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Fimbriata controls flower development by mediating between meristem and organ identity genes
Cell, Vol. 78, 99-107,
July 15, 1994, Copyright
0 1994 by Cell Press
Fimbriata Controls Flower Development by Mediating between Meristem and O rgan Identity Genes Riidiger Simon, Rosemary Carpenter, and Enrico Coen John lnnes Centre Colney Lane Norwich NR4 7UH England
Sandra Doyle,
Summary Two major classes of genes directing flower development have so far been described: early activated genes regulating meristem identity and later acting genes controlling organ identity. Here, we show that the fimbriata (fim) gene acts between these two classes in a sequence of gene activation. The fim gene, originally described in 1930, was cloned by transposon tagging from Antirrhinum majus and encodes a product with no detectable homology to other proteins. Mutations in fim result in partial homeotic transformations of floral organs and in reduced determinacy of the meristem. Expression and function of fim depends on the activity of meristem identity genes, and fim in turn controls the spatial and temporal expression of organ identity genes. The pattern of fim expression defines a new domain of the floral meristem that changes with time in a complementary manner to those of the meristem identity gene f/o&au/a and the organ identity gene plena. Introduction The development of plants depends largely on the behavior of their meristems, groups of rapidly dividing cells that form the growing points. On the periphery of meristems, groups of cells are partitioned off to form either secondary meristems or organ primordia. There are several classes of meristem that are distinguished by the types and arrangements of primordia and secondary meristems they produce. For example, vegetative meristems produce leaf primordia with secondary shoot meristems in their axils. lnflorescence meristems typically produce bract primordia with flower meristems in their axils. Flower meristems produce concentric whorls of four types of organ primordia: whorl 1 (outermost) contains sepals, whorl 2 consists of petals, whorl 3 contains stamens, and whorl 4 contains carpels. Unlike shoot meristems, the growth of flower meristems has a precisely defined determined end point: they do not produce secondary meristems between the whorls (lateral determinacy) and stop initiating organ primordia after the central whorl 4 (apical determinacy). Genetic and molecular studies in Antirrhinum and Arabidopsis have so far defined two main types of genes involved in the control of flower development, meristem identity and organ identity genes (Schwarz-Sommer et al., 1990; Coen and Meyerowitz, 1991; Coen and Carpenter, 1993). Meristem identity genes are involved in initiating the
flower developmental program; mutations in them cause inflorescence meristems to replace floral meristems. Mutations in organ identity genes affect the fate of organ primordia, most often in two adjacent whorls. These mutations can be divided into three classes: a, those that have carpels growing in place of sepals and stamens in place of petals; b, those with sepals instead of petals and carpels instead of stamens; and c, those with a proliferation of petals or sepals instead of stamens and carpels in the center of the flower. Each class of mutation corresponds to a loss in one of three homeotic functions (a, b, or c), which normally act in combination to specify organ type, so that in wild-type floral meristems the combinations in whorls l-4 are a, ab, bc, and c (Carpenter and Coen, 1990; Bowman et al., 1991). Although there is some functional overlap between meristem and organ identity genes, so far genes that mediate between the two classes have not been defined. Here, we describe a novel type of gene, fimbriata (fim), that mediates between meristem and organ identity genes in Antirrhinum. Mutations in fim have effectson the identity of organs in whorls 2, 3, and 4 and also on the lateral determinacy of the floral meristem. Genetic analysis shows that fim interacts with meristem identity genes to initiate the flower developmental program and with organ identity genes to control the fate of primordia. Expression of fim is activated after the meristem identity genes floricaula (f/o) and squamosa (qua) but before deficiens (def) andplena (p/e), genes needed for the b and c organ identity functions, respectively (Coen et al., 1990; Huijser et al., 1992; Sommer et al., 1990; Bradley et al., 1993). Unlike def and p/e, which are expressed in developing organ primordia, fim is expressed transiently in floral meristems and eventually becomes restricted to a region lying at the base of primordia. We propose a sequence of gene activation leading from f/o and sgua through fim to organ identity genes such as def and p/e. This sequence is reflected in both the temporal order of gene activation and in the spatial arrangement of expression domains. Results The Phenotype of fim Mutants Two independent recessive mutations that gave petals with sepal-like tissue, termed the sepaloidea phenotype, were isolated in a large scale transposon mutagenesis program (Carpenter and Coen, 1990). The phenotypes were reminiscent of a classical mutation at the fim locus described previously (Baur, 1930; Harte, 1951), and genetic tests confirmed that both sepaloidea mutations, subsequently called fim-679 and fim-620, were allelic to fim. The most consistent feature of fim-679 mutants was that the petals had longitudinal streaks of green sepaloid tissue, often running the entire length of the petal (Figure 1 a). The streaks were not randomly distributed; they most often ran along the middle or the edges of lower petals. Scanning electron microscopy confirmed that the green
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Figure 1. Phenotypes Mutant Flowers
of fim-679 and fim-620
Flowers of the fim-679 mutant (a) have streaks of green, sepaloid tissue in the petals (arrows). The scanning electron micrograph of the border of a green streak (b) shows the sepaloid cells in the streak (right half) and the adjacent petal cells (left half). Flowers of the fim-620 mutant (c) have petals with larger sepaloid areas, and the stamens are mostly replaced by fused central carpels. Occasionally, a secondary flower is initiated from the second whorl of sepals, as shown by a flower cut open longitudinally (d).
areas contained typical sepaloid cells and revealed that cells lying between these and the adjacent petal cells had intermediate characteristics (Figure 1 b). The longitudinal growth of the green streaks appeared to be less than that of petal tissue, resulting in a distortion in the final shape of the petals. The stamens in whorl 3 were sometimes petaloid, while sepals and carpels developed as in wild me. Plants homozygous for fim-620 had flowers with a variable phenotype, ranging from flowers resembling fim-679 to extreme cases in which flowers consisted of an indeterminate number of sepals. This variability seemed to depend partly on environmental conditions: plants grown in the greenhouse generally produced weaker phenotypes than plants grown in the field in summer. In all cases, the first whorl of sepals was normal but instead of petals, the second whorl contained organs that resembled sepals with varying amounts of petaloid tissue (Figure lc). Organs beyond the second whorl were not usually arranged as discrete whorls, and their identity varied greatly between flowers. In most fim-620 flowers analyzed, there were 510 organs internal to whorl 2, comprising O-5 perianth (sepaloid or petaloid) organs surrounding O-7 central carpeloid organs. The central carpels tended to fuse, often forming a syncarpous and multilocular gynoecium resembling the central structures produced in def mutants of Antirrhinum (Sommer et al., 1990). In some cases, the carpeloid organs lacked a complete style and looked like sepals with stigmatic tips, enclosing ovaries or naked ovules.
Occasionally, fim-620 flowers showed a loss of apical determinacy leading to the production of an indeterminate number of sepals. A partial loss of lateral determinacy was also sometimes observed, secondary flowers or inflorescence shoots being initiated in the axils of second-whorl sepals (Figure 1 d). Isolation and Structure of the fim Gene Genomic DNA from fim-679, fim-620, and their wild-type progenitor was digested with Hindlll and probed with a fragment of Tam3. A novel 4.0 kb fragment was observed in fim-620 that cosegregated with the fim phenotype. The fragment was cloned by isolating a 3-5 kb fraction of Hindlll-digested genomic fim-620 DNA, ligating to a h vector, and screening with a Tam3 probe. The flanking sequence to the left of the Tam3 insertion was then used as a probe against Hindlll-digested DNA from various genotypes. The expected 4.0 kb band was detected in fim-620, whereas the wild-type progenitor gave a 1 .l kb band. The wild-type sequence was cloned by using the flanking probe to screen agenomic library. Further mapping by DNA blots showed that the Tam3 insertion in fim-620 had been accompanied by a genomic rearrangement. To prove that the wild-type clones corresponded to the fim locus, we analyzed the fim-679 mutation. This allele was known t%be slightly unstable because self-pollination of homozygous fim-679 plants gave revertant progeny with a wild-type phenotype at a rate of 1.40/o, indicating that the mutation was caused by a transposon insertion. A 0.8 kb Hindlll fragment (probe A) from the putative fim locus
Fimbriata 101
Mediates between Meristem
and Organ Identity
fim-619
fim
Fim +
E
4.4 kb
\/a
-
a
-iA A
HH
a
E
fim-620
Figure 3. Structure
of the fim Locus
The insertion sites of Tam3 in fim-679 and in fim-620 are shown by closed bars. The DNA sequences to the right of Tam3 in firm620 are rearranged. The stippled arrow indicates the open reading frame of the fim gene. The Hindlll fragment used to identify the insertion in firm619 is labeled A. Restriction sites are EcoRl (E) or Hindlll (H).
