITOCHONDRIAL DNA FROM NORMAL AND CYTOPLASMIC MALE STERILE V A R I E T I E S OF WHEAT
BERt~NICE RICARD *'a, BERNARD LEJEUNEb and ALEJANDRO ARAYA**~ Institut de Biochimie Cellulaire et Neurochimie du CNRS, 1 Rue Camille Saint Saens, 33077 Bordeaux Cedex and bLaboratoire de Biologie Moldculaire Vdgdtale, Universitd de Paris-Sud, Batiment 430, 91405 Orsay Cedex (France)
(Received July 29th, 1985) (Revision received December 5th, 1985) (Accepted December 5th, 1985) The mitochondrial genome of fertile, male-sterile and restored cytoplasm lines of wheat has been studied by means of recombinant DNA and hybridization techniques. Using cloned fragments of mitochondrial DNA (mtDNA) from fertile wheat cytoplasms as probes, about 40% of the genome is shown to have a differential hybridization pattern. The use of wheat rRNA and corn eytochrome oxidase subunit II probes indicates that duplication and rearrangement of genes or parts of genes may account for the differences observed. DNA synthesis in isolated mitochondria showed neither preferential labeling of part of the mtDNA nor the presence of extrachromosomal elements. Key words : wheat; mitochondriai DNA ; cytoplasmic male sterility
Introduction T h e m t D N A of higher plants is unusually variable in size, ranging b e t w e e n 2 0 0 kilo basepairs ( k b p ) and 2 5 0 0 k b p [ 1 , 2 ] . O n e striking feature is the h e t e r o g e n e o u s population of molecules isolated f r o m p l a n t mitoc h o n d r i a , as indicated by t h e c o m p l i c a t e d restriction endonuclease patterns obtained [3]. Studies o f t h e m o l e c u l a r o r g a n i z a t i o n of B r a s s i c a c a m p e s t r i s [4] and Z e a m a y s [5[ m t D N A have led to the idea t h a t m o s t m i t o c h o n d r i a l genes reside in a ' m a s t e r ' c h r o m o some and t h a t a subset o f m t D N A m o l e c u l e s arises f r o m r e c o m b i n a t i o n events. The pres* Present address: Laboratoire de physiologie v6g6tale. I.N.R.A. 33 Pont de la Maye. France. **To whom correspondence should be addressed. Abbreviations: CMS, cytoplasmic male sterility; COII, subunit II of cytochrome c oxidase; kbp, kilo base pairs; PVP, polyvinylpyrolidine; SDS, sodium dodecyl sulfate.
ence o f r e p e a t e d sequences in plant m t D N A s u p p o r t s this h y p o t h e s i s . T h a t t h e r R N A genes are f o u n d on f o u r restriction f r a g m e n t s in w h e a t m t D N A indicates t h a t such a m o d e l m a y also be applicable to w h e a t [6]. I n t r a m o l e c u l a r r e c o m b i n a t i o n as a source o f m t D N A h e t e r o m o r p h i s m has been described in f u n g u s [ 7 ] . A n o t h e r source o f heteromorphism c o u l d be a differential replication process, such as the a m p l i f i c a t i o n o f m i n i c h r o m o s o m e s in P o d o s p o r a a n s e r i n a [8]. T o u n d e r s t a n d the f u n c t i o n i n g o f plant m t D N A , an interesting a p p r o a c h c o u l d be t h e s t u d y o f m t D N A in a h e t e r o l o g o u s c o n t e x t . In w h e a t , c y t o p l a s m i c male sterility (CMS) results f r o m r e p e a t e d interspecific back-crossing, leading t o t h e association o f t h e nucleus o f t h e h e x a p l o i d , T r i t i c u m a e s t i v u m , with t h e c y t o p l a s m o f the tetraploid, T. t i m o p h e e v i . In a variety o f plants in a d d i t i o n t o corn, evidence is a c c u m u l a t i n g t h a t t h e genetic
