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Development 121, 27-35 (1995) Printed in Great Britain © The Company of Biologists Limited 1995 27 Expression of floricaula in single cell layers of periclinal chimeras activates downstream homeotic genes in all layers of floral meristems Sabine S. Hantke, Rosemary Carpenter and Enrico S. Coen John Innes Centre, Colney Lane, Norwich NR4 7UH, UK SUMMARY We show that the flowering sectors on plants mutant for floricaula (flo), a meristem identity gene in Antirrhinum majus, are periclinal chimeras expressing flo in either the L1, L2 or L3 cell layer. Flower morphology is almost normal in L1 chimeras, but altered in L2 and L3 chimeras. Expression of flo in any one cell layer results in the expression of organ identity genes, deficiens (def) and plena (ple) in all three cell layers of the chimeras, showing that flo acts inductively to promote gene transcription. The acti- vation of both def and ple is delayed, and the expression domain of def is reduced, accounting for some of the phenotypic properties of the chimeras. Furthermore, we show that flo exhibits some cell-autonomy with respect to autoregulation. INTRODUCTION may be involved in the genetic control of meristem behaviour. To investigate this possibility on a molecular level, it is important to know the expression pattern of the gene under study, both in wild type and in the chimeras. For example, is the gene normally expressed in one layer or in several, and is its expression pattern within a layer altered in the chimeras? It is also necessary to determine how the activity of other genes might be influenced in the chimeras and understand how this could alter development. The study of flower development provides a suitable system for addressing these questions because many of the key developmental genes have been described. One class of genes affect meristem identity; mutations in them cause shoots to grow in place of flowers. Examples include the floricaula (flo) gene of Antirrhinum (Carpenter and Coen, 1990; Coen et al., 1990) and its homologue, leafy in Arabidopsis (Weigel et al., 1992). A second class of genes affect organ identity; mutations in them result in homeotic replacements of various types of floral organ. Examples include deficiens (def) and plena (ple) of Antirrhinum (Sommer et al., 1990; Bradley et al., 1993) and their counterparts apetala3 and agamous in Arabidopsis (Jack et al., 1992; Yanofsky et al., 1990). In the def mutant, petals are replaced by sepals and stamens are replaced by carpels. Accordingly, def is expressed in whorls 2 and 3 of wild-type flowers (Sommer et al., 1991). In the ple mutant, stamens and carpels are replaced by perianth organs, and the ple gene is expressed in whorls 3 and 4 of wild-type flowers (Bradley et al., 1993). A further class of genes, represented by fimbriata (fim) in Antirrhinum, acts between the meristem and organ identity genes in a sequence of gene activation events (Simon et al., 1994). RNA in situ hybridization experiments show that all of the genes so far analysed are expressed in all three layers of the floral meristem within a given domain. For each gene, Plant meristems are groups of dividing cells that give rise to all postembryonic organs. The shoot meristems of many angiosperms consist of three distinct cell layers: the epidermal layer (L1), the subepidermal layer (L2), and the inner core (L3) (Satina et al., 1940; Huala and Sussex, 1993). Cells of the L1 divide anticlinally throughout development, so that daughter cells remain in the same layer, whereas cells of the L3 divide in all planes. In contrast, cells of the L2 initially divide anticlinally, but can also divide periclinally during organ development. As a result of the planes of division, the three cell layers are clonally distinct (Satina et al., 1940), but cells of all layers participate in the formation of organs. The L1 gives rise to the epidermal structures, the L2 gives rise to the subepidermal mesophyll and the germ cells of the reproductive organs, and the L3 forms most of the internal and vascular tissues of the plant (Satina and Blakeslee, 1941 and 1943; Stewart and Burk, 1970; Dermen and Stewart, 1973). The processes that coordinate the activities of cells in a shoot meristem during development can be analysed by using periclinal