0.8 kb
:_ .:
1
Figure 2. Southern
2
3
Slot of Hindlll-Digested
4
5
6
DNA
Wild type (lane l), fin?-619 (lane 6) and four independent revertants of fim-619 (lanes 2-5) probed with a DNA fragment flanking the transposon insertion in fifn-620 (probe A, see Figure 3). The phenotype of the individual plants is indicated above the lanes, The fim-679 mutant and the (heterozygous) revertants carry the 4.4 kb insertion fragment. Excision of the 3.6 kb Tam3 element from fim-619 restores the 0.6 kb wild-type band.
was used as a probe against homozygous fim-619 mutants and four independent revertants. The fim-619 DNA gave a fragment of 4.4 kb, whereas the revertants had the restored 0.8 kb fragment of wild type as well as the 4.4 kb fragment, as expected from their heterozygous genotype (Figure 2). The fim-679 allele was cloned and shown to contain a Tam3 insertion 0.8 kb from the disruption found in fim-620 (Figure 3). To identify the fim transcript, various fragments from the fim locus were used as probes on blots of poly(A) RNA from wild-type inflorescences. The Hindlll fragment spanning the site of the transposon disruption in fim-620 detected a 1.8 kb transcript, which was specifically expressed in young inflorescences, but not detectable in mature flowers, leaves, or stems (data not shown). The expression level of this transcript was strongly reduced in
inflorescences of fim-619 and fim-620. Four independent cDNA clones corresponding to this transcript were isolated from a cDNA library made from wild-type inflorescences. These cDNAs appeared to originate from the same transcript and mapped to a region immediately to the right of the Tam3 disruption site in fim-620. The disruption points in fim-619 and fim-620 were found to be 976 bp and 196 bp upstream of the 5’ end of the longest cDNA clone, respectively. Further fim mutations were also shown to have deletions or insertions mapping to this transcribed region (G. Ingram, R. C., R. S., and E. C., unpublished data). As deduced from the comparison between the cDNA and the genomic sequence, the transcription unit consisted of one uninterrupted open reading frame of 1287 bp (Figure 4). This had the potential to code for a 429 amino acid protein of 49 kDa M,, which showed no similarity to any other protein sequence in the data bases. Expression Pattern of fim The timing and the histological distribution of fim RNA was determined by in situ hybridization using digoxigeninlabeled fim antisense RNA as a probe against wild-type
Figure 4. Sequence quence
of the fifn cDNA and the Derived
Protein Se-
The most 5’ ATG is assumed to be the start codon. Amino acids are indicated in the standard one letter code.
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Figure 5. In Situ Hybridization
of Wild-Type
and Mutant Sections Probed with firn
Sections of wild type (wt), f/o, and squa inflorescences were probed with digoxigenin-labeled fim antisense RNA and viewed under dark field, which gives the signal a pink color on a bright blue background. The top row shows main inflorescence apices. The bottom row shows later nodes, illustrating either floral meristems (left) or axillary inflorescence meristems (center and right). Scale bar (shown once per row) is 100 pm. b, bracts; s, sepals; p, petals; st, stamen.
inflorescences. Expression of fim was compared with the expression patterns of meristem and organ identity genes (Figures 5 and 6). The earliest stages of flower development were near the top of the inflorescence apex, and progressively later stages were below this. Flower meri-
Figure 6. Wild-Type
Floral Meristems
stems and their subjacent bract primordia (together termed a node) were numbered sequentially starting with node 0 at the top. For plants grown at 20°C, the rate of node initiation was about two per day, so the time interval between node initiation (plastochron) was approximately
Probed with fim or f/o
Adjacent sections of a floral meristem at the pentagon stage (top row) and at the petal-mound stage (bottom row) were probed with fim (left) or f/o (center), showing the complementarity of their expression pattern (see arrows). The sections were not medial and were viewed under light field, which gives the signal a purple color on a mauve background. Scanning electron micrographs show the morphological stages of the meristems at the respective nodes. Scale bar (shown once per row) is 100 pm. s, sepals.
Fimbriata 103
Mediates
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12 hr. We have previously shown that meristem identity genes are activated in the youngest visible node (node 0) and that organ identity genes are expressed by node 1 O11 (Bradley et al., 1993). The earliest stage at which fim transcripts could be detected was in floral meristems at node 9 (Figure 5, top left), before the organ identity genes were expressed. At this stage, no organ primordia were visible and the meristem had the appearance of a loaf of bread (Ft. C. and E. C., unpublished data). Sections through the middle of the loaf showed that fim transcripts first accumulated toward the abaxial side of the central dome, in a region about 4-5 cells wide. By nodes 1 O-l 1, corresponding to a further day of development, the floral meristem had started to adopt a 5-fold symmetry and fim expression had spread out (Figure 6, top left), adopting the shape of a doughnut of 5-8 cells thickness with a central “hole” about 2-3 ceils wide, which could only be seen on medial sections. The peripheral region of the meristem, where sepal primordia were starting to inititiate, lacked fim expression. At this stage, def transcripts could just be detected in the central region of the meristem, overlapping with the domain of fim expression. Expression of f/o seemed to be complementary to that of fim, most transcripts being detected in the peripheral region and absent from the dome (Figure 6, top center), whereas expression of squa was throughout the floral meristem. After another day of development (nodes 12-13) clearly visible sepal primordia surrounded a central dome that was 20 cells across (floritypic stage). Expression of fim had spread further out from the center and was focused in a ring of 5-8 cells thickness, at the base of the dome, adjacent to the sepal primordia (Figure 7, top left). Some weak patchy expression was also observed in the more central region of the dome. Expression of p/e could clearly be detected in the central dome at this stage, and its distribution was complementary to that of fim: the outer boundary of p/e seemed to coincide with the inner boundary of the fim ring (Figure 7, top panel). Transcripts of def could be detected throughout the dome and overlapped with the fim ring but did not extend as far outward (Figure 7, top panel). The outer boundary of fim expression appeared to coincide with the inner boundary of f/o expression, which was mainly restricted to the sepal primordia. During the next day of development, five local areas within the ring of fim expression were cleared of fim transcripts, anticipating the appearance of petal primoridia (see Figure 6, bottom left). By nodes 15-16, when petal primordiawere just visible as mounds (petal mound stage), the expression pattern formed five connected rings, each around the base of a petal primordium and about 3-4 cells in thickness. Expression of f/o had a complementary pattern, being restricted to the sepals and to the central region of each petal primordium that lacked fim (see Figure 6, bottom center). Theple gene was expressed in the central dome of the floral meristem, its outer boundary coinciding with the inner boundary of fim expression. Transcripts of def were restricted to the petal and presumptive stamen primordia, their outer boundary lying within the domain of fim expression. At later stages of development, essentially
the same expression pattern was maintained, fim being restricted to a region about five cells wide lying at the junction of petals with adjacent sepals and stamens (see Figure 5, bottom left). Expression of fim was also studied in inflorescences of fim-619 and fim-620 mutants: it was reduced in fim-679, although the distribution of transcripts was similar to wild type. In fim-620, expression was greatly reduced and only observed sporadically in isolated cells of the floral meristem (Figure 7, bottom left). Interaction between fim and Organ Identity Genes To study the interaction between fim and organ identity genes, the expression patterns of def and p/e in fim-620 mutant inflorescences were analyzed (Figure 7, bottom panel). Transcripts of def were not observed until the floritypic stage, a delay of about 1 day compared with wild type (Sommer et al., 1990; Bradley et al., 1993). Expression was very weak and shifted toward the center of the dome, little or no expression being seen in the presumptive second whorl. At later stages, when def is normally expressed throughout the developing petals and stamens of wild type, patches of def expression were observed in second- and third-whorl organs of fim-620 flowers (data not shown). Similarly, p/e expression was much weaker than in wild type at the floritypic stage and less extensive at later stages. The reciprocal experiment, analyzing fim expression in def and p/e mutants, showed no differences from wild type at early stages of development. In both cases, expression of fim formed rings around the base of second-whorl primordia, even though, in the case of def, these were destined to form sepals rather than petals. However, during later stages of p/e development, fim transcripts were no longer restricted to the base of second-whorl organs but were also detected around the base of all internal organs. The previous results indicated that fim mutations can reduce and delay the expression of def and p/e. Consistent with this, the phenotype of the most extreme fim-620 flowers was very similar to that of the def;p/e double mutant, i.e., flowers with an indeterminate number of sepals, sometimes arranged in a spiral toward the center (Ft. C. and E. C., unpublished data). To separate the genetic interactions of fim with def and p/e and to reveal any possible additional effects of fim, we constructed double mutants among fim, def, and p/e. The fim;def mutant had a similar phenotype to the def single mutant: two whorls of sepals enclosing a whorl of five fused carpels. However, further whorls of sepals were sometimes observed (Figure 8b), and several flowers had an additional, bilocular gynoecium in the center. This phenotype could be explained by a reduction in p/e activity in fim mutants, which might sometimes delay carpel formation and determinacy. The fim;p/e mutant had a normal outer whorl of sepals that enclosed an indeterminate number of petaloid sepals, a phenotype reminiscent of def;p/e, indicating a strong reduction in def activity (Figure 8~). As in the def;p/e double mutant, organs beyond the second whorl could not easily be divided into discrete whorls and tended to be arranged in a spiral. Occasionally, in fim;p/e mutants, the axils of the floral organs
Figure 7. In Situ Hybridization
of Wild-Type
and fim-620 Floral Meristems
Probed with fim, def, and p/e
Adjacent 9 urn sections through node 13 of wild type (top panel) or fim-620 (bottom panel) were probed with digoxigenin labeled RNA and viewed under light field, which gives the signal a purple color on a mauve background. Probes used are indicated in red. The arrows in the top panel indicates the inner or outer boundary of the fim or p/e expression domain, respectively. In fim-620, expression of def and p/e is reduced and less extensive than in wild type. Scale bar is 100 urn.
