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Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding
and autoregulation of its persistent expression throughout flower development
Zsuzsanna Schwarz-Sommer, Isabelle Hue, Peter Huijser, Peter Flor, Rolf Hansen, Frank Tetens, Wolf-Ekkehard Lonnig, Hans Sommer, Heinz Saedler
To cite this version:
Zsuzsanna Schwarz-Sommer, Isabelle Hue, Peter Huijser, Peter Flor, Rolf Hansen, et al.. Characteri- zation of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development. EMBO Journal, EMBO Press, 1992, 1, pp.251 - 263. �hal-02931791�
Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression
throughout flower development
Zsuzsanna Schwarz-Sommer, Isabelle Hue, Peter Huijser, Peter J.Flor, Rolf Hansen, Frank Tetens, Wolf-Ekkehard Lonnig, Heinz Saedler and Hans Sommer
Max-Planck-Institut farZuchtungsforschung, 5000Koin30, FRG Communicated by H.Saedler
Wehavedetermined thestructureofthefloralhomeotic deficiens (defA) gene whose mutants display sepaloid petals and carpelloid stamens, and have analysed its spatial and temporal expression pattern. In addition, several mutantalleles (morphoalleles) were studied. The results ofthese analysesdefinethreefunctional domains of the DEF A protein and identify in the deficiens promoter a possible cis-acting binding site for a transcrip- tion factor which specifically upregulates expression of deficiens in petals and stamens. In vitro DNA binding studies show that DEF A bindsto specific DNA motifs asaheterodimer, together withthe protein product of thefloral homeotic globosagene, thusdemonstratingthat the protein encoded by deficiens is a DNA binding protein. Furthermore, Northern analysis of a temperature sensitive allele at permissive and non- permissive temperatures provides evidence for autoregulation ofthe persistent expression of deficiens throughout flower development. Apossible mechanism ofautoregulation is discussed.
Key words: flower morphology/gene structure/in situ hybridization/MADS-box/transcription factor
Introduction
Flower morphogenesis is acomplex process governed by
asetofregulatorygenes(Meyerowitzetal., 1989; Schwarz- Sommeretal., 1990; Bowmanetal., 1991; Coen, 1991).
The role of these genes in flower development becomes apparentwhenmutationalalteration ofgenefunctionresults in a morphologically altered phenotype. For example, developmentofadifferent organinthe mutant flowerata
position whereinthe wildtype the normalorgandevelops (Meyer, 1966) indicates the function ofahomeoticgenein theprocessofcontrol offloral organidentity. InAntirrhinum andArabidopsis such homeotic genes, or their respective mutants, can be assigned to three different classes. Muta- tions ingenes in each ofthese categories frequently affect differentiation oforgansintwoadjacent whorls of theflower.
Recentmolecular andgenetic studies of suchmutants have led to the proposal of relatively simple models of how homeotic genes, askey regulatory genes ofdevelopmental pathways, may govern floral organogenesis (Haughn and Somerville, 1988; Carpenter and Coen, 1990; Schwarz- Sommer et al., 1990; Bowman et al., 1991; Coen and
Meyerowitz, 1991; Lord, 1991).The molecularmechanisms underlying this control are still unknown.
Thedeficiens(defA)gene is involved in the control ofpetal and stamen development (Klemm, 1927; Sommer etal., 1990). Loss of thedefA function results in sepaloidpetals and carpelloid stamens in deficiens mutants. Sequence analysisof thedefAcDNA cloneindicated that the encoded protein (DEF A)may beatranscriptionfactor,because DEF AcontainsaconservedputativeDNAbinding domain,the MADS-box (Schwarz-Sommeretal., 1990). The MADS- box is also found in proteins known to be transcription factors: the serum response factor(SRF) in human which regulates the c-fos proto-oncogene in response to serum factors(Normanetal., 1988),thesarcomericCArG-binding factor CBF which is involved in the muscle-specific expres- sion of a-actin inhuman (Boxer et al., 1989; Sartorelli et al., 1990) and the yeast regulatory proteins MCM1 and ARGR1 which areinvolved in cell type determination and regulation of the arginine biosyntheticpathway, respectively (Passmoreetal., 1988; Dubois et al., 1987). The MADS- box is alsopresent in AG, the protein product of agamous, ahomeotic gene controlling organogenesis of stamens and carpelsinArabidopsis (Yanofsky etal., 1990).Thegrowing familyofMADS-box proteins meanwhile comprises six AG- like homologues in Arabidopsis (Ma et al., 1991); SRFX, aCArGbinding protein regulatingsomite-specific expres- sion of the cardiac a-actin gene inearlyXenopusembryos (Mohun et al., 1991); several proteins inAntirrhinum, two ofwhich are encoded by the floral homeotic genes squamosa andglobosa (Schwarz-Sommer et al., 1990) and five DEF- likehomologues in tomato(Pnuelyetal., 1991).Thecellular processes controlled by MADS-box proteins differ in the variousorganisms,butdespitethedifferencesin theirspecific action it seems that the function ofMADS-boxgenesis the control of cell differentiation or to mediate thecell'sresponse to environmental signals.