chimeras. These are plants in which the genotype of one layer differs from that of the other layers. In chimeras that are generated experimentally, the affected layer is usually determined by using colour, morphological or cytological markers, which are associated with the gene of interest (Huala and Sussex, 1993). Most of the chimeras have been analysed phenotypically, but the underlying gene interactions have not been studied at the molecular level (for examples, see TilneyBassett, 1986; Sinha and Hake, 1990; Stewart et al., 1972; Szymkowiak and Sussex, 1992 and 1993; Zimmerman and Hitchcock, 1951; Carpenter and Coen, 1994). In some cases, the findings have suggested that interactions between layers Key words: Antirrhinum majus, floricaula, flower development, periclinal chimeras, homeotic genes 28 S. S. Hantke, R. Carpenter and E. S. Coen the expression boundaries appear to pass continuously through different layers, suggesting that expression is coordinated between the layers, although it is unclear how this is achieved. The interactions between floral homeotic genes both within and between layers can be determined through the analysis of their expression patterns in chimeras. In the accompanying paper (Carpenter and Coen, 1994), the genetic and morphological analysis of plants chimeric for flo is described. They arose as flowering sectors on plants carrying the unstable allele flo-613 (Coen et al., 1990), which has a copy of the transposable element Tam3 inserted in the second exon. The genetic behaviour of progeny obtained from flowers on the sectors suggests that each flowering sector arose as the result of a single Tam3 excision event early in meristem development. The flowers observed on the sectors do not have the exact wildtype phenotype. Mainly petal and stamen morphology is affected to various degrees, so that the phenotypes can be divided into three different classes based on their severity: near wild type, intermediate and extreme. The different phenotypes are not transmitted to the progeny of these flowers, but can only be maintained vegetatively through cuttings. Taken together, these results strongly suggest that the flowering sectors are chimeric for flo (Carpenter and Coen, 1994). Here we demonstrate by in situ hybridization and DNA analysis that the plants derived from flowering sectors are periclinal chimeras, expressing the flo gene in only one of the meristematic cell layers: L1, L2, or L3. The flower phenotypes of the chimeras mainly depend on the layer in which flo is expressed; sequence analysis of excision alleles in the chimeras reveals that imprecise excisions can lead to further phenotypic variation. The expression patterns of organ identity genes def and ple reveal that flo acts inductively across cell layers to activate downstream genes. MATERIALS AND METHODS DNA and PCR analysis DNA extraction and Southern blot analysis were carried out as described in Coen et al. (1986). The flo probe used for hybridization is derived from the 1.6 kb flo cDNA clone (Coen et al., 1990). Polymerase chain reactions were carried out under standard buffer conditions based on the method of Rosenthal et al. (1993). The final reaction volume was 100 µl in 1× buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.005% Tween 20 (v/v), 0.01% (w/v) gelatine), containing dNTPs (125 µM each), primers (0.1-0.2 µM each) and 2.5 units of Taq polymerase (AmpliTaq, Perkin Elmer Cetus), using 0.1-1.0 µg of genomic DNA as template. Thermocycling conditions were: 40 cycles of 1 minute at 94°C, 2 minutes at 55°C and 5 minutes at 72°C each, followed by an extension time of 10 minutes at 72°C. Three primers specific for the flo gene upstream of the Tam3 insertion site were used: FloU1 (5′-GCCATCAATTGCATGCATGAAA-3′) FloU2 (5′-GGTTGTCGGAGGAGCCGGTGC-3′) FloU3 (5′-GGAAGACGACGAAAATATCGTAAGCG-3′). Two downstream primers were: FloD1 (5′-GCAAGCTTGCTCGTACAAATGAAACA-3′) FloD2 (5′-CAAATCTCCTACAGTGAG-3′) The primers specific for the left and right end of the transposable element Tam3 were: Tam3L (5′-TGCTCGTGTCCATAAATTGTGA-3′) Tam3R (5′-GAAGACGCAACCATGCCATATG-3′). The combination of FloU2 and FloD1 used on wild-type Antirrhinum DNA yields a product of about 320 bp; the combination of FloU1 and FloD2 gives a product of 1.3 kb (Coen et al., 1990). These PCR products were sequenced using the dsDNA cycle