0168-9452/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland
142 determinants that control CMS are carried by the mitochondrion and not the chloroplast [9]. CMS has been associated with differences in restriction enzyme patterns of mtDNA and identical patterns of cpDNA in wheat [3,10]. In male sterile cytoplasms of Vicia faba, sorghum, sugarbeet, Brassica and corn, episomal DNA can be isolated from mitochondria [11--15], although the available evidence does not suggest that episomes are correlated with CMS. In wheat, the mitochondria are not the sole determinants of CMS since the cytoplasm of T. timopheevi is male fertile in association with the nucleus of T. timopheevi but male sterile in association with the nuclei of T. aestivum. We have searched for episomal-like DNA as well as a modification of mtDNA polymerase which we speculated might be correlated with such DNA in mitochondria isolated from male sterile cytoplasms of wheat. In the second part of our study, we show that the mtDNA from the male sterile cytoplasms is homologous to that in normal cytoplasm of wheat, but organized differently. Such differences in organization may explain in part the incompatibility of T. timopheevi mitochondria with T. aestivum nuclei. Materials and methods
Wheat genetic stocks Seeds of alloplasmic fertile and male sterile lines of wheat (62.2.3 and 62.2.3.T) were kindly provided by P. Auriau (Station d'Am61ioration des Plantes de Versailles, France and P. B6rard (Station d'Am61ioration des Plantes de Clermont-Ferrand, France, who also supplied the fertile hybrid F1 (62.2.3.T × 149 Trf). The fertile hybrid or male-fertile (restored) has the cytoplasm T of T. timopheevi Zhuk of the male-sterile seed parent and the restorer genes of 149 Trf, the pollen parent. One hundred forty-nine Trf combines genes for the restoration of fertility from three different sources T. timopheevi and two T. aestivum varieties.
These restorer genes are present in all three wheat genomes.
Growth and harvesting conditions For etiolated seedlings, seeds were surfacesterilized for 20 min in i.5% Mucocit, washed and germinated for 7--10 days in the dark at 22°C in humid boxes. Isolation o f mitochondria and purification of mitochondrial DNA DNA was isolated from mitochondria prepared as described previously [16] according to the technique of Qu6tier and V6del [3] from etiolated wheat seedlings. Preparation o f mitochondrial extracts Preparation of mitochondrial lysates and synthesis of DNA in whole mitochondria from wheat embryos has been described previously [ 16]. Endonuclease digestion and agarose gel electrophoresis Mitochondrial DNA (0.4 pg) was digested with 4 units of the restriction enzyme Sal I (Genofit) in a 20-ul reaction mixture containing 10 mM Tris--HC1 (pH 7.5), 6 mM MgC12, 0.2 mM EDTA, 150 mM NaC1 for 4 h at 37°C. The reaction was stopped by adding 5 ul of stop buffer (50% glycerol, 0.1% bromphenol blue, 1.25% sodium dodecyl sulfate (SDS), 30 mM EDTA). DNA samples were then loaded on 0.4% agarose gels in 89 mM Tris--borate, 2 mM EDTA buffer containing 0.5 ug/ml of ethidium bromide. Electrophoresis was performed at 40 volts for 20 h. The gel was dried and autoradiography was performed or the DNA was transferred to nitrocellulose filters by the procedure of Southern [ 171. Cloning o f wheat mtDNA Partial or complete Sal I digests of fertile wheat mtDNA were cloned in pHC79 or pBR322 as described [18]. Cytochrome oxidase subunit II gene (mox I) was a generous gift of C.J. Leaver (Edinburgh).