Severalapproacheshave beenappliedtoanalysethe func- tionoftranscriptionfactors. In the case of SRF and MCM1, where the DNA binding sites of the regulatory factors in the promoters of the target genes are known [Serum Response Element(SRE) in the promoter ofc-fosand the P-box in promoters of a- and a-cell type specific genes, respectively], in vitro analysis of the modifiedproteins has revealed functional domains of theprotein (Normanetal., 1988; Christ and Tye, 1991).Forhomeoticgenes likedefi- ciens, naturally occurring mutationsaffectingthe encoded protein and/or mutations which alter thegene's expression pattern can giveclues about its function. We have utilized morphologically distinct mutants, so called morphoalleles, of thedeficiens geneto obtain informationon functionally importantdomainsof the DEF Aprotein.Correlation of the altered gene structure with its modified function allows deeperinsightinto the molecular mechanisms which control deficiens, andthe mechanisms by whichdeficienscontrols organogenesis ofpetals and stamens.
Z.Schwarz-Sommer et al.
Results
Flower morphology ofmutants
Studies of morphological characteristics and temperature dependence of morphoalleles of the deficiens gene were
conducted toget information about the role ofdeficiens in floral development.Inthe following thephenotype of flowers developing at high temperature (25°C) is described first, followed by the phenotype in the cold (15°C).
Wildtype (defA). WildtypeAntirrhinum flowers(Figure 1) consist of four whorls oforgans i.e. the five sepals (first
or outermost whorl), five petals constituting the corolla (second whorl), four stamens and the staminodium (third whorl) andtwocarpels (fourth orinnermostwhorl). In the
matureflower the basalpartsofthe petalsarefused and form
astructure, while thehigherpartsformtheupper(adaxial) and lower (abaxial) lobes. The distinct morphological features of the fourtypes oforgans andtheir development fromorganprimordiatomatureorganshave been described by several authors (Klemm, 1927; Bergfeld, 1956;Awasthi
et al., 1984; Green and Linstead, 1990; Sommer et al., 1990).
Temperature has an influence on flower development.
Decreasing the culture temperature from 25°C to 15°C
resultsinenlarged, intensivelycoloured flowers (Figure 1).
In general, flower primordia and floral organ primordia increase in size in the cold but take longer to develop into mature organs. At 15°C additional organs develop in the thirdwhorl, which flank the filaments of stamens and often showpetaloid characters (Figure 3A). The number ofthese structures is variable, but there are usually two petaloid organs flanking the staminodium. The growth of the upper lobe of the second whorl is promoted in the cold.
chlorantha (defcl?). Chlorantha flowers are smaller and their petals show weak virescence indicating sepaloidfeatures (Figure 1). The stamens are feminized because they develop ovules on the ventral side of the anthers (directed towards the interior of the flower bud in Figure 3A). The dorsal pollen sacs(directed toward the exterior of the flower bud) arealmost normal. The morphological alterations are more distinct for the upper two(adaxial)organsthan for the lower two (abaxial) ones. The ring of trichomes (hair-like struc- tures) surrounding the lower stamens in the wild type is missing in the mutant (Figure 3A). The lower two filaments are not twisted in contrast to the wild type; therefore the positionof their anthers isslightly changed. Carpeldevelop- ment is not affected.