sequencing system from BRL according to the manufacturer’s instructions, with FloD2 or FloU3 (for the opposite strand) as sequencing primers. The above primer combinations (FloU2-FloD1 and FloU1-FloD2) do not yield any PCR product when used on DNA from flo-613 plants. To check the presence of Tam3 in the flo gene, the primer combinations FloU2-Tam3L and FloD1-Tam3R were used, resulting in PCR products of 1.1 kb and 600 bp, respectively, if the transposon was present. In situ hybridization Tissue preparation and in situ hybridization experiments were carried out according to Jackson (1991), with modifications as described in Coen et al. (1990) and Bradley et al. (1993). Digoxigenin-labelled RNA probes were prepared using the Boehringer Mannheim nucleic acid labelling kit, following the manufacturer’s instructions. Bluescript KS(+) and SK(+) vectors containing gene fragments of flo, def or ple (Coen et al., 1990; Sommer et al., 1990; Bradley et al., 1993) were linearized and used as templates for the labelling reaction to produce sense and antisense RNA probes. RESULTS Expression pattern of flo in the periclinal chimeras Twelve plants were derived from flowering sectors through cuttings; three of these plants (plants 1-3) had near wild-type flowers, one (plant 4) had an intermediate flower phenotype, and eight (plants 5-12) had flowers of the extreme class (Carpenter and Coen, 1994). Plant 4 gave rise to progeny that segregated for wild-type and flo mutant plants in a 3:1 ratio, whereas plants 1, 2 and 5-11 only gave rise to flo mutant plants. Plant 3 produced some families that contained only flo mutant plants and some families that segregated 3:1 for wild-type and flo mutant plants. Plant 12 gave no viable seed (Carpenter and Coen, 1994). To determine the relationship between genetic behaviour, flower phenotype and flo expression pattern, the 12 plants derived from flowering sectors were analysed by in situ hybridization. Inflorescences of these plants, as well as those of wild-type and flo-613 mutants, were sectioned and probed with digoxigenin-labelled flo antisense RNA. In these sections, bract primordia and their axillary flower meristems could be seen on either side of the inflorescence (Fig. 1A). Each section showed a series of developmental stages, with the youngest near the top of the inflorescence. Not all of the meristems could be observed in any one section because of their spiral arrangement. However, by analysis of serial sections, the position and number of each meristem Fig. 1. Expression of flo in meristems of wild type and various chimeras. Staining is dark blue and tissue is counterstained with calcofluor (pale blue). The top row shows wild type: (A) inflorescence apex (B) floral meristem at node 10 and (C) floral meristem at node 14. Comparable meristems are shown below wild type for an L1 chimera (plant 2, D-F), L2 chimera (plant 4, G-I) and L3 chimera (plants 8 (J,K) and 9 (L)). In the case of the L2 chimera, a bract primordium rather than a floral meristem is shown in the middle panel (H). Abbreviations are: B, bract; F, flower meristem; S, sepal primordium. Scale bars, 50 µm. floricaula in single cell layers activates downstream genes A B C D E F G H I J K L 29 30 S. S. Hantke, R. Carpenter and E. S. Coen relative to the first visible bract primordium (node 0) could be determined. At early developmental stages (nodes 0-10), expression of flo was seen in young bract primordia and their associated floral meristems. Expression was observed in all three cell layers of wild type (Fig. 1A,B) but was not detected in flo-613 plants (not shown). Two of the plants with near wild-type phenotypes showed strong expression only in the epidermal layer, indicating that they were chimeras in which flo activity had been restored only in L1 (L1 chimeras, plants 1 and 2, Fig. 1D,E). The third near wild-type plant gave variable results: some inflorescences showed expression in L1 alone whereas others showed expression in all three layers. This was consistent with the variable results obtained from progeny testing of this plant (plant 3, Table 1). One possible explanation is that this plant was originally derived from an L1 chimera and that further excisions or layer invasions resulted in part of it expressing flo in all layers. The plant with the intermediate phenotype