Figure 9. Genetic Interactions
between fim and f/o, squa, def, p/e
(a) fim-620 flower; (b) fim-62O;def-627 double mutant, with two or more outer whorls of sepals surrounding fused central carpels; (c) fim-62O;pb624 mutant with indeterminate whorls of sepals or sepaloid petals; (d) fim-619;f/o-662 mutant; the phenotype resembles the fim-620 single mutant flower; (e) axillary structures growing in place of flowers for fim-620 mutant heterozygous for squa-647 (left) and fim-620;squa-641 double mutant (right).
Figure 9. Expression Stage
Domains in the Floral Meristem at the Floritypic
Thediagram representsa longitudinal section through awild-type floral meristem at the floritypic stage. The order of the expression domains from outside to inside reflects the time of activation of the respective genes. The outer f/o domain (black) is restricted to whorl 1, the adjacent ring shaped fim emain (blue) overlaps partially (green) with the domain of def (yellow). The central p/e domain (red) overlaps partially (orange) with def but is separated by a sharp boundary from fim. At later stages, only p/e remains expressed in the center of the floral meristem, which will form whorl 4.
Fimbriata 105
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bore additional flowers, indicating a loss of lateral determinacy, a phenotype not seen in the def;ple mutants. Interactions of fim with Meristem identity Genes The early timing of fim expression, taken together with its effects on def and p/e, suggested that it may act as a mediator between meristem and organ identity genes. We therefore analyzed fim expression in f/o and squa mutant backgrounds. In f/o mutants, axillary meristems were morphologically indistinguishable from wild type up to about node 9, after which they resembled inflorescence meristems rather than the floral meristems of wild type (Coen et al., 1990). Unlike wild type, fim expression was not observed at node 9 or the stages immediately following. However, weak ectopic expression of fim was observed in the main (see Figure 5, top center) and axillary (see Figure 5, bottom center) inflorescence apices in f/o mutants. Axillary meristems mutant for squa also diverged morphologically from wild type at about node 9, after which they became either inflorescence meristems or abnormal flower meristems (Huijser et al., 1992). Expression of fim was not detected in axillary inflorescence meristems (see Figure 5, bottom right) but was observed, after a delay of about six nodes, in floral meristems with a pattern near to wild type (see Figure 5, top right). The functional interactions between fim and the meristem identity genes were analyzed through various double mutants. Because null f/o mutants do not produce flowers, they were expected to be epistatic to fim. This was confirmed by analysisof F2 populations from crosses between f/o nulls and fim that revealed no novel phenotype. We therefore made use of a weak f/o allele, f/o-662, which gave wild-type flowers when homozygous but flowers with split and distorted petals when heterozygous with a null f/o mutant. In a genetic background homozygous for f/o-662, fim-679 conferred a more severe phenotype than usual (Figure 8d). The stamens typically seen in fim-679 single mutants were replaced by carpels, which fused to form a central column. In the second whorl, the sepaloid areas occupied a relatively large proportion of the petals. This indicated that a reduction of f/o activity could reduce the activity of fim. When a fim-620 mutant was crossed with a plant carrying a strong allele of squa, squa-647, five phenotypes were observed in the F2 population in the numbers: 44 wild type, 9 fim, 12 extreme fim, 21 squa, and 7 extreme squa. Comparable ratios were also observed with another squa allele, squa-707. Several plants from each phenotypic class were analyzed by DNA blots to determine their genotype. The plants with the fim phenotype were homozygous for fim and Squa+, whereas those with a squa phenotype were homozygous for squa and homozygous or heterozygous for Fim’. Plants from the extreme fim class produced flowers with two or more outer whorls of sepals, sepaloid petals, and a central, immature carpel. Stamens developed only occasionally and were infertile. Very often secondary flowers or short inflorescences arose from the axils of floral organs, most typically those of whorl 2, and there was sometimes a loss of apical determinacy in the primary flowers (Figure 8e, left). The phenotype was most
extreme in flowers at the base of the main inflorescence. These plants were homozygous for fim but heterozygous for squa, indicating that squa was not fully recessive in a fim mutant background. Plants with the extreme squa phenotype failed to produce any flowers, having only lateral inflorescences instead. These were homozygous fim; squa double mutants, suggesting that fim together with squa has an important role in promoting floral meristem identity (Figure 8e, right). Discussion We show that fim occupies an intermediate position in a sequence of gene activation that starts with the meristem identity genes and leads to expression of the organ identity genes in specific whorls of the floral meristem. The fim gene is activated several days after the meristem identity genes f/o and squa, but before the organ identity genes def and p/e. A combination of genetic and expression studies reveals how fim may interact with upstream and downstream genes. In young axillary meristems of f/o mutants, which develop as inflorescences rather than as flowers, fim expression is not detected, indicating that fim transcription normally depends upon f/o activity. This could explain the phenotype of plants that have a promoter mutation in fim and also carry a weak allele of f/o: both mutations could reduce fim transcription levels, giving a strong overall reduction in fim activity and therefore an enhanced mutant phenotype. However, weak ectopic expression of fim is observed in the apex of the main inflorescence and older axillary inflorescences of f/o mutants, indicating that f/o may normally act as a negative regulator of fim in some regions. This also shows that activation of fim can occur independently of f/o. The meristem identity gene squa may interact with fim in a different manner: strong fim expression is observed in squa mutants but it is delayed, indicating a role for squa in setting the time of fim activation. This expression must have functional importance since fim mutations result in clear phenotypic effects in squa mutant backgrounds. Normally, null squa mutants produce some abnormal flowers (Huijser et al., 1992) but in afim;squa double mutant, only inflorescences proliferate and no flowers are observed. This suggests that squa acts together with fim to promote the transition of meristems from inflorescence to flower development. This transition seems to be poised at a critical or unstable position in fim mutants because in a fim mutant background removal of only one dose of the squa gene results in a shift toward inflorescence identity, whereas in a wild-type background squa mutants are fully recessive. The expression of def and p/e (genes needed for the b and c functions, respectively) is delayed and reduced in fim mutants, showing that fim is normally a positive regulator of these organ identity genes. This is confirmed by the analysis of double mutants. The fim;def mutant resembles the homozygous def single mutant, except that extra whorls of sepals or carpels are sometimes observed. These extra whorls might be expected if fim mutations
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reduce the level of p/e activity and hence delay sex organ development and floral determinacy. The fim;p/e mutant resembles the homozygous p/e single mutant except that petals are replaced by sepaloid organs, as expected if fim mutations reduce the level of def activity. Although def and p/e expression are initially very reduced in fim mutants, both genes are expressed in patches of cells at later stages, indicating either that all of the fim mutants are leaky or that a pathway independent of fim is able to activate def and p/e at later stages. The interactions between fim and the meristem and organ identity genes are sufficient to account for the various flower phenotypes seen in fim mutants. The most consistent effect of fim mutants is sepalody of the petals in whorl 2, which can be accounted for by the reduced transcription of b function genes. In fim-679 mutants, this effect can be seen as longitudinal green streaks of sepal tissue separated by files of intermediate cells from adjoining petal tissue. One possible explanation is that fim function is needed in cells at a very early stage of development to establish the b function. If this fails to occur in one or more cells, their descendants will form a clonal streak of sepal tissue. The identity of organs immediately interior to whorl 2 is consistent with a reduced activity of the b and c function genes as these organs may be transformed from stamens toward petals (loss of c), carpels (loss of b), sepals (loss of b and c), or a mixture of these. The extent of these transformations depends on both the strength of the fim allele and the environmental conditions. The proliferation of whorls that is occasionally observed in fim mutant flowers can also be explained by a reduction in c activity. Finally, the production of additional flowers or inflorescences in the axils of floral organs may reflect the normal role of fim, together with meristem identity genes, in imposing lateral determinacy. Some clues as to how fim may act as a mediator between meristem and organ identity genes come from detailed analysis of its expression pattern in relation to these genes. At a very early stage of development, when the floral meristem is loaf shaped, two domains begin to be defined by fim and f/o expression. A central domain expresses fim, whereas the rest of the meristem expresses f/o. This complementarity between fim and f/o expression is maintained and sharpened as the meristem grows. By the floritypic stage, f/o is restricted to the primordia of whorl 1, whereas fim occupies an adjacent ring-shaped domain that may provide the earliest visible marker for whorl 2 (Figure 9). During the next stages, transcripts of f/o appear to replace those of fim in five equidistant regions of the ring, anticipating the emergence of petal primordia. As the petal primordia grow, the complementarity between f/o and fim is maintained: f/o occupies the central region of each primordium while fim is found around the base. The complementarity in expression patterns might be explained if, in addition to a role of f/o as a general activator of fim, negative regulatory interactions between these genes determine their preciseexpression domains. Alternatively, the patterns of f/o and fim expression may reflect complementary interpretations of an underlying prepattern. Unlike f/o, expression of squa overlaps extensively with