Development in the cold decreases the phenotypic expres-
Fig. 1. Phenotypesofdeficiens mutantsand influence of culturetem-erature onthephenotypic expressionofthe mutations. Thegenotype of the plants homozygous for the indicatedmutantalleles(e.g.defchlldefch = chl) is shown above thepanelsand the culture temperatureis indicatedat the left of eachrow ofphotographs. Wild type morphology isdemonstratedby the flower ofaheterozygote(defgl/defA = gli x wt)toshow that decreasing the wildtype gene dosage hasno morphological effect. Magnifications werethesameforallflowers,except forglobifera flowers, which weremagnified twice (upperrow) and fourtimes (lowerrow) ascompared with the others. Thephotographs show flowers fromanabaxial view;
thus the lower lobe ofthe corolla isatthe front ofthepicture, while theupperlobe isatthe back. Theupper part ofthe globiferaflower(depicted inatopview, butoriented in the samewayasall otherflowers), grownatlow temperature, wasremovedtodemonstrate thestructure of its central gynoecium. Asnoted inthetext,temperature hasnosignificanteffectonthe morphologyofglobifera flowers. Variabilityinthe colour andcolour intensity ofpetalsis due tomutations in colourdetermining genes. Such mutations havenoeffectonthe morphologyof the flowers.
sion of themutation. Petals are larger; the promoted growth of the upper lobe reflectstheeffectof low temperature on developmentofwildtypepetals (Figure 1). Thefeminizing effect of the mutation on stamen development is less pronounced than at 25°C (Figure 3A); the stamens do not develop ovules and the anthers contain fertile pollen, despite their slightly affected morphology.
nicotianoides (deffli). The petals of nicotianoides flowers are reduced further in size and show strong green colour (Figure 1). Theirformis altered and often, particularly when growing in the cold, a third petal-like organ develops between the upper two. The filaments of the stamens are broadened and their dorsal surface is covered with trichomes (Figure 3A). The ventral pollen sacs are deformed and the anthers bear few ovules. At the tip of the male organ, papillate stigmatic tissue,afemalecharacteristic, develops.
The dorsal pollen sacs are less severely affected than the ventral ones and the tendency towards feminization is more pronounced for the upper two stamens than for the lower two. Development of the carpels is hardly affected, but fusion (adnation) of the central gynoecium and parts of the fila- ment of the upper stamens often occurs.
The influence of decreased temperature on petal develop- mentis similar to that observed for wild type or chlorantha flowers. Additional organs develop frequently in the third whorl that are covered with trichomes and hence resemble sepals (Figure 3A).The decreasedcarpellodyof the stamens
(.rJi \E C(21
15 -C
25 C
is similar to the temperature effect on chlorantha stamen developmentsothat ovuleshardly develop. Pollen, however, has not yet been obtained from nicotianoides mutants, although its occasional occurrence has been described (Klemm, 1927).
Insummary, chlorantha and nicotianoides mutants show some commonfeatures with respect to thedifferent strengths of thefeminizingeffects on stamens: (i)in bothmutantsthe upper stamens are more severely affected than the lower stamens and(ii) the morphology ofthe ventral part of the anthers is more strongly distorted thanthat of the dorsal part.
These observations indicate that the deficiens function is unevenly distributed across organ primordia and also between adaxial and abaxial parts of the flower. Further- more, increasing the culture temperature increases the expression of the altered phenotypes, perhaps as a conse- quence of the accelerated differentiation process at the elevated temperature.
globifera (defgli). As described in an earlier report(Sommer etal., 1990), globifera 'petals' are morphologically indis- tinguishablefrom sepals,exceptthat thesesepaloidsecond whorl organs are larger than sepals in the first whorl and that their position in the mature mutant flower resembles theposition of wild type petals of the upper and lower lobes (Figure 1). The broadened filaments fuse laterally and constitutealargecentralgynoeciumcovered withtrichomes on its dorsal surface. Inside this central female organ a
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Fig. 2. Phenotypesofheterozygotes between mutant alleles of deficiens and the influence of culture temperature onphenotypicexpressionof the mutations. Thecrossesusedtogeneratethe allelic heterozygotesareshownabovethe panels and thegrowingtemperature isindicated atthe left of eachrow ofphotographs. Crosseswhich involvedchlorantha plants werecarriedoutwithchlorantha asthemaleparent. Toobtain nicotianoides x
globifera plants, heterozygotes of eachofthe two mutantswith the wildtypedeficiens allelewere generated andtheseheterozygotes were
subsequently crossed.