showed strongest expression in the subepidermal layer of cells, indicating that flo activity had been restored in L2 (L2 chimera, Fig. 1G,H). This was consistent with the progeny testing on this plant, which showed that a wild-type allele was present in its germ line (Table 1). Most of the extreme plants showed relatively little signal in either of the outer two cell layers but strong expression in the remaining core of the meristems, corresponding to L3 (plants 5-11, Fig. 1J,K). One extreme plant showed equally strong expression in both L1 and L3 (plant 12, data not shown). In summary, the phenotype of the chimeras depended on the layer expressing flo: L1 chimeras were near wild type, the L2 chimera was intermediate and L3 chimeras were extreme. By about nodes 13-14, wild-type floral meristems comprised a central dome with sepal primordia on its periphery (Fig. 1). In wild type, flo expression was present in all layers of the sepal primordia and in a small area where petal primordia were initiating (Fig. 1C; Simon et al., 1994). At a comparable morphological stage, the signal in L1 chimeras was in the epidermal cells of the sepal and petal primordia and in some sections extended over the central dome (Fig. 1F). The L2 chimera showed flo expression in almost all of the cells in the sepal, except for the epidermis (Fig. 1I). This is consistent with previous cell lineage studies showing that most internal cells of the sepal are clonal descendants of L2 (Satina and Blakeslee, 1941). The signal in L3 chimeras was largely excluded from sepal primordia and confined to the core cells of the meristem. In some L3 chimeras, expression extended further towards the centre of the dome than in wild type (Fig. 1L). During the next stages of wild-type development (nodes 1520), the expression of flo declined in bracts but was observed in all floral organs except stamens. After node 20, expression declined first in sepals and then in petals and carpels. In the chimeric plants, expression mirrored that in wild type, except that it was restricted to their respective layers. Southern blot analysis of DNA from chimeras and their progeny The genetic, phenotypic and in situ analysis indicated that excision of Tam3 gave rise to revertant sectors having different genetic constitutions in their layers. To test this further, the genomic DNA content of the chimeras was characterised and compared with that of their progeny. Total DNA of each Table 1. Summary of phenotypic, molecular and genetic behaviour of chimeric plants Progeny Flower phenotype Plant number Near wild type 1 L1 flo flo-613 Flo+ 2 L1 flo Flo+ 3 4 L1† L2 flo† 3:1‡ 5 6 L3 L3 flo flo 7 8 9* L3 L3 L3 flo flo flo 10* 11 12 L3 L3 L1+L3 flo flo ND flo-613 and rearrangement Imprecise excision† Flo+ for wt plants flo-640 for flo plants Imprecise excision flo-613 and imprecise excision flo-613 flo-613 flo-613 and rearrangement ND ND ND Intermediate Extreme Layer of flo expr. Phenotype Main excision allele in chimera Genotype ND ND flo-640 flo-640 flo-670 Flo+ flo-640 ND flo-669 ND *Plants 9 and 10 had a particularly extreme phenotype. †Some apices of plant 3 expressed flo in all cell layers and accordingly some of the capsules sown segregated for wild type and flo mutant in a 3:1 ratio. In these cases, the alleles in the progeny were Flo+ for wild type and flo-640 for flo mutant plants. ‡All progeny obtained from plant 4 segregated in a 3:1 ratio for wild type and flo mutant phenotype. ND, Not determined, either because no progeny was available at the time, or because two different excision alleles were present in the original chimera, or because the sequence of the wild type size PCR product was ambiguous. chimera should reflect the combined genotype of all layers, whereas the DNA of its progeny should reflect that of L2. To distinguish flo-613 from alleles lacking Tam3, Southern blots of genomic DNA digested with EcoRI were probed with flo (Fig. 2). This gave a 9.0 kb band for flo-613 and a 5.5 kb band for wild-type plants that lack the 3.5 kb Tam3 element (Fig. 2A, left two lanes). Most of the chimeras had a 9.0 kb band, showing that they carried Tam3 in the flo gene. In addition, all chimeras had a 5.5 kb band of varying intensity, presumably because some of their cells lacked Tam3 at the flo gene, as a consequence of excision (Fig. 2A). In