fim, as might be expected if these two genes act together. The product of squa belongs to the MADS box family of
transcription factors (Huijser et al., 1992; Schwarz-Sommer et al, 1992). Two other members of this family, yeast MCMl and mammalian serum response factor, have been shown to recruit accessory proteins into functional DNAbinding complexes (Lydall et al., 1991; Dalton and Treisman, 1992). The F/M protein might therefore interact with MADS box proteins as an accessory protein, potentiating or specifying their function as transcriptional activators. Alternatively, the interaction between fim and squa products could be more indirect. By the time that the domains of fim and f/o have defined the outer whorls of the meristem, p/e expression starts to appear in the center of the meristem and spreads outward. By the late floritypic stage, the sharp outer boundary of p/e seems to coincide with the inner boundary of the ringshaped fim domain. The position of the fim boundary is not determined by p/e because fim expression appears to be normal in p/e mutants at this stage. However, a role for fim in setting the boundary of p/e between whorls 2 and 3 is possible because the outer boundary of p/e is less sharp and closer to the center of the meristem of fim mutants. Shortly before activation of the p/e gene, def expression starts in the center of the floral meristem and spreads outward during the following stages. The outer boundary of the def domain lies within the area of fim expression between whorls 1 and 2, but is shifted toward the center of the meristem in fim mutants, indicating that fim influences the expression domains of both p/e and def. One result of fim activity is therefore the correct positioning of the expression domains of organ identity genes in register with the morphological boundaries between organs in different whorls. However, this positioning by fim might involve different mechanisms for defandple, because unlike p/e, def expression overlaps with fim. The effect of fim on the outer boundary of b function activity may be compared with that of f/o-l0 or superman of Arabidopsis, which is thought to be involved in positioning the inner boundary (Schultz et al., 1991; Bowman et al., 1992). The superman gene acts as a repressor of b gene expression in whorl 4, but in contrast with fim, is not thought to be involved in activation of b. The expression patterns of fim, together with the meristem and organ identity genes, allow four partially overlapping regions to be defined at the floritypic stage (Figure 9). The spatial arrangement of gene expression mirrors the temporal sequence of gene activation: the domains starting from the outer whorl and moving inward are in the order f/o, fim, def, p/e, which is also the temporal order in which the transcripts of these genes appear. This could indicate that sequential processes underly the establishment of specific spatial domains. For example, gene activities may move outward from the central region of meristems as a series of consecutive waves that are somehow brought into rigister with waves of primordium initiation. The same process may be recapitulated in each organ primordium, as evidenced by the early activation of fim in presumptive whorl 2 cells, followed by its elimination from
Fimbriata 107
Mediates between Meristem
the central on there. Experimental
and Organ Identity
region of each petal primordium
as f/o comes
Procedures
Antirrhinum Stocks and Double Mutants The origins of fim-679 and fim-620 have been described previously under the names Sep.679 and sep-620, respectively (Carpenter and Coen, 1990). The lines def-627, p/e-624, and f/o-640, which were used for expression studies and the construction of double mutants, have been described previously (Carpenter and Coen. 1990; Coen et al.. 1990). Line f/o-662 was derived from line f/o-613 (Coen et al., 1990; R. C. and E. C., unpublished data). Lines squa-647 and squa-707 were obtained in a screen for transposon induced mutations and were therefore in the same genebc background as the other mutations. The genotype of all double mutants was analyzed by DNA blots. DNA Analysis The methods for DNAextraction and DNA blot analysiswere described previously (Bradley et al., 1993). The 4.0 kb fragment of the fim-620 mutant was cloned by digesting genomic DNA from pooled plants homozygous for the fim-620 mutation with Hindlll, fractionating the DNA by agarose gel electrophoresis and purification of the fraction between 3 kb and 5 kb by electroelution and ion-exchange chromatography using NACS columns (Bethesda Research Laboratories). The purified fraction was ligated into Hindlll digested 1NM1149, and phages carrying Tam3 sequences were identified using a BslEll-Xbal fragment of Tam3 as a probe. Sequences flanking the Tam3 insertion were subcloned as Smal-Hindlll into pGEM4Z (Pharmacia) to give pJAM136R. This fragment was then used to screen a IEMBL4 library of wild-type Antirrhinum DNA, partially digested with SauSA, and 24 overlapping clones with an average insert size of 16 kb were isolated. These clones were further characterized by Southern hybridization to obtain a detailed map of the fim locus. After the fim-679 mutation had been shown lo be caused by the insertion of a Tam3 element into a 0.8 kb Hindlll fragment (probe A) of the fim locus, the corresponding 4.4 kb Hindlll fragment from fim-679 was cloned by ligating a size fraction of Hindlll digested genomic DNA from a fim-679 mutant plant into lNMll49 and screening the recombinant phages with probe A. Five independent 1 clones, containing identical inserts were identified, and the inserts were subcloned to give pJAMl28. A 7.5 kb EcoRl fragment from the fim locus was subcloned into pGEM4.Z to give pJAMl37, and was sequenced by the dideoxynucleotide method. Subfragments of pJAMl37 were used as probes on Northern blots against poly(A) RNA from wild-type and mutant inflorescence tips. A 1 .l kb Hindlll fragment of pJAM137 (probe C) detected a 1.8 kb mRNA in Northern blots. A cDNA library in lNMll49 was synthesized using poly(A) RNA from very young wild-type inflorescence tips and screened with probe C. Four independent cDNA clones were isolated and subcloned into the EcoRl site of pGEM4Z. The longest cDNA clone was fully sequenced; the three shorter ones were sequenced from each end to confirm that they derived from the same mRNA species. The sequence of the cDNA clones was identical to the sequence obtained from the genomic clones, except for few base differences which are due to the fact that genomic and cDNA clones were obtained from different Antirrhinum lines. In Situ Hybridization The methods for digoxigenin labeling of RNA probes, tissue preparation, and in situ hybridization were as described in Bradley et al., 1993. The probes used lo detect p/e, def, and f/o transcripts were prepared as described in Bradley et al., 1993. A 1.2 kb fragment from the genomic clones, covering the open reading frame of fim, was subcloned Into pGEM4.Z to give plasmid pCTW6. Linearization with EcoRl or Hindlll provided templates for T7 or SP6 polymerases to generate sense or antisense RNA, respectively. Acknowledgments We are grateful to Hans Sommer for providing a genomic library of wild-type Antirrhinum and to Zsusanna Schwarz-Sommer for stimulating discussions. We thank Lucy Copsey for scanning electron microscopy, Coral Vincent for help with in situ hybridization, and the photogra-
phy department of the John lnnes Centre for their work. For critical discussion of the manuscript and comments. we thank Desmond Bradley, Elisabeth Schultz. Sabine Hantke. Gwyneth Ingram, and Sylvie Pouteau. We are grateful to the Biotechnology Program of the European Community for a postdoctoral fellowship lo R. S., and for support from the Agriculture and Food Research Council Plant Molecular Biology program and the European Community Biotechnology Research for Innovation, Development, and Growth in Europe (BRIDGE) program. Received January 31. 1994; revised May 13, 1994 References Baur, E. (1930). MutationsauslBsung 23, 676-702.
bei Antirrhinum
majus. Z. Bat.
Bowman, J. L., Sakai, I. I., Jack, T., Weigel, D.. Mayer, U., and Meyerowitz, E. M. (1992). SUPERMAN, a regulator of floral homeotic genes in Arabidopsis. Development 7 74, 599-615. Bowman, J. L., Smyth. D. R., and Meyerowitz, E. M. (1991). Genetic interactions among floral homeotic genes of Arabidopsis. Development 772, I-20. Bradley, D., Carpenter, R., Sommer, H.. Hartley, N., and Coen, E. S. (1993). Complementary floral homeotic phenotypes result from opposite orientations of a transposon at the plena locus of Antirrhinum. Cell 72, 85-95. Carpenter, R., and Coen, E. S. (1990). Floral homeotic mutations produced by transposon-mutagenesis in Antirrhinum majus. Genes Dev. 4, 1483-1493. Coen, E. S., Romero, J. M., Doyle, S., Elliott, R., Murphy, G., and Carpenter, R. (1990). floricaula: a homeotic gene required for flower development in Antirrhinum majus. Cell 63, 1311-1322. Coen. E. S., and Meyerowitz, E. M. (1991). The war of the whorls: genetic interactions controlling flower development. Nature 353, 3137. Coen, E. S., and Carpenter, Plant Cell 5, 1175-l 181
R. (1993). The metamorphosis
of flowers.