143
Labeling and hybridization of cloned fragme n ts Approximately 1 pg of cloned m t D N A was added to a nick translation mixture which contained 50 mM Tris--HC1 (pH 7.2), 10 mM MgSO4, 0.1 mM dithiothreitol, 100 uM dATP, dGTP, dCTP and 20 uCi [a_32p]. TTP (Amersham France, 3000 Ci/mmol). Five units of DNA polymerase I were added and the reaction was allowed to proceed for 1 h at 15°C. Reaction was stopped by the addition of EDTA to 20 mM. After purification by gel filtration on Sephadex {G-50 medium), the DNA was heated at 100°C for 5 min, quick-cooled and added to hybridization buffer at a radioactive concentration of 106 cpm/ml. Hybridizations were carried out at 37°C for 40 h in 50% formamide, 3 × SSC (1 × SSC = 0.15 M NaC1, 0.015 M sodium citrate), 0.02% polyvinylpyrolidone (PVP)--Ficoll, 0.1% SDS, 1 mM EDTA, 50 mM sodium phosphate buffer (pH 6.5), 50 pg/ml denatured, sonicated salmon sperm DNA. After annealing, filters were washed at 37°C once in 3 × SSC, 0.02% PVP--Ficoll, 0.1% SDS, 1 mM EDTA, 50 mM sodium phosphate buffer (pH 6.5), twice in 50% formamide, 3 × SSC, 0.1% SDS, 1 mM EDTA, twice in 3 ×SSC, 0.1% SDS and twice in 0.1 ×SSC, 0.1% SDS. Filters were dried and autoradiography was performed. Reverse transcription of total mtRNA Total RNA was prepared by two successive phenol/chloroform (1 : 1) extractions of mitochondria lyzed 30 min at r o o m temperature with sodium N-lauryl sarcosine (0.2%, w/v) and 200 ug/ml of proteinase K. Ten micrograms of total RNA was incubated for 1 h at 37°C in a 50-~1 reaction mixture containing 25 mM Tris--HC1 (pH 8.3), 6 mM MgC12, 8 mM dithiothreitol, 200 t~M dATP, dGTP, TTP, 75 uCi [a-32P]dCTP (3000 Ci/mmol), 500 t~g/ml calf thymus random primer DNA, 20 ug/ml actinomycin D, 17 units of AMV reverse transcriptase {Life Sciences). The reaction was stopped with 5 ~l of 0.1 M
EDTA and 3 ul of 5% SDS. RNA templates were removed by adding NaOH to 0.3 M and incubating overnight at 37°C. After neutralization, the labeled cDNA was separated from unincorporated nucle~tides by chromatography through a Sephadex G-100 column equilibrated with 10 mM Tris--HC1 (pH 9), 100 mM NaC1, 1 mM EDTA. Hybridization conditions were the same as described above for DNA labeled by nick translation. Results arid discussion
Replication of DNA in mitochondria from male sterile and fertile cytoplasms Mitochondria from both male sterile and fertile cytoplasms are capable of incorporating TTP into the entire mitochondrial genome. A comparison of Fig. 1B and C shows that the Sal I restriction enzyme profile of DNA labeled in organello {Fig. 1B) is indistinguishable from that of unlabeled mtDNA (Fig. 1C). In particular, the variable intensities of restriction fragments are reproduced, indicating that the entire genome is involved in DNA synthesis. No preferential synthesis of particular fragments is evident, as has been shown to occur with episomal elements in corn {V. Walbot, pers. commun.). This is supported by the autoradiograph of undigested mtDNA synthesized in organello (Fig. 1A): only chromosomal DNA is labeled. Likewise, no episomal elements were found, either by analysis of unlabeled mtDNA or by labeling of mtDNA in organello. The mitochondrial DNA polymerases from male sterile and fertile cytoplasms of alloplasmic lines were isolated as described previously [16]. No differences were evident in the template specificities, cation activation profiles, and inhibitor sensitivities. The specific activities correlated with the percentage of embryos capable of germination and not with type of cytoplasm (male sterile or fertile) {data not shown). These results are not surprising if we assume a nuclear
144
B
A
C
¢ ,G ,H ~i: ¸ ~"
i
"
"
.K ,L =
i
M
0 Q S
U V W
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1
2
3
4
5
6
7
Fig. 1. A, B; Autoradiogram of 32P-labeled DNA synthesized in mitochondria isolated from malesterile (lanes 1, 3) and fertile (lanes 2, 4) cytoplasms of wheat without digestion (A) and following digestion (B) with restriction enzyme Sal I. The lettering of fragments in fertile m t D N A of wheat is that of Lejeune and Qu~tier (unpublished data). (C) restriction enzyme analysis of mtDNA isolated from fertile
cytoplasms of T. aestivurn (lane 5), male-sterile T. aestivum (cytoplasm T. tirnopheevi) (lane 6) and restored male-fertile T. aestivum (cytoplasm T. timopheevi) (lane 7). origin of mtDNA polymerases, as has been shown in the case of yeast [19]. Organization o f m t D N A from normal and male sterile cytoplasms o f wheat When Sal I restriction enzyme fragments
