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How the snapdragon chooses its color In some snapdragons, a yellow spot in a field of magenta shows the bee the best place to go. Flowers of a related subspecies are mainly yellow with magenta veins marking the target. Bradley et al. analyzed a locus that regulates the pattern of color. The locus contains an inverted gene duplication that encodes small RNAs that repress pigment biosynthesis. Analysis of flowers derived from a region of the Pyrenees where the subspecies coexist indicates that natural selection is operating upon the locus. Science , this issue p. 925

A fossil plant of Eocene age from Antarctica was studied using X-ray and neutron tomography to reveal the three-dimensional plant structures encased within carbonate nodules. The fossil was identified as a branch and leaves of an araucarian conifer, which grew on the volcanic highlands of the Antarctic Peninsula region approximately 50 million yr ago. Both X-ray and neutron imaging techniques successfully exposed the full three-dimensional structure of the fossil without destroying the original specimen, revealing that most of the fossil was present as voids in the concretion and little organic matter was present. However, neutron tomography was found to produce images with superior quality and detail.

To understand how genes control floral asymmetry, we have isolated and analyzed the role of the RADIALIS ( RAD ) gene in Antirrhinum . We show that the RAD gene encodes a small MYB-like protein that is specifically expressed in the dorsal region of developing flowers. RAD has a single MYB-like domain that is closely related to one of the two MYB-like domains of DIV, a protein that has an antagonistic effect to RAD on floral development. Interactions between RAD and other genes indicate that floral asymmetry depends on the interplay between two pairs of transcription factors. First, a pair of TCP proteins is expressed in dorsal regions of the floral meristem, leading to the activation of RAD in the dorsal domain. The RAD MYB-like protein then antagonizes the related DIV MYB-like protein, preventing DIV activity in dorsal regions. In addition to its role in dorsal regions, RAD acts nonautonomously on lateral regions either directly, through RAD protein movement, or indirectly, through a signaling molecule.

Comparative studies on the ABC model of floral development have revealed extensive conservation of B and C class genes, but have failed to identify similar conservation for A class genes. Using a reverse genetic approach, we show that the previous inability to obtain Antirrhinum mutants corresponding to the A class gene AP2 of Arabidopsis reflects greater genetic redundancy in Antirrhinum . Antirrhinum has two genes corresponding to AP2, termed LIP1 and LIP2, both of which need to be inactivated to give a mutant phenotype. Analysis of interactions between LIP and class B/C genes shows that unlike AP2 in Arabidopsis, LIP genes are not required for repression of C in outer whorls of the flower. However, like AP2, LIP genes play a role in sepal, petal and ovule development, although some of their detailed effects are different, reflecting the diverse morphologies of Antirrhinum and Arabidopsis flowers. The dual functions for which AP2 is required in Arabidopsis are therefore separate in Antirrhinum, showing that the genetic basis of some aspects of organ identity have undergone major evolutionary change.

The DEFICIENS (DEF) gene is required for establishing petal and stamen identity in Antirrhinum and is expressed in all three layers of the floral meristem in whorls 2 and 3. Expression of DEF in a subset of meristem layers gives rise to organs with characteristic shapes and cell types, reflecting altered patterns and levels of DEF gene activity. To determine how the contributions of layers and gene activity interact, we exploited a DEF allele which carries a transposon insertion in the MADS box region to generate periclinal chimeras expressing alleles with different activities. By comparing the phenotype, development and expression patterns of these chimeras we show that expression of DEF in L1 makes a major contribution to morphology in whorl 2, irrespective of the allele. By contrast L1 expression is largely unable to rescue whorl 3, possibly because of a non-autonomous inhibitor of DEF activity in this whorl.

Summary In Antirrhinum majus, one proposed role of the gene fimbriata (fim) is as a mediator between the floral meristem identify gene floricaula (flo) and floral organ identity genes such as deficiens (def) and plena (ple). The mechanism of fim activity is probably unique as, while the other genes in the hierarchy are thought to be transcription factors, fim is thought to target proteins to a ubiquitin-mediated destruction pathway. Both flo and def have been shown to act non-cell autonomously. We tested the hypotheses that (i) fim acts in a non-cell autonomous manner; and (ii) non-cell autonomy of flo might be through activation and subsequent non-cell autonomous activity of fim. Plants bearing an unstable fim allele were monitored for revertant shoots. Analysis of fim RNA expression in plants derived from revertant shoots, and segregation of revertant phenotype in progeny from revertant plants, indicated that all were periclinal chimeras with wild-type fim expression only in subepidermal layers. Despite the absence of fim in the epidermal layer, expression of downstream genes was normal, suggesting non-cell autonomous activity of fim. Subsequently, we tested the hypothesis that fim is the mediator of flo non-cell autonomy by examining fim expression in flo periclinal chimeras. In these chimeras, fim is activated in cells where flo is not expressed, indicating that fim cannot be the sole mediator of flo non-cell autonomy.

