Expression levels of meristem identity and homeotic genes are modified by nuclear–mitochondrial

Expression levels of meristem identity and homeotic genes are modified by nuclear–mitochondrial interactions in alloplasmic male-sterile lines of Brassica napusRita Teresa Teixeira,Isabelle Farbos and Kristina Glimelius*Department of Plant Biology and Forest Genetics,Swedish University of Agricultural Sciences,Box7080,SE-75007Uppsala, SwedenReceived1November2004;revised17February2005;accepted7March2005.*For correspondence(faxþ4618673279;e-mail kristina.glimelius@vbsg.slu.se).SummaryHomeotic conversions of anthers were found in cytoplasmic male sterile(CMS)plants of Brassica napusderived from somatic hybrids of B.napus and Arabidopsis thaliana.CMS lineflowers displayed petals reducedin size and width and stamens replaced by carpelloid structures.In order to investigate when thesedevelopmental aberrations appeared,flower development was analysed histologically,ultrastructurally andmolecularly.Disorganized cell divisions were detected in thefloral meristems of the CMS lines at stage4.AsCMS is associated with mitochondrial aberrations,ultrastructural analysis of the mitochondria in thefloralmeristems was performed.Two mitochondrial populations were found in the CMS lines.One type haddisrupted cristae,while the other resembled mitochondria typical of B.napus.Furthermore,expressionpatterns of genes expressed in particularfloral whorls were determined.In spite of the aberrant developmentof the third whorl organs,BnAP3was expressed as in B.napus during thefirst six stages of development.However,the levels of BnPI were reduced.At later developmental stages,the expression of both BnAP3andBnPI was strongly reduced.Interestingly the expression levels of genes responsible for AP3and PI activationsuch as LFY,UFO and ASK1were higher in the CMS lines,which indicates that activation of B-genes in the CMSlines does not occur as in B.napus.Disrupted and dysfunctional mitochondria seem to be one of thefirstaberrations manifested in CMS which result in a retrograde influence of the expression levels of genesresponsible for the second and third whorl organ differentiation.Keywords:Arabidopsis thaliana,Brassica napus,cytoplasmic male sterility,homeotic conversion.IntroductionFloral organogenesis is influenced by nuclear–mitochond-rial interactions as most notably demonstrated by cyto-plasmic male sterility(CMS),which is a mitochondrially inherited trait manifested by failure to produce pollen (reviewed by Budar and Pelletier,2001;Schnable and Wise,1998).Besides the absence of viable pollen,abnor-mal development of anthers and otherfloral organs is often observed in CMS plants.For example,homeotic conversion of stamens into carpelloid structures has been described for CMS lines of rapeseed(Leino et al.,2003), tobacco(Farbos et al.,2001;Kofer et al.,1991),carrot (Linke et al.,2003)and wheat(Murai et al.,2002).These phenotypes resemble those of Arabidopsisfloral homeotic gene mutants APETALA3(AP3)and PISTILLATA(PI),in which stamens are replaced by carpelloid organs in the third whorl.However,in these mutants the petals are also converted to sepal-like organs in whorl2(Bowman et al., 1989;Jack et al.,1992,1994).Although petals in CMS plants retain their identity,they can be reduced in size and changed in colour(Farbos et al.,2001;Leino et al.,2003). In this respect,flowers formed in most CMS plants resemble weak double mutant plants for the UNUSUAL FLORAL ORGANS gene(UFO)and one of the SCF UFO complex genes such as the Arabidopsis SKP1-LIKE(ASK1) gene or the AtCUL1gene in which anthers are replaced by carpelloid orfilamentous structures,and petals are often reduced in size(Ni et al.,2004;Wang et al.,2003;Zhao et al.,1999).