of mtDNA from normal T. aestivum cytoplasm are fractionated by 0.4% agarose gel electrophoresis, the pattern obtained is complex but characteristic. It is easily distinguished from that of mtDNA from male sterile cytoplasms, even though the majority of the fragments have similar mobilities (Fig. 1C). Approximately 33 Sal I fragments can be visualized by EtBr staining. Twentynine appear to be shared by the two types of mtDNA. T h e Sal I restriction profiles of male sterile cytoplasm is indistinguishable whether in association with T. timopheevi or T. aestivum nuclei, in the presence or absence of the genes for the restoration of fertility (Ref. 10, Fig. 1). These results suggest that no apparent alterations at the mtDNA level occur as a consequence of the introduction of a T. aestivum nucleus to a T. timopheevi cytoplasm. In T. timopheevi induced male sterility, mtDNA alterations due to cytoplasmic mutations cannot be invoked to explain male sterility. It seems more probable that the defect lies at the level of nucleo-cytoplasmic interactions. These interactions may be affected by a change in the molecular organization of certain genes, perhaps at the level of leader sequences involved in transport, initiation/termination sites, precursor/ product relationships of RNA transcripts or time sequence of transcription. It therefore seemed interesting to us to analyze the region of the mitochondrial genome which differs in both cytoplasms. It is at these regions that potential defects in nucleo-cytoplasmic interactions m a y originate. We sought first to evaluate the degree of similarity between the two mt genomes and second, to identify that part of the mt genome which was different. This was done using cloned fragments of mtDNA from fertile wheat cytoplasms. The cloned fragments ranged in size from 0.85 to 29 kbp and summed to a total of 170 kbp. This corresponds to 39% of the estimated total molecular weight of the wheat mt genome {unit genome size estimated to be 430 kbp, B. Lejeune and F. Qu~tier, pers.
145
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qb
B
C
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Cloned fragments with identical hybridization patterns in mtDNA from fertile and male sterile cytoplasms. Eleven cloned fragments, summing to 71 kbp, hybridized with a single band of similar mobility in m t D N A from fertile and male sterile cytoplasms {Fig. 2). This result shows that no major homology differences exist in these mitochondrial fragments and that they have been conserved in the two cytoplasms. In the case of clone H2 (Fig. 2A), hybridization appears less intense with the homologous normal mtDNA of fertile wheat than with the mtDNA of male sterile cytoplasms. This seems at first surprising since the restriction profiles indicate that band H is of comparable intensities in the three mt genomes. According to Qu~tier et al., however (pers. commun.), band H is composed of two fragments of identical size (clone H1 and H2) in fertile wheat mtDNA, so that it is possible that H2 is present in different amounts in the m t D N A from male sterile and fertile cytoplasms. Cloned fragments hybridizing differently in mtDNA from fertile and male-sterile cytoplasms. Six cloned fragments, summing to 84 kbp, hybridized with a band of similar mobility b u t also to different bands in m t D N A from fertile and male sterile cytoplasms (Fig. 3A). This result shows redundancy of at least a part of the cloned fragment. One of the fragments is conserved in the two cytoplasms, while the other may be located in a different chromosomal environment. Three cloned fragments, summing to 21 kbp, hybridized with a single band of different mobilities in m t D N A from fertile and male-sterile cytoplasms (Fig. 3C). In each case, hybridization with the m t D N A from male-sterile cytoplasms was weak, suggesting that these fragments are not present in equimolar amounts. One cloned fragment, of 22.7 kbp, hybridized with its band of origin in fertile wheat m t D N A and with t w o bands of different mobilities in malesterile wheat mtDNA. It is difficult to explain I
1
2
3
4
5
6
7
8
9
Fig. 2. Hybridization of cloned and labeled Sal I fragments H2 (A) and X1, Y, AB (C) to a Southern transfer (B) of Sal I-digested mtDNA male-sterile (lane 4), fertile (lane 5), and male-fertile (restored) (lane 6) wheat cytoplasms.