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Fig.3. Effect of mutationsinthedeficiensgeneonstamenmorphologyand influence ofculturetemperatureonexpressionof themutations. The genotypeof theplants,homozygous (A)orheterozygous (B)for the indicatedmutantalleles, is shownabove thepanels. Forscanningelectron
microscopy (SEM)0.5cmlongyoung buds ofplantsgrown atlow andhightemperature(indicatedat the left of eachrow) werefixed and driedas
describedpreviously (Sommeretal., 1990). The SEMphotographsshowoneof the abaxial andoneof the adaxial stamens after removing sepals, petals, carpelsand the otherpairofstamens. Additional organswerealso removed unlesstheydid notcoveressentialpartsofstamens(seefor example, gli x wtin the lowerpanel inpartA. The staminodium (indicated byanarrowhead), locatedbetween thetwoadaxial(upper)stamensin theflower, is orientedtotherightoneachphotograph.
variable number of loculi, filled with ovules,canbeobserved (Figure 1). The genuine gynoecium eitherdoesnotdevelop,
orit shows variablefusions with the carpelloidstamensof the third whorl. The chimney-like structure, typical of globifera flowers, arises by elongation of the filamentsand resembles the style ofa gynoecium. Occasionally a 'style withinastyle' structuredevelops, probably by development ofthe genuine style inside the 'chimney'.
Exceptfortheirincreasedsize, globiferaflowersarenot altered morphologically when developing in the cold.
defA-23.DefA-23flowers(Figure 4) display greenish petals with a distorted shape. The carpelloid stamens form a
chimney-like structurecomparablewith thecentral female
organofglobiferaflowers. Additionalpetal-like, elongated structuresoftendevelopbetweenthesecond and third whorls.
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Fig. 4. Phenotype of flowers of the defA-23 mutant. Thephotographs atthetop show the flower from the abaxial side. The alteredstructure ofthe reproductive organs is documented at the right where the lower lobe ofthe flower andadditional petal-like structureswere removed.
The inner structure of the centralgynoecium is depictedatthe bottom.
They probably represent displaced parts of the filaments and often fuse with the petals or with the central gynoecium.
Besides stronger growth, decreased temperature has no visible effect on the morphology of defA-23 flowers.
defA-101. The second whorl organs ofdefA-101 flowers (Figure 5) are sepaloid and strongly resemble those of globiferaflowers.The broadenedfiamentsinthe thirdwhorl aregenerallyshorter than thecomparable organs ofglobifera ordefA-23 flowers; their lateral fusion, resultinginloculi, is often incomplete. The central female organ is complex in structure (Figure 5). Placentae with ovules develop at the base of the fused filaments. The number of placentae is variable, although the number of loculi seems to be five.
Three long, incompletely fused, rod-like structures which resemble thegenuine style of the gynoecium are located in the centre ofthe flower.
Atalower culture temperature, defA-101 plants develop flowers which often possess six deformed petals in their second whorl (Figure5). The third whorl organs are also deformed, but represent morphologically almost normal stamenswith broadenedfilaments which carry anthers with dorsal and ventral pollensacs. Mutantplantsgrown inthe coldproducefertilepollen. The flower containsseveral addi- tionalsepaloidandpetaloidstructureswhich allseem tobe lateral protrusions of the filaments in the third whorl. The dorsal part of filaments is often split at the tip. The gynoecium contains usually more than two loculi with placentaeand theshapeof thestigmaalso indicates thatmore than two carpels may constitute the female organ.
Fig. 5. Phenotype of flowers of thedefA-101 mutant. Photographs in theupper rowwere made from the side ofintact flowers. Photographs inthe lowerrow show the structure of thereproductive organs after removing the lowerlobe of the flower or after dissection of thecentral female organ (at the right). The growing temperature is indicated belowthepanels.
Intheglasshouse, underintermediatetemperaturecondi- tions, defA-101 flowers show an intermediate phenotype (Figure 5). Preliminary temperature-shift experiments indicatethat,afterinitiationofdevelopmentat25°C, petals ultimatelybecome restoredto acertainextentwhen theplant is returned to the cold. Restoration ofthird whorl organs is less pronounced.