Dalton, S., and Treisman, R. (1992). Characterization of SAP-l, a protein recruited by serum response factor lo the c-fos serum response element. Cell 68, 597-612. Harte, C. (1951). Untersuchungen ijber labile Gene: Selektionsversuche an Antirrhinum majus mut. fimbriata. Z. Indukt. Abstammungs Vererbungslehre 83, 392-413. Huijser, P., Klein, J., Ltinnig, W.-E., Meijer, H., Saedler, H., and Sommer. H. (1992) Bracteomania, an inflorescence anomaly, is caused by the loss of function of the MADS-box gene squamosa in Antirrhirwm majus. EMBO J. 77, 1239-1250. Lydall. D., Ammerer, G., and Nasmyth, K. (1991). A new role for MCMl in yeast: cell cycle regulation of SW15 transcription. Genes Dev. 5, 2405-2419. Schultz. E. A., Pickett, F. B., and Haughn. G. W. (1991). The FL070 gene product regulates the expression domain of homeotic genesAP and PI in Arabidopsis flowers. Plant Cell 3, 1221-l 237. Schwarz-Sommer, Z., Huijser, P.. Nacken, W., Saedler. H., and Sommer. H. (1990). Genetic control of flower development by homeotic genes in Antirrhinum majus. Science 250, 931-936. Schwarz-Sommer, Z., Hue, I., Huijser, P., Flor, P. J., Hansen, R., Tetens, F., Lbnnig, W.-E., Saedler, H., and Sommer, H. (1992). Characterization of the Antirrhirwm floral homeotic MADS-box gene de& ciens: Evidence for DNA binding and autoregulation of its persistent expression throughout flower development. EMBO J. 77, 251-263. Sommer, H., Beltran, J-P., Huijser, P., Pape, H., LBnnig, W.-E., Saedler, H., and Schwarz-Sommer, Z. (1990). Deficiens, a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus: the protein shows homology to transcription factors. EM60 J. 9, 605-613.
July 15, 1994, Copyright
0 1994 by Cell Press
Fimbriata Controls Flower Development by Mediating between Meristem and O rgan Identity Genes Riidiger Simon, Rosemary Carpenter, and Enrico Coen John lnnes Centre Colney Lane Norwich NR4 7UH England
Sandra Doyle,
Summary Two major classes of genes directing flower development have so far been described: early activated genes regulating meristem identity and later acting genes controlling organ identity. Here, we show that the fimbriata (fim) gene acts between these two classes in a sequence of gene activation. The fim gene, originally described in 1930, was cloned by transposon tagging from Antirrhinum majus and encodes a product with no detectable homology to other proteins. Mutations in fim result in partial homeotic transformations of floral organs and in reduced determinacy of the meristem. Expression and function of fim depends on the activity of meristem identity genes, and fim in turn controls the spatial and temporal expression of organ identity genes. The pattern of fim expression defines a new domain of the floral meristem that changes with time in a complementary manner to those of the meristem identity gene f/o&au/a and the organ identity gene plena. Introduction The development of plants depends largely on the behavior of their meristems, groups of rapidly dividing cells that form the growing points. On the periphery of meristems, groups of cells are partitioned off to form either secondary meristems or organ primordia. There are several classes of meristem that are distinguished by the types and arrangements of primordia and secondary meristems they produce. For example, vegetative meristems produce leaf primordia with secondary shoot meristems in their axils. lnflorescence meristems typically produce bract primordia with flower meristems in their axils. Flower meristems produce concentric whorls of four types of organ primordia: whorl 1 (outermost) contains sepals, whorl 2 consists of petals, whorl 3 contains stamens, and whorl 4 contains carpels. Unlike shoot meristems, the growth of flower meristems has a precisely defined determined end point: they do not produce secondary meristems between the whorls (lateral determinacy) and stop initiating organ primordia after the central whorl 4 (apical determinacy). Genetic and molecular studies in Antirrhinum and Arabidopsis have so far defined two main types of genes involved in the control of flower development, meristem identity and organ identity genes (Schwarz-Sommer et al., 1990; Coen and Meyerowitz, 1991; Coen and Carpenter, 1993). Meristem identity genes are involved in initiating the
flower developmental program; mutations in them cause inflorescence meristems to replace floral meristems. Mutations in organ identity genes affect the fate of organ primordia, most often in two adjacent whorls. These mutations can be divided into three classes: a, those that have carpels growing in place of sepals and stamens in place of petals; b, those with sepals instead of petals and carpels instead of stamens; and c, those with a proliferation of petals or sepals instead of stamens and carpels in the center of the flower. Each class of mutation corresponds to a loss in one of three homeotic functions (a, b, or c), which normally act in combination to specify organ type, so that in wild-type floral meristems the combinations in whorls l-4 are a, ab, bc, and c (Carpenter and Coen, 1990; Bowman et al., 1991). Although there is some functional overlap between meristem and organ identity genes, so far genes that mediate between the two classes have not been defined. Here, we describe a novel type of gene, fimbriata (fim), that mediates between meristem and organ identity genes in Antirrhinum. Mutations in fim have effectson the identity of organs in whorls 2, 3, and 4 and also on the lateral determinacy of the floral meristem. Genetic analysis shows that fim interacts with meristem identity genes to initiate the flower developmental program and with organ identity genes to control the fate of primordia. Expression of fim is activated after the meristem identity genes floricaula (f/o) and squamosa (qua) but before deficiens (def) andplena (p/e), genes needed for the b and c organ identity functions, respectively (Coen et al., 1990; Huijser et al., 1992; Sommer et al., 1990; Bradley et al., 1993). Unlike def and p/e, which are expressed in developing organ primordia, fim is expressed transiently in floral meristems and eventually becomes restricted to a region lying at the base of primordia. We propose a sequence of gene activation leading from f/o and sgua through fim to organ identity genes such as def and p/e. This sequence is reflected in both the temporal order of gene activation and in the spatial arrangement of expression domains. Results The Phenotype of fim Mutants Two independent recessive mutations that gave petals with sepal-like tissue, termed the sepaloidea phenotype, were isolated in a large scale transposon mutagenesis program (Carpenter and Coen, 1990). The phenotypes were reminiscent of a classical mutation at the fim locus described previously (Baur, 1930; Harte, 1951), and genetic tests confirmed that both sepaloidea mutations, subsequently called fim-679 and fim-620, were allelic to fim. The most consistent feature of fim-679 mutants was that the petals had longitudinal streaks of green sepaloid tissue, often running the entire length of the petal (Figure 1 a). The streaks were not randomly distributed; they most often ran along the middle or the edges of lower petals. Scanning electron microscopy confirmed that the green
Cell 100
Figure 1. Phenotypes Mutant Flowers
of fim-679 and fim-620
Flowers of the fim-679 mutant (a) have streaks of green, sepaloid tissue in the petals (arrows). The scanning electron micrograph of the border of a green streak (b) shows the sepaloid cells in the streak (right half) and the adjacent petal cells (left half). Flowers of the fim-620 mutant (c) have petals with larger sepaloid areas, and the stamens are mostly replaced by fused central carpels. Occasionally, a secondary flower is initiated from the second whorl of sepals, as shown by a flower cut open longitudinally (d).
areas contained typical sepaloid cells and revealed that cells lying between these and the adjacent petal cells had intermediate characteristics (Figure 1 b). The longitudinal growth of the green streaks appeared to be less than that of petal tissue, resulting in a distortion in the final shape of the petals. The stamens in whorl 3 were sometimes petaloid, while sepals and carpels developed as in wild me. Plants homozygous for fim-620 had flowers with a variable phenotype, ranging from flowers resembling fim-679 to extreme cases in which flowers consisted of an indeterminate number of sepals. This variability seemed to depend partly on environmental conditions: plants grown in the greenhouse generally produced weaker phenotypes than plants grown in the field in summer. In all cases, the first whorl of sepals was normal but instead of petals, the second whorl contained organs that resembled sepals with varying amounts of petaloid tissue (Figure lc). Organs beyond the second whorl were not usually arranged as discrete whorls, and their identity varied greatly between flowers. In most fim-620 flowers analyzed, there were 510 organs internal to whorl 2, comprising O-5 perianth (sepaloid or petaloid) organs surrounding O-7 central carpeloid organs. The central carpels tended to fuse, often forming a syncarpous and multilocular gynoecium resembling the central structures produced in def mutants of Antirrhinum (Sommer et al., 1990). In some cases, the carpeloid organs lacked a complete style and looked like sepals with stigmatic tips, enclosing ovaries or naked ovules.