commun.). Every clone hybridized with its fragment of origin in fertile wheat mtDNA and with either the same or a different fragment in male sterile mtDNA. None of the clones studied hybridized differently to male fertile (restored) m t D N A as compared to male sterile mtDNA. Thus, our studies so far do not permit us to distinguish any differences in the m t D N A of male fertile (restored) and male sterile cytoplasms.
146
B
A
C
Recent sequence data for different plant mtDNA genes has shown that the coding regions are highly conserved [21,22]. It is interesting to speculate that the differences observed in wheat mtDNA may occur in non-coding or control regions of the mitochondrial genome.
W
O
1
2
3
4
5
6
7
8
9
Fig. 3. Hybridization of cloned ai~d labeled Sal I fragments O (A) and K2 (C) to a Southern transfer (B) o f DNA from mitochondria o f male-sterile (lane 4), fertile (lane 5), and male-fertile (restored) (lane 6) wheat cytoplasms.
these differences as involving a sequence change in a single Sal I cleavage site. To explain the complex patterns, one could evoke multiple mutational events that create and/or abolish several restriction recognition sites. Alternatively, these patterns could be explained by sequence rearrangements. It has been shown that most plant mitochondrial genomes contain repeated sequences. The precise role of these potential recombination regions, that are different in different species, is n o t clear [20]. A summary of the hybridization results is presented in Table I. Of 169 kbp of mtDNA analyzed (the sum of the molecular weights of the cloned fragments), nearly 100 kbp showed an identical pattern of hybridization in the two cytoplasms, while 69 kbp displayed a different pattern.
Organization o f r R N A genes and the cytochrome oxidase H gone in m t D N A from normal and male sterile cytoplasms Falconet et al. [6] have shown that in normal wheat mtDNA, the 18--5 S rRNA genes are located on four Sal I fragments: F2, G2, R1, T (corresponding to fragments 5a, 5b, 19, 21 in their notation). Sal I fragm e n t T, 5.5 kbp, contains the 18--5 S rRNA genes in a structural unit at least 4 kbp long. This probe was used to detect the rRNA genes on mtDNA from male sterile cytoplasms. Figure 4, lane 5, confirms Falconet's results: fragment T hybridizes to three bands in the Sal I digest of fertile mtDNA (FG, R, and T). On our gels, bands F and G are not resolved. Fragment T also hybridizes to bands R and T in male sterile mtDNA but, in addition, to three other bands (Fig. 4, lanes 4 and 6). Likewise fragm e n t M, used to detect the 26 S rRNA gone on Sal I fragments o f mtDNA from malesterile cytoplasms, co-hybridizes to band M in both cytoplasms but also to different bands. In order to confirm that the multiple hybridization is indeed due to the presence of multiple rRNA genes, we synthesized labeled cDNA from total RNA by reverse transcription, as described in Materials and methods. Ribosomal RNA comprises the bulk of total RNA so that the probe prepared detects essentially ribosomal RNA genes. Figure 4, lane 8, shows that the rRNA probe hybridizes to the same five bands as fragments M and T in the Sal I digest of fertile wheat mtDNA. In male sterile wheat mtDNA seven Sal I bands were homologous to the rRNA probe; these were the same as those detected by fragments M and T.