several cases, the intensity of the 5.5 kb band was quite weak, as would be expected if only one layer was heterozygous for the excision allele (plants 2,7,8,10,11). The progeny of several L1 or L3 chimeras did not show the excision band, demonstrating that the excision allele had not been transmitted through the germ line (plants 1,2,7,8). However, the excision allele was present in the progeny of both plants that gave germ line revertants (plants 3,4). In addition to the flo-613 allele and excision alleles restoring flo wild-type activity, two further types of allele were observed in some chimeras and their progeny. In several cases, flo mutant progeny were observed to have the 5.5 kb excision band instead of the expected 9.0 kb size (plants 3,4,5). Furthermore, the intensity of the excision band in the total DNA from several of the chimeras was much greater than expected if only one layer had an excision allele (plants 3,4,5,6,9). This was particularly striking for plant 5, in which all layers appeared to have an excision allele even though it was an L3 chimera. These floricaula in single cell layers activates downstream genes 31 A B results were most likely due to imprecise excisions restoring the wild-type fragment size but not flo function (see below). More complex rearrangements were present in the flo gene of plants 2 and 9. Both of these plants had an additional band at about 13 kb which was transmitted to their progeny. Many types of rearrangements involving Tam3 have been described previously (Martin et al., 1988; Robbins et al., 1989; Bollmann et al., 1991), but the precise nature of those in plants 2 and 9 have not been determined. Sequence of the excision alleles in the chimeras Although there was a correlation between the layer expressing flo and the phenotype of the flowers, the layer of expression did not account for all aspects of phenotypic variation. Considerable variation in phenotype was observed between different plants of the extreme class. Furthermore, plant 12 expressed flo in both L1 and L3, yet it had an extreme phenotype rather than near wild type. One possible explanation for these discrepancies was that imprecise excision of Tam3 left footprints that restored flo function to various degrees. To test this, excision alleles were amplified by PCR from total DNA of chimeras and their progeny, and the sequence of the PCR products was determined (Table 1 and Fig. 2. (A) Southern analysis of chimeras and their progeny. Genomic DNA was isolated from each individual chimera (C) and from a pool of 15 progeny plants (P) of each chimera. Samples of 5 µg DNA were digested with EcoRI, blotted and probed with flo. The type of chimera (L1, L2, L3) and plant numbers (1-11) are given above the lanes. Controls are shown in the left two lanes: DNA of wild-type plants (wt) gives a 5.5 kb band and DNA of flo-613 plants (flo-613) gives a 9.0 kb band. Note that flo-613 also has a weak band at 5.5 kb because the DNA was derived from plant material grown in cool outside conditions which favour somatic excision of Tam3. (B) Map of the flo-613 allele to illustrate the origin of fragments observed in Fig. 2A. The flo-613 allele carries an insertion of the transposon Tam3 (3.5 kb) in the second exon of the flo gene. Restriction enzyme digest with EcoRI results in a 9.0 kb fragment if Tam3 is present, and in a 5.5 kb fragment if Tam3 is absent. A 1.5 kb fragment from the 3′-end of the gene is detected in EcoRI digests when blots were probed with the flo cDNA, irrespective of the presence of Tam3. The thin line indicates the flo genomic sequence, filled boxes represent exons, the Tam3 insertion is indicated by a triangle, EcoRI restriction sites are marked with E and the transcription start site is indicated by a wavy arrow. Fig. 3). Precise excisions, giving the wild-type sequence, were observed in L1 and L2 chimeras and in one L3 chimera. This confirmed that the phenotype of these plants was a direct consequence of the layer of Flo+ expression. Plant 7 carried an imprecise excision, flo-670, that restored the original reading frame but resulted in a conservative amino acid substitution. Plant 11 carried the flo-669 footprint, which could code for a protein with a three amino acid insertion relative to wild type. Plants predicted on the basis of Southern blots to carry additional