Small GTPases have diverse roles in animals and yeast, including signal transduction, regulation of secretion, organisation of the cytoskeleton, and control of cell division. Similar GTPases have also been found in bacteria, such as the Escherichia coli GTPase ERA, which is involved in regulating metabolism and cell division [1,2]. Many small GTPases have been cloned from plants but their functional analysis has largely been limited to complementation of mutations in corresponding yeast genes, and antisense experiments which have implicated these proteins in processes such as root nodulation [3,4]. No mutations in plant GTPases have been reported, and thus their true importance in plant growth and development is unknown. Here we report the isolation of a gene from Antirrhinum majus encoding a protein from an entirely novel class of eukaryotic GTPases showing strongest similarity to the prokaryotic protein ERA. We have named this gene ERG (for ERA-related GTPase). The ERG gene is expressed in dividing or metabolically active cells. We generated a deletion allele of ERG by site-selected transposon mutagenesis and have shown that seeds containing embryos and endosperm homozygous for this deletion arrest soon after fertilisation. We conclude that ERG has a crucial role in plant growth and development, possibly by influencing mitochondrial division.

The development of reproductive organs in Antirrhinum depends on the expression of an organ identity gene, plena, in the central domain of the floral meristem. To investigate the mechanism by which plena is regulated, we have characterised three mutants in which the pattern of plena expression is altered. In polypetala mutants, expression of plena is greatly reduced, resulting in a proliferation of petals in place of reproductive organs. In addition, polypetala mutants exhibit an altered pattern of floral organ initiation, quite unlike that seen in loss-of-function plena mutants. This suggests that polypetala normally has two roles in flower development: regulation of plena and control of organ primordia formation. In fistulata mutants, plena is ectopically expressed in the distal domain of petal primordia, resulting in the production of anther-like tissue in place of petal lobes. Flowers of fistulata mutants also show a reduced rate of petal lobe growth, even in a plena mutant background. This implies that fistulata normally has two roles in the distal domain of petal primordia: inhibition of plena expression and promotion of lobe growth. A weak allele of the floral meristem identity gene, floricaula, greatly enhances the effect of fistulata on plena expression, showing that floricaula also plays a role in repression of plena in outer whorls. Taken together, these results show that genes involved in plena regulation have additional roles in the formation of organs, perhaps reflecting underlying mechanisms for coupling homeotic gene expression to morphogenesis.

The overall aerial architecture of flowering plants depends on a group of meristematic cells in the shoot apex. We demonstrate that the Arabidopsis TERMINAL FLOWER 1 gene has a unified effect on the rate of progression of the shoot apex through different developmental phases. In transgenic Arabidopsis plants which ectopically express TERMINAL FLOWER 1, both the vegetative and reproductive phases are greatly extended. As a consequence, these plants exhibit dramatic changes in their overall morphology, producing an enlarged vegetative rosette of leaves, followed by a highly branched inflorescence which eventually forms normal flowers. Activity of the floral meristem identity genes LEAFY and APETALA 1 is not directly inhibited by TERMINAL FLOWER 1, but their upregulation is markedly delayed compared to wild-type controls. These phenotypic and molecular effects complement those observed in the tfl1 mutant, where all phases are shortened. The results suggest that TERMINAL FLOWER 1 participates in a common mechanism underlying major shoot apical phase transitions, rather than there being unrelated mechanisms which regulate each specific transition during the life cycle.

The presentation of flowers on a modified stem, the inflorescence, requires the integration of several aspects of meristem behaviour. In Antirrhinum, the inflorescence can be distinguished by its flowers, hairy stem, modified leaves, short internodes and spiral phyllotaxy. We show, by a combination of physiological, genetical and morphological analysis, that the various aspects of the inflorescence are controlled by three pathways. The first pathway, depends on expression of the floricaula gene, and is rapidly and discretely induced by exposure to long daylength. Activation of this pathway occurs in very young axillary meristems, resulting in a floral identity. In addition, the length of subtending leaves and hairiness of the stem are partially modified. The second pathway affects leaf size, internode length, and stem hairiness, but does not confer floral meristem identity. This pathway is induced by long daylength, but not as rapidly or discretely as the floricauladependent pathway. The third pathway controls the switch in phyllotaxy from decussate to spiral and is activated independently of daylength. The coordination of these three programmes ensures that apical and axillary meristem behaviour is integrated.