147 Table I.
Summary of hybridization experiments.
A. Cloned fragments with identical hybridization patterns in Sal I restricted m t D N A from male-sterile and fertile cytoplasms o f wheat
Cloned fragment used as probe
kbp
Hybridization in Sal I digest of fertile wheat mtDNA to band :
Hybridization to co-migrating band in Sal I digest of male sterile wheat mtDNA
C3 H: L~ U: W X~ Y AA AB AC AE/AF
24 16.8 9.8 4.6 3.6 3.2 3.1 1.8 1.7 1.5 0.85
C H L U W X Y AA AB AC AE/AF
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
B. Cloned fragments with different hybridization patterns in Sal I restricted m t D N A from male sterile and fertile cytoplasms o f wheat
Cloned fragment used as probe
kbp
Hybridization in Sal I digest of fertile wheat mtDNA to band:
Hybridization to comigrating band in Sal I digest of male sterile wheat mtDNA: number of non-co-migrating bands
kbp of comigrating band(s)
A D, K~ K: O R: T X2
29 22.7 11.7 11.7 7.6 6.2 5.5 3.2
A + C D K ~ K O + R F +G X
Yes (Q):2 No : 2 Yes (M}:I No :1 Yes ( Z ) : I No :1 Yes(R + T ) : 3 No :1
6.4
~ Q M Z + R + T
The mox I clone corresponding to subunit II o f c y t o c h r o m e c o x i d a s e ( C O I I ) f r o m c o r n mitochondria [ 2 2 ] was u s e d t o i d e n t i f y homologous s e q u e n c e s in m t D N A from male-sterile a n d -fertile cytoplasms. Figure 5 shows that mox I hybridizes to an identical b a n d o f 3.9 k b p i n b o t h c y t o p l a s m s b u t also t o d i f f e r e n t b a n d s . R e c e n t l y B o n e n e t al. [21] have published the complete sequence o f t h e w h e a t c y t o c h r o m e o x i d a s e s u b u n i t II g e n e . T h e g e n e c o n t a i n s a Sal I site i n t h e i n t r o n v e r y n e a r t h e 3' e x o n . B o n e n e t al.
8.6 2.3 6.2 4- 5.5
[211 also d e m o n s t r a t e d t h e p r e s e n c e o f a repeated sequence corresponding to an i n t e r n a l p o r t i o n o f t h e 5' e x o n . T h e r e s u l t s s h o w n i n Fig. 5 m a y b e i n t e r p r e t e d i n t h e l i g h t o f t h e s e r e s u l t s . T h e m o x I p r o b e hybridizes to two b a n d s of m t D N A f r o m n o r m a l c y t o p l a s m s ( 7 . 6 a n d 3.9 k b p ) ; v e r y f a i n t h y b r i d i z a t i o n can be d e t e c t e d on the original a u t o r a d i o g r a m ( b u t n o t in t h e f i g u r e ) t o a t h i r d b a n d o f 13 k b p . T h e t w o b a n d s o f 19 a n d 3.9 k b p s h o win g the most in te n s e hybridization probably correspond to the cyto-
148
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4
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7
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8
Fig. 4. Identification of restriction fragments of mtDNA from male-sterile cytoplasms containing rRNA gene sequences. Labeled Sal I fragment T (B) was hybridized to a Southern blot (A) of Sal Idigested mtDNA from male-sterile (lanes 1,4), fertile (lanes 2,5), and male-fertile (restored) (lanes 3,6) wheat cytoplasms. Labeled mtRNA (C) was hybridized to a Southern blot of Sal I-digested mtDNA from male-sterile (lane 7) and fertile (lane 8) wheat cytoplasms.
c h r o m e o x i d a s e II gene separated on 2 fragm e n t s by t h e Sal I site. This site is a p p a r e n t l y c o n s e r v e d in t h e c y t o c h r o m e oxidase gene in male-sterile w h e a t m t D N A . T h e 5' e x o n a p p e a r s t o be l o c a t e d o n a Sal I f r a g m e n t of identical size in t h e t w o c y t o p l a s m s . The 3' end o f t h e gene is o n a f r a g m e n t o f larger size in male sterile w h e a t m t D N A . T h e b a n d with v e r y w e a k h y b r i d i z a t i o n (11 k b p ) c o u l d r e p r e s e n t the r e p e a t e d sequence.