The effects of temperature on flowers ofplants carrying thedefA-101 allele suggest thatdefA-101 is a temperature sensitive allele. Incontrast, all otherdeficiens morphoalleles displaynormal temperatureeffectsondevelopment, likewild type flowers.
Dominance relationship between different deficiens morphoalleles
Allmorphoallelesdescribed abovearerecessivetowild type but some alleles, when combined asheterozygotes, reveal incomplete dominance relations among each other.
Ingeneral,thechloranthaalleledominatesoverall other mutantalleles with respect topetaldevelopment because the heterozygotes resemble the chlorantha homozygote (see nic x chl in Figure 2). The globifera x chlorantha heterozygote, however, is closertowild type morphology thanthechloranthahomozygote(comparecurvatureof upper and lowerpetalsof the correspondingflowers inFigures 1 and2). Themodificationseems tobespecificforthis allelic combination because combination ofchlorantha withnico- tianoidesorglobifera with nicotianoides doesnot seemto have a similar effect on petal development. Also, phenotypicallyandgenotypically wildtyperevertantsofthe germinally unstable globifera allele are indistinguishable from other wild type forms. Since theunexpected phenotype
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Fig.6Strutureotheeficinstrascripion uit. Th sequnce rspresets pat of 7 kb coRI ragmetcontiningthe etire wld tye def locus(thissequencehas been deposited in the EMBL data library under theaccessionAA1 number X62810).qr0ATTT" TheAexonsAareAC boxesL'andin encodedTthe
amino acids areshown below theDNAsequence. Consensussequences, such asthe TATA box andpolyadenylation signalsare in inverted boxes.
Both polyadenylationsignals arefunctional, becausecDNAspolyadenylated attwodifferent sites werefound. Theinvertedrepeat upstreamof the TATAboxisunderlinedand a consensus SRE motif isindicated. Nucleotide exchanges anddeletions foundindeficiens mutants are shown above the nucleotidesequence andthose which generate amino acidexchangesordeletions are indicated below the amino acidsequence. Theinsertion sites of the transposons Tam7 and Tam8 areindicated bythe three underlined nucleotides which areduplicatedin the mutants and flank the inserts.
ofglobifera x chlorantha heterozygotes was observed in a population of about ten thousand individual Fl hetero- zygotes, it seems that the (dominant) modifying effect on chlorantha is linked to theglobifera allele.
With respect to stamen development the dominance rela- tionships between alleles are complex. In general, allelic heterozygotes display some phenotypic characteristics of the more severely feminizing allele (Figure 3B). Forexample, combinations of chlorantha with globifera and nicotianoides possess ovuleswhose development persists in the cold. The morphologyof third whorl organs in the globifera x nico- tianoidesheterozygote is interesting: at 25°C the lateral parts of thebroadened filaments show a tendency to form atube- like structure, indicating the formation of loculi which become completely closed in globifera flowers (Figure 1).
Our analyses of allelic combinations with defA-101 and defA-23 are not yet complete. Preliminary results indicate thatchlorantha dominates both alleles with respect to petals.
The combination globifera x defA-23 reveals increased sepalody of the petals whose morphology is still distinctively different from those of the globifera flowers.
The structure of the deficiens transcription unit We have determined the structure of the genomicdeficiens
transcriptionunit bysequencingcDNAclones and - 5.6 kb of a genomic wild type clone comprising 3 kb of upstream region and 2.6 kb containing the coding region (Figure 6).
Similarly,sequenceanalysisofgenomic clones of five mutant alleles ofdeficiens was carried out to identify the sites of mutations in the respective alleles. To exclude ambiguities the entire 5.6 kb ofgenomic sequence, shown in part in Figure 6 for wild type, was also determined for all mutant alleles, except for defA-23.
Consensus sequences, such as exon-intron boundaries, startoftranslation, polyadenylation signals and the high AT contentofintrons correspond to rules obtained from other eukaryotic genes (Messing etal., 1983; Joshi, 1987).
The deficiens promoter region. SI mapping and primer extension experiments (data not shown) revealed several transcription initiation sites. Thestrongestsignal, although notexperimentally proven to represent the genuine start of transcription initiation,wasarbitrarily designated as position
+1 in the sequence shown in Figure 6. At position -19 to-32 anunusuallylong putative TATA box is found whose location, with respect to the site of transcription initiation, corresponds to that found in other plant genes (Messing etal., 1983; Joshi, 1987). Further upstream, the defA
promoterregion contains a 244 bp, almost perfect inverted repeat (Figure 6). The significance of this potential stem-loopstructure inthetranscriptional regulation of the gene is not known.