Occasionally, fim-620 flowers showed a loss of apical determinacy leading to the production of an indeterminate number of sepals. A partial loss of lateral determinacy was also sometimes observed, secondary flowers or inflorescence shoots being initiated in the axils of second-whorl sepals (Figure 1 d). Isolation and Structure of the fim Gene Genomic DNA from fim-679, fim-620, and their wild-type progenitor was digested with Hindlll and probed with a fragment of Tam3. A novel 4.0 kb fragment was observed in fim-620 that cosegregated with the fim phenotype. The fragment was cloned by isolating a 3-5 kb fraction of Hindlll-digested genomic fim-620 DNA, ligating to a h vector, and screening with a Tam3 probe. The flanking sequence to the left of the Tam3 insertion was then used as a probe against Hindlll-digested DNA from various genotypes. The expected 4.0 kb band was detected in fim-620, whereas the wild-type progenitor gave a 1 .l kb band. The wild-type sequence was cloned by using the flanking probe to screen agenomic library. Further mapping by DNA blots showed that the Tam3 insertion in fim-620 had been accompanied by a genomic rearrangement. To prove that the wild-type clones corresponded to the fim locus, we analyzed the fim-679 mutation. This allele was known t%be slightly unstable because self-pollination of homozygous fim-679 plants gave revertant progeny with a wild-type phenotype at a rate of 1.40/o, indicating that the mutation was caused by a transposon insertion. A 0.8 kb Hindlll fragment (probe A) from the putative fim locus
Fimbriata 101
Mediates between Meristem
and Organ Identity
fim-619
fim
Fim +
E
4.4 kb
\/a
-
a
-iA A
HH
a
E
fim-620
Figure 3. Structure
of the fim Locus
The insertion sites of Tam3 in fim-679 and in fim-620 are shown by closed bars. The DNA sequences to the right of Tam3 in firm620 are rearranged. The stippled arrow indicates the open reading frame of the fim gene. The Hindlll fragment used to identify the insertion in firm619 is labeled A. Restriction sites are EcoRl (E) or Hindlll (H).
0.8 kb
:_ .:
1
Figure 2. Southern
2
3
Slot of Hindlll-Digested
4
5
6
DNA
Wild type (lane l), fin?-619 (lane 6) and four independent revertants of fim-619 (lanes 2-5) probed with a DNA fragment flanking the transposon insertion in fifn-620 (probe A, see Figure 3). The phenotype of the individual plants is indicated above the lanes, The fim-679 mutant and the (heterozygous) revertants carry the 4.4 kb insertion fragment. Excision of the 3.6 kb Tam3 element from fim-619 restores the 0.6 kb wild-type band.
was used as a probe against homozygous fim-619 mutants and four independent revertants. The fim-619 DNA gave a fragment of 4.4 kb, whereas the revertants had the restored 0.8 kb fragment of wild type as well as the 4.4 kb fragment, as expected from their heterozygous genotype (Figure 2). The fim-679 allele was cloned and shown to contain a Tam3 insertion 0.8 kb from the disruption found in fim-620 (Figure 3). To identify the fim transcript, various fragments from the fim locus were used as probes on blots of poly(A) RNA from wild-type inflorescences. The Hindlll fragment spanning the site of the transposon disruption in fim-620 detected a 1.8 kb transcript, which was specifically expressed in young inflorescences, but not detectable in mature flowers, leaves, or stems (data not shown). The expression level of this transcript was strongly reduced in
inflorescences of fim-619 and fim-620. Four independent cDNA clones corresponding to this transcript were isolated from a cDNA library made from wild-type inflorescences. These cDNAs appeared to originate from the same transcript and mapped to a region immediately to the right of the Tam3 disruption site in fim-620. The disruption points in fim-619 and fim-620 were found to be 976 bp and 196 bp upstream of the 5’ end of the longest cDNA clone, respectively. Further fim mutations were also shown to have deletions or insertions mapping to this transcribed region (G. Ingram, R. C., R. S., and E. C., unpublished data). As deduced from the comparison between the cDNA and the genomic sequence, the transcription unit consisted of one uninterrupted open reading frame of 1287 bp (Figure 4). This had the potential to code for a 429 amino acid protein of 49 kDa M,, which showed no similarity to any other protein sequence in the data bases. Expression Pattern of fim The timing and the histological distribution of fim RNA was determined by in situ hybridization using digoxigeninlabeled fim antisense RNA as a probe against wild-type
Figure 4. Sequence quence
of the fifn cDNA and the Derived
Protein Se-
The most 5’ ATG is assumed to be the start codon. Amino acids are indicated in the standard one letter code.
Cell 102
Figure 5. In Situ Hybridization
of Wild-Type
and Mutant Sections Probed with firn
Sections of wild type (wt), f/o, and squa inflorescences were probed with digoxigenin-labeled fim antisense RNA and viewed under dark field, which gives the signal a pink color on a bright blue background. The top row shows main inflorescence apices. The bottom row shows later nodes, illustrating either floral meristems (left) or axillary inflorescence meristems (center and right). Scale bar (shown once per row) is 100 pm. b, bracts; s, sepals; p, petals; st, stamen.
inflorescences. Expression of fim was compared with the expression patterns of meristem and organ identity genes (Figures 5 and 6). The earliest stages of flower development were near the top of the inflorescence apex, and progressively later stages were below this. Flower meri-
Figure 6. Wild-Type
Floral Meristems
stems and their subjacent bract primordia (together termed a node) were numbered sequentially starting with node 0 at the top. For plants grown at 20°C, the rate of node initiation was about two per day, so the time interval between node initiation (plastochron) was approximately
Probed with fim or f/o
Adjacent sections of a floral meristem at the pentagon stage (top row) and at the petal-mound stage (bottom row) were probed with fim (left) or f/o (center), showing the complementarity of their expression pattern (see arrows). The sections were not medial and were viewed under light field, which gives the signal a purple color on a mauve background. Scanning electron micrographs show the morphological stages of the meristems at the respective nodes. Scale bar (shown once per row) is 100 pm. s, sepals.
Fimbriata 103
Mediates
between Meristem and Organ Identity
12 hr. We have previously shown that meristem identity genes are activated in the youngest visible node (node 0) and that organ identity genes are expressed by node 1 O11 (Bradley et al., 1993). The earliest stage at which fim transcripts could be detected was in floral meristems at node 9 (Figure 5, top left), before the organ identity genes were expressed. At this stage, no organ primordia were visible and the meristem had the appearance of a loaf of bread (Ft. C. and E. C., unpublished data). Sections through the middle of the loaf showed that fim transcripts first accumulated toward the abaxial side of the central dome, in a region about 4-5 cells wide. By nodes 1 O-l 1, corresponding to a further day of development, the floral meristem had started to adopt a 5-fold symmetry and fim expression had spread out (Figure 6, top left), adopting the shape of a doughnut of 5-8 cells thickness with a central “hole” about 2-3 ceils wide, which could only be seen on medial sections. The peripheral region of the meristem, where sepal primordia were starting to inititiate, lacked fim expression. At this stage, def transcripts could just be detected in the central region of the meristem, overlapping with the domain of fim expression. Expression of f/o seemed to be complementary to that of fim, most transcripts being detected in the peripheral region and absent from the dome (Figure 6, top center), whereas expression of squa was throughout the floral meristem. After another day of development (nodes 12-13) clearly visible sepal primordia surrounded a central dome that was 20 cells across (floritypic stage). Expression of fim had spread further out from the center and was focused in a ring of 5-8 cells thickness, at the base of the dome, adjacent to the sepal primordia (Figure 7, top left). Some weak patchy expression was also observed in the more central region of the dome. Expression of p/e could clearly be detected in the central dome at this stage, and its distribution was complementary to that of fim: the outer boundary of p/e seemed to coincide with the inner boundary of the fim ring (Figure 7, top panel). Transcripts of def could be detected throughout the dome and overlapped with the fim ring but did not extend as far outward (Figure 7, top panel). The outer boundary of fim expression appeared to coincide with the inner boundary of f/o expression, which was mainly restricted to the sepal primordia. During the next day of development, five local areas within the ring of fim expression were cleared of fim transcripts, anticipating the appearance of petal primoridia (see Figure 6, bottom left). By nodes 15-16, when petal primordiawere just visible as mounds (petal mound stage), the expression pattern formed five connected rings, each around the base of a petal primordium and about 3-4 cells in thickness. Expression of f/o had a complementary pattern, being restricted to the sepals and to the central region of each petal primordium that lacked fim (see Figure 6, bottom center). Theple gene was expressed in the central dome of the floral meristem, its outer boundary coinciding with the inner boundary of fim expression. Transcripts of def were restricted to the petal and presumptive stamen primordia, their outer boundary lying within the domain of fim expression. At later stages of development, essentially
the same expression pattern was maintained, fim being restricted to a region about five cells wide lying at the junction of petals with adjacent sepals and stamens (see Figure 5, bottom left). Expression of fim was also studied in inflorescences of fim-619 and fim-620 mutants: it was reduced in fim-679, although the distribution of transcripts was similar to wild type. In fim-620, expression was greatly reduced and only observed sporadically in isolated cells of the floral meristem (Figure 7, bottom left). Interaction between fim and Organ Identity Genes To study the interaction between fim and organ identity genes, the expression patterns of def and p/e in fim-620 mutant inflorescences were analyzed (Figure 7, bottom panel). Transcripts of def were not observed until the floritypic stage, a delay of about 1 day compared with wild type (Sommer et al., 1990; Bradley et al., 1993). Expression was very weak and shifted toward the center of the dome, little or no expression being seen in the presumptive second whorl. At later stages, when def is normally expressed throughout the developing petals and stamens of wild type, patches of def expression were observed in second- and third-whorl organs of fim-620 flowers (data not shown). Similarly, p/e expression was much weaker than in wild type at the floritypic stage and less extensive at later stages. The reciprocal experiment, analyzing fim expression in def and p/e mutants, showed no differences from wild type at early stages of development. In both cases, expression of fim formed rings around the base of second-whorl primordia, even though, in the case of def, these were destined to form sepals rather than petals. However, during later stages of p/e development, fim transcripts were no longer restricted to the base of second-whorl organs but were also detected around the base of all internal organs. The previous results indicated that fim mutations can reduce and delay the expression of def and p/e. Consistent with this, the phenotype of the most extreme fim-620 flowers was very similar to that of the def;p/e double mutant, i.e., flowers with an indeterminate number of sepals, sometimes arranged in a spiral toward the center (Ft. C. and E. C., unpublished data). To separate the genetic interactions of fim with def and p/e and to reveal any possible additional effects of fim, we constructed double mutants among fim, def, and p/e. The fim;def mutant had a similar phenotype to the def single mutant: two whorls of sepals enclosing a whorl of five fused carpels. However, further whorls of sepals were sometimes observed (Figure 8b), and several flowers had an additional, bilocular gynoecium in the center. This phenotype could be explained by a reduction in p/e activity in fim mutants, which might sometimes delay carpel formation and determinacy. The fim;p/e mutant had a normal outer whorl of sepals that enclosed an indeterminate number of petaloid sepals, a phenotype reminiscent of def;p/e, indicating a strong reduction in def activity (Figure 8~). As in the def;p/e double mutant, organs beyond the second whorl could not easily be divided into discrete whorls and tended to be arranged in a spiral. Occasionally, in fim;p/e mutants, the axils of the floral organs
Figure 7. In Situ Hybridization
of Wild-Type
and fim-620 Floral Meristems
Probed with fim, def, and p/e
Adjacent 9 urn sections through node 13 of wild type (top panel) or fim-620 (bottom panel) were probed with digoxigenin labeled RNA and viewed under light field, which gives the signal a purple color on a mauve background. Probes used are indicated in red. The arrows in the top panel indicates the inner or outer boundary of the fim or p/e expression domain, respectively. In fim-620, expression of def and p/e is reduced and less extensive than in wild type. Scale bar is 100 urn.