imprecise excisions (plants 3,4,5,6,9) contained an 8 bp footprint whose sequence was identical to that of the previously described stable flo-640 allele, which had a frameshift and encoded a truncated protein. Unfortunately, it was not possible to determine the sequence of the functional flo alleles in plants 5, 6 and 9 because of the abundance of the flo-640 sequence that was always present in an excess after PCR. Expression of organ identity genes in the chimeras Expression of flo in only one layer seemed to be sufficient to activate the floral genetic programme, resulting in the formation of flowers. However, the phenotypes observed indicated that this activation was less effective than in wild type. To determine whether this might have been due to altered 32 S. S. Hantke, R. Carpenter and E. S. Coen Fig. 3. Sequence of flo alleles detected in chimeras compared to Flo+ and flo-613. The target duplication caused by insertion of Tam3 (triangle) in flo-613 is indicated by capital letters and by an arrow above the sequence. Alterations relative to the Flo+ sequence are highlighted in bold. The predicted amino acid sequences are shown under each allele in the one-letter code. expression of later acting genes, we analysed the expression of the organ identity genes, deficiens (def) and plena (ple) (Sommer et al., 1990; Bradley et al., 1993) in the chimeras. As controls, adjacent sections were also probed with flo to confirm which layer expressed flo (Fig. 4A,D,G,J). The results were compared with those obtained with wild type and flo mutants. In wild type, expression of def was first detected at about node 11, and by node 13 def was strongly expressed in all cell layers of the central dome, resembling two inverted peaks in median sections (Fig. 4C). After this stage, the def gene continued to be expressed strongly in the developing petal and stamen primordia (Fig. 4F,L). In flo-613 and flo-640 mutants, no expression of def was observed (data not shown), showing that flo activity is normally required to switch on def. In the chimeras, def was expressed in all three cell layers but the timing and distribution was different from wild type. Detailed analysis was carried out on L1 and L3 chimeras only, since insufficient amounts of L2 material were available. In L1 chimeras, expression of def was first observed at about nodes 14-15 (Fig. 4B) corresponding to a delay of about three nodes compared to wild type. By node 16, def was strongly expressed in two inverted peaks, similar to the pattern seen in node 13 of wild type (Fig. 4E). However, the def domain appeared to be more restricted than wild type and did not extend all the way to the centre or outer edges of the dome (compare Fig. 4C with E). A significant delay in morphological development was observed in L1 chimeras by the analysis of serial sections as well as by scanning electron microscopy (Carpenter and Coen, 1994). Due to this delay, the overall appearance of node 16 meristems of the L1 chimera was similar to node 13 of wild type: in both cases, sepal primordia surrounded a central dome (compare Fig. 4C with E). However, node 16 meristems of the L1 chimeras were larger than node 13 of wild type. The reduced domain of def expression may therefore reflect the increased meristem size at the time of def activation. In contrast to node 16 of L1 chimeras, node 15 meristems of wild type showed distinct petal and stamen primordia expressing def and a flattened central region void of def expression (Fig. 4F). During later stages, from about node 20 onwards, morphology of meristems and def expression in L1 chimeras was similar to wild type. In L3 chimeras, def expression was first detected in nodes 13-14, corresponding to a delay of two to three nodes as compared to wild type, correlating with their retarded development observed by scanning electron microscopy (Carpenter and Coen, 1994). The def domain in nodes 14-15 resembled that of wild-type node 13 meristems, forming two inverted peaks with no gap in the centre (Fig. 4H). However, the boundaries of the def domain did not extend to the outer edges of the central dome. In some of L3 chimeras the delay in def expression was greater than two to three nodes and could be as much as eight nodes in plants with the most extreme phenotypes (plants 9 and 10). At later stages of L3 chimera