ª2005Blackwell Publishing Ltd731 The Plant Journal(2005)42,731–742doi:10.1111/j.1365-313X.2005.02407.xThe identity of organs in whorl three is dependent upon the correct expression of AP3,PI and AGAMOUS(AG) (Bowman et al.,1991a;Coen and Meyerowitz,1991).More recently,the action of other genes and co-factors was shown to be necessary for correct AP3and PI activation(Jack,2004; Lamb et al.,2002).The expression of AP3and PI has two distinct phases.Initially,during thefirst sixfloral stages, AP3/PI activation is directly and indirectly dependent on the activity of the meristem identity genes LFY and AP1(Lamb et al.,2002;Ng and Yanofsky,2001;Weigel and Meyerowitz, 1993).After stage6,when sepals completely enclose the floral bud(Smyth et al.,1990),AP3and PI activation is facilitated by an autoregulatory pathway that is dependent on the relative concentrations of AP3and PI proteins(Hill et al.,1998;Jack et al.,1992,1994).In this later phase,when AP3and PI mainly are involved in the regulation of organ growth in whorls2and3,the expression of AP3and PI is also regulated by UFO and ASK1(Laufs et al.,2003;Samach et al.,1999;Zhao et al.,2001)by interacting genetically with LEAFY(LFY),a direct activator of AP3and PI(Samach et al., 1999;Wang et al.,2003).Precise control of cell division is important for normal floral organogenesis.Floral organ initiation results from patterned control of the numbers,loci,and planes of cell division,in accordance with the regulation of cell expansion (Meyerowitz,1997).In the CMS lines of Brassica napus and Nicotiana tabacum,the second and third whorl organs are often smaller due to fewer cells(Farbos et al.,2001;Leino et al.,2003).In addition,modified cell division patterning in the third whorl organ primordia was reported(Farbos et al., 2001)similar to modifications in the size of the organ primordia(Linke et al.,2003).Organ identity genes are not required for the initiation offloral organogenesis.They rather alter the identity of organs but do not perturb the number or position offloral primordia within the whorl. The CMS B.napus lines included in this study have the nuclear genome of B.napus but mitochondria with DNA derived mainly from Arabidopsis thaliana(Leino et al., 2003).These lines are characterized by homeotic conver-sions of stamens into carpelloid structures and smaller petals.These phenotypic aberrations suggest that the genes responsible for anther identity and formation are misregulated.Thus,genes known to be upstream of the B-genes AP3and PI such as LFY,UFO and ASK1,as well as the B-genes and AG gene were analysed by determining their levels and patterns of expression.The results revealed that the expression patterns of all the genes in the CMS lines were analogous to those in B.napus, although the levels of AP3and PI decreased drastically after stage6in the CMS lines.In contrast,the levels of LFY and UFO transcripts in the CMS lines increased at these laterfloral stages in comparison with B.napus.Structural alterations were observed in the mitochondria very early in flower development and seem to be one of thefirst phenotypic manifestations of the rearranged mtDNA between B.napus and A.thaliana.ResultsFloral developmentAllflowers in the CMS lines4:19and41:17investigated in this study were phenotypically similar to each other.For morphological overviews we refer to Leino et al.(2003). Stamens in B.napus were composed of afilament and an anther(Figure1a).In contrast,third whorlfloral organs in the CMS lines resembled an unfused carpel with ovule-like structures appearing at the internal margins of the organ (Figure1b,c).The tip of these organs was covered with stigmatic-like cells(Figure1b).The stages offloral development in B.napus that were determined by Polowick and Sawhney(1986)resembled the stages defined by Smyth et al.(1990)for A.thaliana.We used the stage definitions by Smyth et al.