Figure 9. Genetic Interactions
between fim and f/o, squa, def, p/e
(a) fim-620 flower; (b) fim-62O;def-627 double mutant, with two or more outer whorls of sepals surrounding fused central carpels; (c) fim-62O;pb624 mutant with indeterminate whorls of sepals or sepaloid petals; (d) fim-619;f/o-662 mutant; the phenotype resembles the fim-620 single mutant flower; (e) axillary structures growing in place of flowers for fim-620 mutant heterozygous for squa-647 (left) and fim-620;squa-641 double mutant (right).
Figure 9. Expression Stage
Domains in the Floral Meristem at the Floritypic
Thediagram representsa longitudinal section through awild-type floral meristem at the floritypic stage. The order of the expression domains from outside to inside reflects the time of activation of the respective genes. The outer f/o domain (black) is restricted to whorl 1, the adjacent ring shaped fim emain (blue) overlaps partially (green) with the domain of def (yellow). The central p/e domain (red) overlaps partially (orange) with def but is separated by a sharp boundary from fim. At later stages, only p/e remains expressed in the center of the floral meristem, which will form whorl 4.
Fimbriata 105
Mediates
between Meristem and Organ Identity
bore additional flowers, indicating a loss of lateral determinacy, a phenotype not seen in the def;ple mutants. Interactions of fim with Meristem identity Genes The early timing of fim expression, taken together with its effects on def and p/e, suggested that it may act as a mediator between meristem and organ identity genes. We therefore analyzed fim expression in f/o and squa mutant backgrounds. In f/o mutants, axillary meristems were morphologically indistinguishable from wild type up to about node 9, after which they resembled inflorescence meristems rather than the floral meristems of wild type (Coen et al., 1990). Unlike wild type, fim expression was not observed at node 9 or the stages immediately following. However, weak ectopic expression of fim was observed in the main (see Figure 5, top center) and axillary (see Figure 5, bottom center) inflorescence apices in f/o mutants. Axillary meristems mutant for squa also diverged morphologically from wild type at about node 9, after which they became either inflorescence meristems or abnormal flower meristems (Huijser et al., 1992). Expression of fim was not detected in axillary inflorescence meristems (see Figure 5, bottom right) but was observed, after a delay of about six nodes, in floral meristems with a pattern near to wild type (see Figure 5, top right). The functional interactions between fim and the meristem identity genes were analyzed through various double mutants. Because null f/o mutants do not produce flowers, they were expected to be epistatic to fim. This was confirmed by analysisof F2 populations from crosses between f/o nulls and fim that revealed no novel phenotype. We therefore made use of a weak f/o allele, f/o-662, which gave wild-type flowers when homozygous but flowers with split and distorted petals when heterozygous with a null f/o mutant. In a genetic background homozygous for f/o-662, fim-679 conferred a more severe phenotype than usual (Figure 8d). The stamens typically seen in fim-679 single mutants were replaced by carpels, which fused to form a central column. In the second whorl, the sepaloid areas occupied a relatively large proportion of the petals. This indicated that a reduction of f/o activity could reduce the activity of fim. When a fim-620 mutant was crossed with a plant carrying a strong allele of squa, squa-647, five phenotypes were observed in the F2 population in the numbers: 44 wild type, 9 fim, 12 extreme fim, 21 squa, and 7 extreme squa. Comparable ratios were also observed with another squa allele, squa-707. Several plants from each phenotypic class were analyzed by DNA blots to determine their genotype. The plants with the fim phenotype were homozygous for fim and Squa+, whereas those with a squa phenotype were homozygous for squa and homozygous or heterozygous for Fim’. Plants from the extreme fim class produced flowers with two or more outer whorls of sepals, sepaloid petals, and a central, immature carpel. Stamens developed only occasionally and were infertile. Very often secondary flowers or short inflorescences arose from the axils of floral organs, most typically those of whorl 2, and there was sometimes a loss of apical determinacy in the primary flowers (Figure 8e, left). The phenotype was most
extreme in flowers at the base of the main inflorescence. These plants were homozygous for fim but heterozygous for squa, indicating that squa was not fully recessive in a fim mutant background. Plants with the extreme squa phenotype failed to produce any flowers, having only lateral inflorescences instead. These were homozygous fim; squa double mutants, suggesting that fim together with squa has an important role in promoting floral meristem identity (Figure 8e, right). Discussion We show that fim occupies an intermediate position in a sequence of gene activation that starts with the meristem identity genes and leads to expression of the organ identity genes in specific whorls of the floral meristem. The fim gene is activated several days after the meristem identity genes f/o and squa, but before the organ identity genes def and p/e. A combination of genetic and expression studies reveals how fim may interact with upstream and downstream genes. In young axillary meristems of f/o mutants, which develop as inflorescences rather than as flowers, fim expression is not detected, indicating that fim transcription normally depends upon f/o activity. This could explain the phenotype of plants that have a promoter mutation in fim and also carry a weak allele of f/o: both mutations could reduce fim transcription levels, giving a strong overall reduction in fim activity and therefore an enhanced mutant phenotype. However, weak ectopic expression of fim is observed in the apex of the main inflorescence and older axillary inflorescences of f/o mutants, indicating that f/o may normally act as a negative regulator of fim in some regions. This also shows that activation of fim can occur independently of f/o. The meristem identity gene squa may interact with fim in a different manner: strong fim expression is observed in squa mutants but it is delayed, indicating a role for squa in setting the time of fim activation. This expression must have functional importance since fim mutations result in clear phenotypic effects in squa mutant backgrounds. Normally, null squa mutants produce some abnormal flowers (Huijser et al., 1992) but in afim;squa double mutant, only inflorescences proliferate and no flowers are observed. This suggests that squa acts together with fim to promote the transition of meristems from inflorescence to flower development. This transition seems to be poised at a critical or unstable position in fim mutants because in a fim mutant background removal of only one dose of the squa gene results in a shift toward inflorescence identity, whereas in a wild-type background squa mutants are fully recessive. The expression of def and p/e (genes needed for the b and c functions, respectively) is delayed and reduced in fim mutants, showing that fim is normally a positive regulator of these organ identity genes. This is confirmed by the analysis of double mutants. The fim;def mutant resembles the homozygous def single mutant, except that extra whorls of sepals or carpels are sometimes observed. These extra whorls might be expected if fim mutations
Cdl 106
reduce the level of p/e activity and hence delay sex organ development and floral determinacy. The fim;p/e mutant resembles the homozygous p/e single mutant except that petals are replaced by sepaloid organs, as expected if fim mutations reduce the level of def activity. Although def and p/e expression are initially very reduced in fim mutants, both genes are expressed in patches of cells at later stages, indicating either that all of the fim mutants are leaky or that a pathway independent of fim is able to activate def and p/e at later stages. The interactions between fim and the meristem and organ identity genes are sufficient to account for the various flower phenotypes seen in fim mutants. The most consistent effect of fim mutants is sepalody of the petals in whorl 2, which can be accounted for by the reduced transcription of b function genes. In fim-679 mutants, this effect can be seen as longitudinal green streaks of sepal tissue separated by files of intermediate cells from adjoining petal tissue. One possible explanation is that fim function is needed in cells at a very early stage of development to establish the b function. If this fails to occur in one or more cells, their descendants will form a clonal streak of sepal tissue. The identity of organs immediately interior to whorl 2 is consistent with a reduced activity of the b and c function genes as these organs may be transformed from stamens toward petals (loss of c), carpels (loss of b), sepals (loss of b and c), or a mixture of these. The extent of these transformations depends on both the strength of the fim allele and the environmental conditions. The proliferation of whorls that is occasionally observed in fim mutant flowers can also be explained by a reduction in c activity. Finally, the production of additional flowers or inflorescences in the axils of floral organs may reflect the normal role of fim, together with meristem identity genes, in imposing lateral determinacy. Some clues as to how fim may act as a mediator between meristem and organ identity genes come from detailed analysis of its expression pattern in relation to these genes. At a very early stage of development, when the floral meristem is loaf shaped, two domains begin to be defined by fim and f/o expression. A central domain expresses fim, whereas the rest of the meristem expresses f/o. This complementarity between fim and f/o expression is maintained and sharpened as the meristem grows. By the floritypic stage, f/o is restricted to the primordia of whorl 1, whereas fim occupies an adjacent ring-shaped domain that may provide the earliest visible marker for whorl 2 (Figure 9). During the next stages, transcripts of f/o appear to replace those of fim in five equidistant regions of the ring, anticipating the emergence of petal primordia. As the petal primordia grow, the complementarity between f/o and fim is maintained: f/o occupies the central region of each primordium while fim is found around the base. The complementarity in expression patterns might be explained if, in addition to a role of f/o as a general activator of fim, negative regulatory interactions between these genes determine their preciseexpression domains. Alternatively, the patterns of f/o and fim expression may reflect complementary interpretations of an underlying prepattern. Unlike f/o, expression of squa overlaps extensively with