Background: Homeotic genes controlling the identity of flower organs have been characterized in several plant species. To determine whether cells expressing these genes are specified to follow particular developmental fates, we have studied the pattern of cell lineages in developing flowers of Antirrhinum . Each flower has four whorls of organs, and progenitor cells of these can be marked at particular stages of development using a temperature-sensitive transposon. This allows the cell lineages in the flower to be followed, as well as giving information about rates of cell division. Results We show here that, prior to the emergence of organ primordia, cells in the floral meristem have not been allocated organ identities. After this time, lineage restrictions arise between whorls, correlating with the onset of expression of genes that control organ identity. A further lineage restriction appears slightly later on, between the dorsal and ventral surfaces of the petal. Our results further suggest that the rates of cell division fluctuate during key stages of meristem development, perhaps as a consequence of meristem-identity gene expression. Conclusion The patterns of lineage restriction and organ-identity gene expression in early floral meristems are consistent with some cells being allocated specific identities at about this stage of development. Plant cells cannot move relative to each other, so lineage restrictions in plants may reflect particular orientations and/or rates of growth at boundary regions.

Flowers can be classified into two basic types according to their symmetry: regular flowers have more than one plane of symmetry and irregular flowers have only a single plane of symmetry. The irregular condition is thought to have evolved many times independently from the regular one: most commonly through the appearance of asymmetry along the dorso-ventral axis of the flower. In most cases, the irregular condition is associated with a particular type of inflorescence architecture. To understand the molecular mechanism and evolutionary origin of irregular flowers, we have been investigating genes controlling asymmetry inAntirrhinum. Several mutations have been described inAntirrhinum, a species with irregular flowers, that reduce or eliminate asymmetry along the dorso-ventral axis. We describe the nature of these mutations and how they may be used to analyse the molecular mechanisms underlying floral evolution.

Summary Delila (del), a regulatory gene of Antirrhinum, alters anthocyanin pigmentation when introduced into two Solanaceous species. In tomato, pigmentation in vegetative tissues is strongly increased while in tobacco, intensification of pigment is restricted to flowers. Although del transcripts are ubiquitous in the transgenic plants, transcript levels of host anthocyanin biosynthetic genes are only increased in pigmented regions. Constructs carrying the maize transposon Ac, inserted at the 3′ end of the 35S promoter prior to the start of translation of the del gene, give variegated leaves in tomato, suggesting that del acts cell-autonomously and that it may be used as a phenotypic marker. In Arabidopsis, del has no strong phenotypic effects, suggesting that del may not be able to function effectively in all plant hosts.

Flower meristems comprise several distinct cell layers. To understand the role of cell interactions between and within these layers, we have generated plants chimeric for a key floral homeotic gene, floricaula (flo). These chimeras arose in Antirrhinum by excision of a transposon, restoring flo gene function. Activity of flo in a subset of cell layers gives fertile flowers with an abnormal morphology. This shows that flo can act non-autonomously between layers, although some aspects of its function are impaired. In addition, we show that flo exhibits some cell-autonomy within layers.

Septic shock is the leading cause of death in critical care units. New research in the management of septic shock is focused on the use of immune-based therapy such as antiendotoxin monoclonal antibodies. The object of this therapy is to neutralize endotoxin and prevent the progression of the pathophysiologic events of sepsis. To date, the promise of this therapy has not been realized, but hopefully, ongoing research will provide optimism for treatment of this syndrome. Two agents, E5 and HA-1A, are discussed.