(1990).By scanning electron microscopy(SEM)studies we found thatfloral bud phyllotaxis did not differ between the CMS plants and B.napus(Figure1d–f).No differences were found between B.napus and the two CMS lines until stage4 (Figure1g–i).The size of thefloral meristem enclosed between the lateral sepals was the same(98–100l m)in both B.napus and the two CMS lines(Figure1g–i).However, by stage9,when allflower organs were established, including thefinal shape of the anthers,phenotypic differ-ences betweenflowers of the CMS lines and B.napus were detected.Even though the length of thefloral buds of B.napus and the two CMS lines was similar throughout flower development(Figure1j–l),the petals of the CMS lines were narrower and shorter compared with those of B.napus (Figure1j).By stage9when the anthers in B.napus reach the stigma,the CMS lines displayed carpelloid organs with ovule-like structures differentiating at the margins (Figure1r),instead of anthers.Furthermore,the petals were narrower and shorter than in B.napus(Figure1j)and at stage12,the CMS petals had a curly appearance (Figure1k,q,r).Even though whorl2and3organs were morphologically smaller and different in the CMS plants,the overallfloral bud length was the same because both the pistil and the sepals were of the same size and shape as in B.napus(Figure1j–l)(Leino et al.,2003).Despite the fact that the carpelloid structures are not stamens,their arrangement in theflowers was the same as the arrangement of stamens in B.napus,that is four upper and two lower(Figure1m–o,s–u)and nectaries were present at their bases.The spatial distribution of all otherfloral organs inflowers of the CMS lines was similar to that found in B.napus.Additional phenotypic changes were found in CMS flowers arising later in the same inflorescence.The CMS732Rita Teresa Teixeira et al.ªBlackwell Publishing Ltd,The Plant Journal,(2005),42,731–742Nuclear–mitochondrial interactions duringflower development in B.napus733Figure1.Phenotypic characterization offlower development in Brassica napus and the two CMS lines.(a)Fully developed B.napus stamen.(b)Third whorl organ of the CMS line4:19.(c)Magnification of the ovule-like structure formed at the unfused margin of the carpel-like organ.(d–f)Inflorescences showing the phyllotaxis. Numbers relate the sequence offloral bud appearance in(d)B.napus,(e)CMS line4:19,(f)CMS line41:17.(g–i)Size of thefloral meristem enclosed between the lateral sepals of(g)B.napus,(h)CMS line4:19and(i)CMS line41:17.(j)Flower buds at stage9.From left to right:B.napus,CMS line4:19,CMS line41:17.(k)Flower buds at stage12(petals are as long as the length of the lower stamen).From left to right:B.napus,CMS line4:19,CMS line41:17.(l)Flower buds at stage12.Two sepals and two petals were removed.From left to right:B.napus,CMS line4:19,CMS line41:17.(m)Openflower of B.napus.(n)Openflower of the CMS line4:19.(o) Openflower of the CMS line41:17.Sepals were removed in(n)and(o).Nectaries are indicated by arrows in the base of the third whorl organs.SEM visualization of flower buds and petal epidermal surfaces.(p)Flower bud of B.napus at stages9and10.At stage12,petals completely cover the anthers,therefore,in order to visualize anthers,petals and sepals,flower buds at stage10were chosen for B.napus.(q)Flower bud of the CMS line4:19at stage12.(r)Flower bud of the CMS line 41:17at stage12.Ovule-like structures appear at the margin of the third whorl organs(arrow).Detailed view of the base of theflower buds with the nectaries indicated by arrows:(s)B.napus,(t)CMS line4:19and(u)CMS line41:17.Adaxial surfaces of petals:(v)B.napus and(w)CMS line4:19.Abaxial surface in petal:(x) B.napus and(y)CMS line4:19.Bar(b,j–l):1mm,(m–o)2mm(s–u):100l m.an,anther;cl,carpelloid structure;fl,filament;ls,lower sepal;ol,ovule-like structure;p, petal;pi,pistil;s,sepal;sl,stigmatic-like cell;S4,floral stage4;us,upper sepal.