fim, as might be expected if these two genes act together. The product of squa belongs to the MADS box family of
transcription factors (Huijser et al., 1992; Schwarz-Sommer et al, 1992). Two other members of this family, yeast MCMl and mammalian serum response factor, have been shown to recruit accessory proteins into functional DNAbinding complexes (Lydall et al., 1991; Dalton and Treisman, 1992). The F/M protein might therefore interact with MADS box proteins as an accessory protein, potentiating or specifying their function as transcriptional activators. Alternatively, the interaction between fim and squa products could be more indirect. By the time that the domains of fim and f/o have defined the outer whorls of the meristem, p/e expression starts to appear in the center of the meristem and spreads outward. By the late floritypic stage, the sharp outer boundary of p/e seems to coincide with the inner boundary of the ringshaped fim domain. The position of the fim boundary is not determined by p/e because fim expression appears to be normal in p/e mutants at this stage. However, a role for fim in setting the boundary of p/e between whorls 2 and 3 is possible because the outer boundary of p/e is less sharp and closer to the center of the meristem of fim mutants. Shortly before activation of the p/e gene, def expression starts in the center of the floral meristem and spreads outward during the following stages. The outer boundary of the def domain lies within the area of fim expression between whorls 1 and 2, but is shifted toward the center of the meristem in fim mutants, indicating that fim influences the expression domains of both p/e and def. One result of fim activity is therefore the correct positioning of the expression domains of organ identity genes in register with the morphological boundaries between organs in different whorls. However, this positioning by fim might involve different mechanisms for defandple, because unlike p/e, def expression overlaps with fim. The effect of fim on the outer boundary of b function activity may be compared with that of f/o-l0 or superman of Arabidopsis, which is thought to be involved in positioning the inner boundary (Schultz et al., 1991; Bowman et al., 1992). The superman gene acts as a repressor of b gene expression in whorl 4, but in contrast with fim, is not thought to be involved in activation of b. The expression patterns of fim, together with the meristem and organ identity genes, allow four partially overlapping regions to be defined at the floritypic stage (Figure 9). The spatial arrangement of gene expression mirrors the temporal sequence of gene activation: the domains starting from the outer whorl and moving inward are in the order f/o, fim, def, p/e, which is also the temporal order in which the transcripts of these genes appear. This could indicate that sequential processes underly the establishment of specific spatial domains. For example, gene activities may move outward from the central region of meristems as a series of consecutive waves that are somehow brought into rigister with waves of primordium initiation. The same process may be recapitulated in each organ primordium, as evidenced by the early activation of fim in presumptive whorl 2 cells, followed by its elimination from
Fimbriata 107
Mediates between Meristem
the central on there. Experimental
and Organ Identity
region of each petal primordium
as f/o comes
Procedures
Antirrhinum Stocks and Double Mutants The origins of fim-679 and fim-620 have been described previously under the names Sep.679 and sep-620, respectively (Carpenter and Coen, 1990). The lines def-627, p/e-624, and f/o-640, which were used for expression studies and the construction of double mutants, have been described previously (Carpenter and Coen. 1990; Coen et al.. 1990). Line f/o-662 was derived from line f/o-613 (Coen et al., 1990; R. C. and E. C., unpublished data). Lines squa-647 and squa-707 were obtained in a screen for transposon induced mutations and were therefore in the same genebc background as the other mutations. The genotype of all double mutants was analyzed by DNA blots. DNA Analysis The methods for DNAextraction and DNA blot analysiswere described previously (Bradley et al., 1993). The 4.0 kb fragment of the fim-620 mutant was cloned by digesting genomic DNA from pooled plants homozygous for the fim-620 mutation with Hindlll, fractionating the DNA by agarose gel electrophoresis and purification of the fraction between 3 kb and 5 kb by electroelution and ion-exchange chromatography using NACS columns (Bethesda Research Laboratories). The purified fraction was ligated into Hindlll digested 1NM1149, and phages carrying Tam3 sequences were identified using a BslEll-Xbal fragment of Tam3 as a probe. Sequences flanking the Tam3 insertion were subcloned as Smal-Hindlll into pGEM4Z (Pharmacia) to give pJAM136R. This fragment was then used to screen a IEMBL4 library of wild-type Antirrhinum DNA, partially digested with SauSA, and 24 overlapping clones with an average insert size of 16 kb were isolated. These clones were further characterized by Southern hybridization to obtain a detailed map of the fim locus. After the fim-679 mutation had been shown lo be caused by the insertion of a Tam3 element into a 0.8 kb Hindlll fragment (probe A) of the fim locus, the corresponding 4.4 kb Hindlll fragment from fim-679 was cloned by ligating a size fraction of Hindlll digested genomic DNA from a fim-679 mutant plant into lNMll49 and screening the recombinant phages with probe A. Five independent 1 clones, containing identical inserts were identified, and the inserts were subcloned to give pJAMl28. A 7.5 kb EcoRl fragment from the fim locus was subcloned into pGEM4.Z to give pJAMl37, and was sequenced by the dideoxynucleotide method. Subfragments of pJAMl37 were used as probes on Northern blots against poly(A) RNA from wild-type and mutant inflorescence tips. A 1 .l kb Hindlll fragment of pJAM137 (probe C) detected a 1.8 kb mRNA in Northern blots. A cDNA library in lNMll49 was synthesized using poly(A) RNA from very young wild-type inflorescence tips and screened with probe C. Four independent cDNA clones were isolated and subcloned into the EcoRl site of pGEM4Z. The longest cDNA clone was fully sequenced; the three shorter ones were sequenced from each end to confirm that they derived from the same mRNA species. The sequence of the cDNA clones was identical to the sequence obtained from the genomic clones, except for few base differences which are due to the fact that genomic and cDNA clones were obtained from different Antirrhinum lines. In Situ Hybridization The methods for digoxigenin labeling of RNA probes, tissue preparation, and in situ hybridization were as described in Bradley et al., 1993. The probes used lo detect p/e, def, and f/o transcripts were prepared as described in Bradley et al., 1993. A 1.2 kb fragment from the genomic clones, covering the open reading frame of fim, was subcloned Into pGEM4.Z to give plasmid pCTW6. Linearization with EcoRl or Hindlll provided templates for T7 or SP6 polymerases to generate sense or antisense RNA, respectively. Acknowledgments We are grateful to Hans Sommer for providing a genomic library of wild-type Antirrhinum and to Zsusanna Schwarz-Sommer for stimulating discussions. We thank Lucy Copsey for scanning electron microscopy, Coral Vincent for help with in situ hybridization, and the photogra-
phy department of the John lnnes Centre for their work. For critical discussion of the manuscript and comments. we thank Desmond Bradley, Elisabeth Schultz. Sabine Hantke. Gwyneth Ingram, and Sylvie Pouteau. We are grateful to the Biotechnology Program of the European Community for a postdoctoral fellowship lo R. S., and for support from the Agriculture and Food Research Council Plant Molecular Biology program and the European Community Biotechnology Research for Innovation, Development, and Growth in Europe (BRIDGE) program. Received January 31. 1994; revised May 13, 1994 References Baur, E. (1930). MutationsauslBsung 23, 676-702.
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