development, def expression remained low in some regions of petal primordia, particularly in the outer cells. In most cases, def expression eventually attained the wild-type pattern by nodes 16-22. However, in plants with particularly extreme phenotypes, abnormally restricted streaks of def expression could be observed along the axis of petals as late as nodes 23 or 24 (Fig. 4K). In stamens, def seemed to be expressed normally. Expression of ple in wild type was first detected at about node 12 and was observed in all cell layers of the central dome of the floral meristem by nodes 13-14. After this stage, ple continued to be strongly expressed in the developing primordia of whorls 3 and 4. In flo-613 and flo-640 mutants, no expression of ple was observed (data not shown), demonstrating that flo activity is normally required to switch on ple. In chimeras, expression of ple was observed in all cell layers but was delayed by about 2-3 nodes. Once activated, ple expression had a similar pattern to wild type (Fig. 4I). DISCUSSION We show by in situ hybridization, DNA and sequence analysis Fig. 4. Expression of flo, def and ple in periclinal chimeras. Consecutive sections were probed with digoxigenin-labelled flo, def or ple antisense RNA and viewed as in Fig. 1. An L1 chimera (plant 1) is shown probed with flo and def at node 15 (A and B resepectively) and node 16 (D and E respectively). For comparison, expression of def in a wild-type node 13 (C) and node 15 (F) are shown. Note the differences in def expression pattern and signal strength between B and C even though the morphologies appear comparable. In comparing E and F, note the more advanced floral organ primordia in the wild type and the difference in the def expression domain. An L3 chimera node 14 floral meristem (plant 5) is shown probed with flo (G), def (H) and ple (I). The def domain is similar to that in node 13 of wild type (compare H and C) but does not extend as far towards the sepal primordia. A node 23 floral meristem of an L3 chimera (plant 10) showing expression of flo (J) and def (K) compared to a node 21 of wild type probed with def (L). Expression of flo can hardly be detected in the chimera at this stage whereas that of def is reduced to narrow streaks in petals, but is apparently normal in stamens (compare K and L). Abbreviations: c, carpel; p, petal; s, sepal; st, stamen. Scale bars (shown once per row) is 100 µm. floricaula in single cell layers activates downstream genes that the flowering sectors on flo-613 mutant plants are periclinal chimeras for flo caused by excision of Tam3. The excision events occur early in meristem development, restoring the 33 expression of flo in only one cell layer. Among the 12 plants derived from flowering sectors (Carpenter and Coen, 1994) three main types of chimera were identified: L1 chimeras A B C D E F G H I J K L 34 S. S. Hantke, R. Carpenter and E. S. Coen express flo in the epidermal layer of the meristem, the L2 chimera expresses flo in the subepidermal layer and shows germ line transmission of the revertant allele, and L3 chimeras express flo in meristem core. The types of chimera correlate with the flower phenotypes: L1 chimeras are near wild type, the L2 chimera are intermediate, and L3 chimeras have an extreme phenotype. Tam3 excision events can be detected in the chimeras both by Southern blots and DNA sequencing, confirming that they arose by transposition. These results demonstrate that flo expression in single cell layers can promote flower development in a non-autonomous manner, coordinating cell division and differentiation in all layers. However, some aspects of flo function are hampered when it is restricted to one layer, evidenced by two observations. Firstly, the flower phenotypes of the chimeras are not normal: L1 chimeras give near wild-type flowers, the L2 chimeras are intermediate and L3 chimeras give an extreme phenotype. Secondly, the chimeras with near wild-type or intermediate phenotypes all carry Flo+ alleles caused by precise excisions, even though such events are likely to be rare (Coen et al., 1989, 1990). This suggests that when flo function is hampered by restriction to one layer, the requirements to produce nearly normal flowers are so stringent that precise excisions are selected for. This is supported by the observation of imprecise