ªBlackwell Publishing Ltd,The Plant Journal,(2005),42,731–742line4:19developed5.8Æ0.5(n¼30)carpelloid structures, while B.napus displayed a constant number of stamens (6.0Æ0.0,n¼30).Furthermore,in CMS line41:17,the carpelloid structures had a more curly feature(Figure1o) than those formed in earlierflowers(Figure1r).Scanning electron microscopy analysis of B.napus petal surfaces revealed that the adaxial cells are conical in shape(Figure1v),while the abaxial cells are more dome shaped(Figure1x).Within each surface,all cells were uniform in size and shape and all revealed epicuticular ridges.In the CMS petals,on the contrary,the polarity between the adaxial and the abaxial side was not noticeable and instead,the two epidermal surfaces resembled each other with a tendency for abaxialization of the adaxial side(Figure1w,y).On both surfaces of the CMS petals,cells of different sizes were found(Fig-ure1w,y).Petals in the CMS lines were smaller as a result of the reduced number of cells.In conclusion,the phenotypic analysis throughoutflower development showed that CMS lines displayfloral alterations only in the second and third whorls.Histological and ultrastructural analyses of the earlyfloral meristemIn the two CMS lines investigated in this study,the second and third whorl organs were reduced in size,a feature also found in CMS lines of tobacco(Farbos et al.,2001)and in carrot CMS plants(Linke et al.,2003).To determine if this was due to aberrant cell division,we performed studies of cell division patterns in earlyfloral meristems.Disorganiza-tion in the planes of cell division was found in youngfloral buds(stages4and5)at the sites of the putativefloral whorls 2and3from which the petals and stamens will arise.Thus, the well established L1–L3meristematic cell layers typical for B.napus(Figure2a,b)were not truly maintained in the two CMS lines(Figure2c–f).Another feature was the re-duced cytoplasmic density due to higher numbers of small vacuoles(Figure2a–f).These early alterations indicate that the CMS condition affects mechanisms that regulate the cell patterning in areas of theflower meristem that later on will give rise to second and third whorl organs. Observations of young developing anthers in thin cross sections revealed that in B.napus,anther compartments started to differentiate with archesporial cells acquiring their identity at stages7and8(Figure2g,h),as shown by the density of the cytoplasm and the large nucleus present in the cells(Figure2h).The archesporial cells that originated from the L2layer were surrounded by more vacuolated cells that give rise to the epidermis of the anther(Figure2h).In contrast to true anther primordia,the carpelloid primordia formed in the CMS lines were not as wide and the cells were uniform throughout the organ(Figure2i,j). Later infloral development,all different tissues that constitute the anthers in B.napus had differentiated and the two carpels were formed(data not shown).However, in the CMS lines,third whorl organs mainly elongated and the only further tissue differentiation was the forma-tion of ovule-like structures at the margins of the organ followed later on by the development of stigmatic cells at the tip of the organs(Figures1b and2i–l).These histo-logical studies reveal that CMS lines are incapable of sporogenous tissue differentiation followed by the home-otic conversion of male organs into carpelloid tissue. Such phenotypic alterations in the CMS lines are closely related to the fact that CMS-associated novel mitochond-rial genes accumulate more in sporogenous initial cells as has been the case in CMS sunflower plants(Smart et al., 1994).In situ hybridization of B.napus(nap)CMS lines revealed that transcripts from the CMS-associated mito-chondrial orf were preferentially localized to microsporan-gia(Geddy et al.,2005).As the CMS trait is regulated by the mitochondria,it was of interest to investigate the mitochondrial structures in floral tissues.Ultrastructural studies revealed that mito-chondria in B.napusflower buds at stages4and6had a large number of mitochondria that displayed well-develo-ped cristae(Figure3a,d).This is in contrast to the two types of mitochondria found in the CMS cells of the putative whorls2and3and of the carpelloid organ primordia.One type resembled B.napus mitochondria,although they were reduced in size and in