A major change in the development of plants occurs upon floral induction. Meristems in certain positions become organized to form flowers. We are studying this process using a combination of genetic, molecular and physiological approaches in Antirrhinum . In particular, we are exploiting transposon-induced mutations in genes controlling early switches in floral development. These mutations cause homeotic and heterochronic phenotypes and three categories of genes have been identified. The first includes floricaula ( flo ), which is required to switch inflorescence meristems to a floral state. This gene has been isolated and shown to be expressed transiently in bract, sepal, petal and carpel primordia. The second group of genes controls the identity (and sometimes the number) of organs in a whorl. These genes affect overlapping whorls and their mutant phenotypes suggest a co binatorial model for gene action in determining the fate of floral primordia. Some of the regulatory interactions between these genes have been revealed by studying cis -or trans -acting mutations which have resulted in ectopic gene expression. Genes of the third category determine the identity of organs within one whorl and thus affect the symmetry of the flower. We propose that the interactions of these homeotic genes control the basic patterns of inflorescence and flower development not only in Antirrhinum , but also in a diverse range of plant species.

Recessive mutations at the plena (ple) locus result in a homeotic conversion of sex organs to sterile perianth organs in flowers of Antirrhinum majus. A complementary phenotype, in which sex organs replace sterile organs, is conferred by semidominant ovulata mutations. The ple locus was identified and isolated using a homologous gene, agamous from Arabidopsis, as a probe. The expression of ple is normally restricted to the inner two whorls of the flower, where sex organs develop. However, in ovulata mutants, ple is expressed ectopically in the outer two whorls of the flower and in vegetative organs. These mutants correspond to gain-of-function alleles of ple, suggesting that ple is sufficient for promoting sex organ development within the context of the flower. The plena and ovulata phenotypes result from opposite orientations of the transposon Tam3 inserted in the large intron of ple.

New pigmentation mutants were generated by transposon mutagenesis in Antirrhinum majus, in three previously described loci, nivea, delila and incolorata, and two new loci, daphne and olive. The wild-type olive gene is required for the production of dark-green leaves, and the daphne gene for the synthesis of flavones. Five out of the six mutants were both germinally and somatically unstable, indicating that they resulted from transposon insertions. Molecular analysis of the mutant at nivea (niv-600) showed that it was caused by insertion of a new transposon, Tam4. The sequence of Tam4 suggests that it is unable to transpose autonomously and that it is related to Tam1 and Tam2. All three of these transposons have identical inverted repeats, produce 3 bp target duplications, leave similar excision footprints and share at one end a 600-700 bp region containing many palindromic copies of a motif sequence, possibly required in cis for transposition. The somatic excision of Tam4 in niv-600 is at a very low rate compared to germinal excision but it can be activated by crossing to lines carrying derivative alleles of a Tam1 insertion at niv. Molecular analysis of four different pigmentation mutants has shown that insertions of Tam1, Tam2, Tam3 and Tam4 have been obtained, illustrating the potential of general transposon mutagenesis for trapping and isolating new transposons as well as for tagging genes.

We describe two novel ways in which changes in gene expression in Antirrhinum majus may arise as a consequence of the Tam3 transposition mechanism. One involves excision of Tam3 from the nivea gene promoter and insertion of two new Tam3 copies 3.4 kb and 2.1 kb away, on either side of the excision site. One of the new insertions is in the nivea coding region and completely blocks production of an active gene product. This allele probably arose by a symmetrical double transposition, following chromosome replication. The second case involves a small deletion at one end of Tam3 in the pallida gene, flanked by a sequence typical of a Tam3 excision footprint. This suggests that the end of Tam3 was cleaved at an early step in an attempted transposition and re-ligated back to its original flanking sequence. The alteration restores some expression to the pallida gene, suggesting that the ends of the intact Tam3 element contain components which can actively inhibit gene expression. The implications of these findings for the mechanism of Tam3 transposition and for the effects of Tam3 on host gene expression are discussed.

Phytochemicals and plant extracts, present in fruit, vegetables, plants, herbs, and beverages, have been shown to have antioxidant potential that may modulate the etiology of certain chronic diseases. The objective of the present study was to determine the concentration of compound that inhibited cell growth by 50% (IC(50)) of a range of phytochemicals and plant extracts and to investigate their antioxidant and genoprotective effects under conditions of oxidative stress in U937 cells. Two phytochemicals-resveratrol and citroflavan-3-ol-and four plant extracts-grapeseed polyphenols, olive leaf extract, bearberry, and Echinacea purpurea-were examined. Viability was assessed by the fluorescein diacetate/ethidium bromide assay. The IC(50) was calculated. To examine their antioxidant and genoprotective effects, U937 cells were pretreated with the test compounds at levels below the IC(50) and then exposed to oxidants: 0.5 microM etoposide or 100 microM hydrogen peroxide (H(2)O(2)) or 400 microM tert-butylhydroperoxide (tBOOH). Cellular reduced glutathione levels were measured as an indicator of oxidative stress. DNA damage was assessed by the alkaline single-cell gel electrophoresis assay or comet assay. Resveratrol demonstrated the highest IC50 value of 13.7 microg/mL, with Echinacea the lowest at 9,400 microg/mL. Etoposide-induced oxidative stress was strongly reduced by olive leaf extract and bearberry. Grapeseed polyphenols and bearberry strongly protected against H2O2- and tBOOH-induced DNA damage. In conclusion, these results provide evidence that non-nutrient dietary constituents may act as significant bioactive compounds and that plant extracts, such as bearberry, grapeseed polyphenols, and olive leaf extract, strongly protect against oxidative stress.