Asearch forconsensusbindingsites of known transcrip- tion factors revaled a CArG-box [CC(A/T)6GG, see SRE box inFigure 6] 1.2 kbupstream of the transcription start.
This motif represents the consensus of thebinding sites for MADS-boxcontaining transcriptionfactors (Passmoreetal., 1989;Boxeretal., 1989). Inthechlorantha allele,a muta- tionaffecting four bases (CGG instead of CCCCTG) is found 32 bp upstream of this box (Figure 6), indicating the func- tional importanceof thisregionforregulationofdefAgene expression. Becausethe site affected inthe chlorantha allele (CCCCTG) slightly resembles the consensus binding
sequenceof MyoD(CANNTG), it isinterestingto notethat inthe promoter of thecardiaca-actin gene several CArG- boxes, representing the binding site ofan SRF-like factor (CBF), are located near the binding site of MyoD1 (GustafsonandKedes, 1989). Twoof theCArG-boxes, posi- tioned50 and 90bp upstream of theCANNTG-box,aswell asthebinding site for MyoD 1 (and Spi)wereshowntobe required formuscle-specificexpression of the gene(Sartorelli et al., 1990).
Thedeficienscodingregion and the DEFA protein. The 294 bpfirst exon contains the MADS-box whose features, such as the presence of a potential phosphorylation site for calmodulin-dependent kinases, a hydrophobic putative dimerization domain and the overall conservation ofastretch of57 amino acids, also found in the known transcription factors MCM1 and SRF and in other floral MADS-box proteins (Yanofsky etal., 1990; Ma etal., 1991), have been described indetail elsewhere(Sommeretal., 1990;Schwarz- Sommeretal., 1990). In thenicotianoides allele aGtoA substitution at position 185 (Figure 6), within the DNA binding domain of the MADS-box, results in aglycine to aspartic acid exchange.
The short third and fourth exons ofdefAcontributetothe K-box, a region with somehomologytokeratin (Maetal., 1991). A small deletion of three basepairsattheN-terminal endof theputativeK-box causes loss of alysineintheprotein of the mutant defA-101 allele (Figure 6).
Analysis of the defA-23 allele (Figure 4) revealed the insertion of the Tam8 transposable element (see below) within the last large exon of the defA gene (Figure 6).
Sequence analysis of the cDNA derived from defA-23 mRNAshowed that, at the 3' end - 300bp of the wild type cDNA sequence is replaced in the mutant by a sequence derived from the Tam8 element. Asaconsequence,the last 12aminoacids ofDEF A(GGSDLTTFALLE*)arereplaced by seven element-encoded amino acids (HYNKNGL*) in the mutant protein. Co-transcription and co-translation of plant genes with insertionelementswasalso observed in Zea mays (Gierl etal., 1985; Schwarz-Sommer et al., 1987).
Intheglobiferaalleleatransposon, Tam7(Sommer etal., 1990), is inserted into the third intron of the defA gene (Figure 6). Inaddition,theallelecontainsseveralpointmuta- tionsin exons(Figure6) andnumeroussmall alterations in the introns (not shown), in comparison with wild type and all othersequenceddeficiensmutantalleles. Thehighnumber ofdifferences in theglobiferaallele isnotsurprisingbecause thismutant was isolated from aline with differentgenetic background (see Materials andmethods). Becauseglobifera is agenetically unstable allele which reverts germinallyto wildtype (Sommeretal., 1990), aminoacidexchangesin the DEFA protein encoded by this alleledefine positions not important for wild type functions. Such apoint muta- tionis observed inthe fourth exon, resultingin alysineto glutamine exchange (Figure 6). Thepositive charge of the lysine residueatthispositionthus doesnotseem tobeessen- tial for proper wild type DEF A function.
Transposable elements. The altered phenotypesof flowers of globifera and defA-23 plants are due to transposable elementinsertions, Tam7 andTam8, respectively, into the defAgene. The insertion sites of the elements (Figure6), and in part their sequence (not shown), have been deter-