number,while the other type lacked the cristae and the dense matrix(Figure3b,c,e,f).In some of the mitochondria in the CMS lines,the outer membrane was ruptured(Figure3e).The disrupted assembly of the inner membrane system present in the CMS mitochondria could reflect the presence of recombined mtDNA in the CMS lines (Leino et al.,2003).Transcript levels determined by real-time RT-PCRPhenotypically,flower development in the CMS plants resembles that in its B.napus counterpart until stage6/7is reached but by the time third whorl organs begin to differ-entiate,homeotic modifications appear.We therefore determined the expression levels of several relevant genes such as BnLFY,BnUFO,BnASK,BnAG,BnAP3and BnPI.All these genes are known for their involvement in petal and stamen formation(Bowman et al.,1991a;Coen and Mey-erowitz,1991;Zhao et al.,2001).Because of alterations related to cell division in second and third whorlfloral organs(Figure1)and cell patterning inflower meristems (Figure2)of the CMS lines,transcription levels of BnTON1 and BnCycB1were also analysed as both genes are mitosis-related(Capron et al.,2003;Torres-Ruiz and Ju¨rgens,1994). We performed expression level studies using two inde-pendent samples,one representingfloral stages from1to6 and one representing stages7–9.734Rita Teresa Teixeira et al.ªBlackwell Publishing Ltd,The Plant Journal,(2005),42,731–742Stages1–6The genes BnLFY,BnUFO and BnASK formed group I,as they act upstream of the homeotic genes BnAG,BnAP3and BnPI that formed group II.Their expression levels were analysed in the CMS lines in comparison with B.napus during thefirst six stages offlower development.ASK1and UFO,together with other components,form the SCF UFO complex that interacts genetically with LFY and regulate AP3 and PI gene expression(Samach et al.,1999;Wang et al., 2003).Interestingly,the CMS line4:19revealed significantly higher transcript levels of these three genes than did B.na-pusflowers of stages1–6(Figure4a).Of the group II genes,the expression levels of BnAG showed no significant difference between B.napus and the CMS lines(Figure4b).This was also the case for BnAP3in stages1–6,while the expression was significantly reduced in floral bud stages7–9(Figure4b).The expression levels of BnPI mRNA in the CMS lines were significantly reduced compared with the levels of B.napusfloral buds in all stages (Figure4b).Histological studies of youngflower meristems revealed alterations in the plane and order of cell divisions (Figure2a–f).We therefore tested the transcription levels of two mitosis-related BnTON1and BnCycB1genes(group III).Their expression levels were higher in CMS line4:19 compared with B.napus,while for line41:17,the level was only slightly higher for BnTON1,whereas no statistical difference was found for either of the genes when compared with B.napus(Figure 4c).Figure2.Histological studies offlower development.Flower buds at floral stages4and5of(a)Brassica napus.(b)Magnification of the presumptive whorls2and3.(c)Flower bud at stages4and5of the CMS line4:19.(d)Magnification of the presumptive whorls2and3.(e)Section offlower bud at stages4and5of the CMS line41:17.(f)Magnification of the presumptive whorls2and3.(g)Section offlower bud at stages7and 8of B.napus.(h)Magnification of the anther primordium containing densely stained cells in the archesporial cells(star)surrounded by less stained cells that will give rise to the epidermis(arrow).(i)Thin section of flower bud at stages7and8of the CMS line4:19.(j)Magnification of the third whorl organ primordium.(k)Thin section offlower bud at stage9of the CMS line41:17.