1
2
3
Fig. 5. Identification of restriction fragments of mtDNA from male-sterile cytoplasms containing sequences homologous to the maize cytochrome oxidase II gene (pZmE1 clone). Labeled 2.4 kbp Eco RI fragment from pZmE1 recombinant plasmid was hybridized to a Southern blot of Sal I-digested mtDNA from restored (lane 1), male-sterile (lane 2) and fertile (lane 3) wheat cytoplasm.
Conclusion M i t o c h o n d r i a l D N A p o l y m e r a s e f r o m normal a n d male-sterile w h e a t c y t o p l a s m s are capable o f replicating t h e entire m i t o c h o n drial g e n o m e . N o episomes or preferential synthesis o f p a r t o f the g e n o m e or o f extrac h r o m o s o m a l e l e m e n t s c o u l d be detected.
149 M i t o c h o n d r i a l D N A s f r o m n o r m a l and male sterile w h e a t c y t o p l a s m s are largely h o m o logous and conserve the same sequence o r g a n i z a t i o n over nearly 60% o f the g e n o m e . H y b r i d i z a t i o n studies revealed differential h y b r i d i z a t i o n p a t t e r n s for a b o u t 40% o f the g e n o m e . These differences are unlikely t o be t h e result o f single m u t a t i o n a l events creating or abolishing a single restriction e n z y m e site. Detailed m a p p i n g studies o f r R N A genes f r o m fertile w h e a t m t D N A [6] as well as o u r h y b r i d i z a t i o n studies using w h e a t r R N A and c o r n c y t o c h r o m e oxidase II p r o b e s indicate t h a t d u p l i c a t i o n and r e a r r a n g e m e n t events are likely to a c c o u n t for m u c h o f the h y b r i d i z a t i o n differences d e t e c t e d . R e c e n t l y Isaac et al. [23] have s h o w n t h a t in mitoc h o n d r i a l D N A f r o m the male sterile c y t o plasm o f maize, the c y t o c h r o m e oxidase s u b u n i t ! gene is present o n several restriction f r a g m e n t s p r o b a b l y as t h e result o f rearrangem e n t s u p s t r e a m o f t h e c o d i n g region. App a r e n t l y m o s t o f these events do n o t lead t o f u n c t i o n a l differences in the m i t o c h o n d r i a . B o u t r y et al. [24] have studied the p r o t e i n s s y n t h e s i z e d in m i t o c h o n d r i a f r o m n o r m a l fertile lines o f T. aestivum and male-sterile lines c o n t a i n i n g T. timopheevi c y t o p l a s m . Many o f the p o l y p e p t i d e s were identical b u t one or t w o a d d i t i o n a l p o l y p e p t i d e s were f o u n d to be s y n t h e s i z e d in male-sterile c y t o p l a s m s . We have r e p e a t e d these experim e n t s and o b t a i n e d similar results. We are c o n t i n u i n g o u r studies of the s e q u e n c e o r g a n i z a t i o n of identified m t genes with the h o p e of correlating differential e~/pression with varying s e q u e n c e organizations. Acknowledgements The a u t h o r s are e x t r e m e l y grateful t o Drs. F. Quetier (Orsay) and S. Litvak ( B o r d e a u x ) for their c o n s t a n t s u p p o r t a n d
e n c o u r a g e m e n t . This w o r k was s u p p o r t e d b y t h e C N R S (ATP Biologic Mol~culaire V~g~tale 4 5 9 5 t o A.A. and S. Litvak) a n d t h e University o f B o r d e a u x II. References
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