excisions in some of the chimeras with extreme phenotypes. Interactions of flo with downstream organ identity genes At least two organ identity genes, def and ple, are activated in all three layers of flo chimeras, even though they are not activated in stable flo mutants. This indicates that the nonautonomy of flo is effective very early in floral development and that activation of organ identity genes by flo involves cell communication between layers. Such communication may also be involved in coordinating the expression pattern of organ identity genes between the layers. Although extending to all layers, the def expression domain is reduced, and the activation of both def and ple appears to be delayed in the chimeras. The effects on def are even greater in L3 chimeras with particularly extreme phenotypes. These effects on def and ple gene expression may partly explain the phenotype of the chimeric flowers. For example, def mutations result in sepals replacing petals in whorl 2 and carpels replacing stamens in whorl 3. The altered pattern of def activity, particularly in whorl 2, may therefore account for the petals in some chimeras being split, small, and having streaks of green, sepaloid tissue. The expression domain of ple, a gene controlling sex organ identity, is not markedly affected, consistent with the ability of the chimeras to produce fully functional sex organs. These results suggest that expression of def, particularly in whorl 2, is relatively sensitive to changes in flo activity. This may reflect the properties of fimbriata, a gene known to mediate between flo and def (Simon et al., 1994). A close dependence of def on flo is also suggested from the analysis of counterpart genes in Arabidopsis, which shows that apetala3 expression is reduced in leafy mutants (Weigel and Meyerowitz, 1993). However, it is unlikely that all aspects of chimera phenotype can be accounted for by altered def and ple activity. The altered morphology of first whorl organs (Carpenter and Coen, 1994), for example, suggests a role of flo in the activation of genes involved in specifying sepal identity. Autoregulation of flo Analysis of plants carrying the flo-640 allele indicates that flo increases its own expression (unpublished data). The flo-640 allele carries a frameshift caused by imprecise excision of Tam3 and therefore has the potential to be transcribed normally. Nevertheless, when compared to wild type, plants homozygous for the stable flo-640 allele express flo at low levels, providing evidence for flo autoregulation. This raises the question of whether restoring flo activity in one layer can increase flo expression in other layers. This could be tested because several of the chimeras contain alleles with the same sequence as flo-640 in layers other than the one with restored flo function. For example, two of L3 chimeras contain substantial amounts of the flo-640 sequence, and this appears to be transmitted to their progeny, showing that L2 and possibly other layers carry this sequence. However, flo transcripts are mainly observed in L3 of both of these chimeras, reflecting the location of the functional allele. This suggests that flo activity in L3 cannot increase expression of flo in other layers, otherwise strong expression would also be seen in the L1 and L2 of these chimeras. These results indicate that flo autoregulation is largely cell-autonomous with respect to the layers. Another aspect of flo autoregulation is suggested by the more extensive flo expression pattern observed in some of the chimeras at early stages of development. For example, flo expression in wild-type meristems at about node 13 is not observed in the main part of the central dome, destined to form whorls 3 and 4. However, at a comparable stage in L1 and L3 chimeras, flo expression sometimes extends further into the central dome than in wild type. One explanation for this is that flo normally activates organ identity genes, which are responsible for inhibiting flo activity in the central dome, at an early stage (Coen et al., 1990). In the chimeras, there may be a delay in the activation of such genes and hence ectopic expression of flo. This is supported by the observed delay in expression of organ identity genes such as def and ple in the chimeras. 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