(l)Magnification of the third whorl organ with the ovule-like structure beginning to differentiate(arrow).Bar:(a,c and e) 20l m;(b,d and f)8l m;(g,i and k)100l m;(h,j and l)25l m.anp,anther primordia;ca,carpel;clp,carpelloid primordium;cl,carpelloid structure;L1–3,meristematic cell layer.Nuclear–mitochondrial interactions duringflower development in B.napus735ªBlackwell Publishing Ltd,The Plant Journal,(2005),42,731–742Stages 7–9In flower buds,the difference in expression levels of BnLFY and BnUFO between the CMS lines and B.napus was more accentuated (Figure 4a)during stages 7–9.However,no significant difference in BnASK expression level was noted between the CMS lines and B.napus at later stages of flower development (Figure 4a).For the second group of genes,BnAP3,BnPI and BnAG ,the expression levels were reduced in the CMS lines,especially for BnAP3and BnPI (Figure 4b).Furthermore,the group III genes composed of BnTON1and BnCycB1exhibited reduced expression levels in the CMS lines (Figure 4c).In conclusion,the transcription levels of the B-genes in the CMS lines do not correspond to the levels of the transcripts of the genes directly responsible for BnAP3and BnPI activation.As this activation does not occur properly,a response by upregulation of the upstream genes takes place.The exact reason for such blockage of the B-gene activation is,however,not known.Spatial expression of LFY ,AG ,PI and AP3genes during floral developmentIn order to determine whether homeotic conversions were associated only with differences in levels of gene expres-sion or also with the pattern of gene expression,in situhybridizations with BnLFY ,BnAG ,BnAP3and BnPI were performed to visualize their expression patterns in flower meristems and young flower buds.In wild-type B.napus the mRNA of BnLFY was first detected in stage 2during flower development (data not shown).Expression was present throughout flower development until stage 5.From this stage on,the BnLFY mRNA signal was excluded from the sepals but still present in petals,stamens and carpels (Figure 5a–c,data not shown).The expression pattern of BnLFY observed in the CMS lines was indistinguishable from B.napus (Figure 5a–c).BnAG expression was detected in stages 2–4in the centre of the meristem (data not shown).Later on,BnAG expres-sion was confined to whorls 3and 4from which stamen and carpel primordia develop.No differences in the BnAG expression pattern in the CMS lines from the pattern in B.napus were found (Figure 5d–f).BnAP3was expressed as early as stage 3(data not shown).Upon sepal differentiation (stage 4),transcripts were detected in the presumptive second and third whorls (Figure 5g–i)and from stage 5onwards,BnAP3was expressed throughout the developing petals and stamens in all three lines (data not shown).Until stamens and carpel-like organs were differentiated,the BnAP3expression pat-tern in young CMS flower buds was similar to that of B.napus (Figure 5g–i).Later on,the accumulation of BnAP3mRNA was similar to expression in the pistil.Signals were detected in the tip of the organ and in areas thatresembledFigure parison of mitochondria in Brassica napus and in the CMS lines.(a)Ultrastructural view of putative whorl 3from a B.napus flower bud at stage 4.Mitochondria occupy a large portion of the cell.(b)Ultrastructural view of the putative whorl 3from a CMS line 4:19flower bud at stage 4.Mitochondria are small,lack cristae and most of the matrix content.(c)Ultra-thin section of putative whorl 3from CMS line 41:17flower bud at stage 4.(d)Ultra-thin section of stamen primordium from B.napus at stage 7.(e)Ultra-thin section of third whorl primordium from CMS line 4:19at stage 6.(f)Ultra-thin section of third whorl primordia from CMS line 41:17at stage 6of flower development.mt,mitochondria;pl,plastid;v,vacuole.Bar:1l m.736Rita Teresa Teixeira et al.ªBlackwell Publishing Ltd,The Plant Journal ,(2005),42,731–742ovule-like structures (data not shown).This expression pattern is in accordance with the finding that the AP3protein was expressed in the ovules of A.thaliana wild-type plants (Jack et al.,1994).BnPI displayed an expression profile similar to BnAP3but with an area more centred in the meristem (Figure 5j–l).As in the case of other genes,there was no difference in the expression pattern between B.napus and the two CMS lines.Spatial expression of BnCycB1and BnTON1genes in young flower budsDue to the disorganized plane of cell division formed mainly in presumptive whorls 2and 3,in situ hybridizations were performed using probes for BnCycB1and BnTON1.BnCycB1mRNA was expressed in all mitotically active cells of flowertissues (Figure 5m–o).The expression level was high from stage 2to stage 7.The carpel and stamen primordia showed a similar expression pattern in all the lines (Figure 5m–o).Later on in development,microsporogenous tissues and ovules showed the highest accumulation of transcripts in B.napus .The two CMS lines displayed the same pattern except for the microsporogenous tissue that was replaced by ovule-like structures (data not shown).As expected,BnTON1expression resembled BnCycB1expression (Fig-ure 5p–r).A higher magnification image of the inflorescence area from B.napus and the CMS line 4:19shows disorgan-ized cell division in the CMS line (Figure 5s,t).In situ experiments showed that the formation of the homeotic organs in the CMS lines was not due to an incorrect expression of the homeotic genes in the floral whorls but rather due to differences in expressionlevels.Figure 4.Results from real-time RT-PCR amplification of RNA isolated from Brassica napus ,CMS lines 4:19and 41:17inflorescences containing floral buds from stages 1–6and 7–9.Amplifications were performed with specific primers for:(a)BnASK1,BnUFO and BnLFY genes.(b)BnAG ,BnAP3and BnPI genes.(c)BnTON1and BnCycB1genes.Results were normalized to B.napus expression levels.Standard deviations levels are shown (n ¼4).Letters indicate significant difference at the probability level P <0.05in comparison with B.napus .Nuclear–mitochondrial interactions during flower development in B.napus 737ªBlackwell Publishing Ltd,The Plant Journal ,(2005),42,731–742DiscussionEven though the CMS flower phenotypes resemble home-otic mutant phenotypes,homeotic genes in the CMS lines are not mutated.Rather,the aberrant phenotype is the result of the alloplasmic combination of a nucleus from one spe-cies with mitochondria from another species (Budar et al.,2003;Hanson and Bentolila,2004).In those CMS systems that have been characterized molecularly,the different CMS-associated mitochondrial ORFs show very few similarities but still the products of the novel mtDNA regions result in similar effects (Hanson and Bentolila,2004;Schnable andWise,1998).Furthermore,in most CMS systems,the new polypeptides encoded by CMS-induced mitochondrial genes are found in the mitochondrial membrane fractions or inside the mitochondria (Abad et al.,1995;Budar and Pelletier,2001;Grelon et al.,1994;Song and Hedgcoth,1994).Ultrastructural studies of the CMS lines revealed that the inner-membrane integrity of most mitochondria was disrupted in the flower tissues,while mitochondria in the fertile B.napus cultivar exhibited ordinary morphology.Farbos et al.(2001)reported similar results from studies of alloplasmic male-sterile lines of N.tabacum .Moreover,disrupted mitochondria were found in male-steriletobaccoFigure 5.Spatial mRNA expression using BnLFY ,BnAG ,BnPI ,BnAP3,BnCycB1and BnTON 1Dig-labelled probes.BnLFY expression of (a)Brassica napus ,(b)CMS line 4:19and (c)CMS line 41:17.BnAG expression of (d)B.napus ,(e)CMS line 4:19and (f)CMS line 41:17.BnAP3expression of (g)B.napus (arrowhead),(h)CMS line 4:19(arrowhead)and (i)CMS line 41:17(arrowhead).BnPI expression of (j)B.napus ,(k)CMS line 4:19and (l)CMS line 41:17.BnCycB1expression of (m)B.napus ,(n)CMS line 4:19and (o)CMS line 41:17.BnTON1expression of (p)B.napus ,(q)CMS 4:19and (r)CMS 41:17.BnTON1expression in an inflorescence contain-ing a stage 2flower meristem:(s)B.napus and (t)CMS line 4:19.Flower stages are marked at the bottom of each figure.Bar:(a–r)100l m;(s,t)20l m.an,anther;cl,carpelloid structure;st,stamen primordium;w3–4,whorls 3and 4.738Rita Teresa Teixeira et al.ªBlackwell Publishing Ltd,The Plant Journal ,(2005),42,731–742。