To understand how differentiation and growth may be coordinated during development, we have studied the action of the CINCINNATA (CIN) gene of Antirrhinum. We show that in addition to affecting leaf lamina growth, CIN affects epidermal cell differentiation and growth of petal lobes. Strong alleles of cin give smaller petal lobes with flat instead of conical cells, correlating with lobe-specific expression of CIN in the wild type. Moreover, conical cells at the leaf margins are replaced by flatter cells, indicating that CIN has a role in cell differentiation of leaves as well as petals. A weak semidominant cin allele affects cell types mainly in the petal but does not affect leaf development, indicating these two effects can be separated. Expression of CIN correlates with expression of cell division markers, suggesting that CIN may influence petal growth, directly or indirectly, through effects on cell proliferation. For both leaves and petals, CIN affects growth and differentiation of the more distal and broadly extended domains (leaf lamina and petal lobe). However, while CIN promotes growth in petals, it promotes growth arrest in leaves, possibly because of different patterns of growth control in these systems.

Although curvature of biological surfaces has been considered from mathematical and biophysical perspectives, its molecular and developmental basis is unclear. We have studied the cin mutant of Antirrhinum , which has crinkly rather than flat leaves. Leaves of cin display excess growth in marginal regions, resulting in a gradual introduction of negative curvature during development. This reflects a change in the shape and the progression of a cell-cycle arrest front moving from the leaf tip toward the base. CIN encodes a TCP protein and is expressed downstream of the arrest front. We propose that CIN promotes zero curvature (flatness) by making cells more sensitive to an arrest signal, particularly in marginal regions.

Organ asymmetry is thought to have evolved many times independently in plants. In Antirrhinum, asymmetry of the flower and its component organs requires cyc and dich gene activity. We show that, like cyc, the dich gene encodes a product belonging to the TCP family of DNA-binding proteins that is first expressed in the dorsal domain of early floral meristems. However, whereas cyc continues to be expressed throughout dorsal regions, expression of dich eventually becomes restricted to the most dorsal half of each dorsal petal. This correlates with the effects of dich mutations and ectopic cyc expression on petal shape, providing an indication that plant organ asymmetry can reflect subdomains of gene activity. Taken together, the results indicate that plant organ asymmetry can arise through a series of steps during which early asymmetry in the developing meristem is progressively built upon.

Flowering plants exhibit one of two types of inflorescence architecture: indeterminate, in which the inflorescence grows indefinitely, or determinate, in which a terminal flower is produced. The indeterminate condition is thought to have evolved from the determinate many times, independently. In two mutants in distantly related species,terminal flower 1inArabidopsisandcentroradialisinAntirrhinum, inflorescences that are normally indeterminate are converted to a determinate architecture. TheAntirrhinumgeneCENTRORADIALIS(CEN) and theArabidopsisgeneTERMINAL FLOWER 1(TFL1) were shown to be homologous, which suggests that a common mechanism underlies indeterminacy in these plants. However, unlikeCEN,TFL1is also expressed during the vegetative phase, where it delays the commitment to inflorescence development and thus affects the timing of the formation of the inflorescence meristem as well as its identity.

Dorsoventral asymmetry in flowers is thought to have evolved many times from a radially symmetrical ancestral condition. The first gene controlling floral asymmetry, cycloidea in Antirrhinum, has been isolated. The cycloidea gene is expressed at a very early stage in dorsal regions of floral meristems, where it affects growth rate and primordium initiation. Expression continues through to later stages in dorsal primordia to affect the asymmetry, size and cell types of petals and stamens.

Flowering plants exhibit two types of inflorescence architecture: determinate and indeterminate. The centroradialis mutation causes the normally indeterminate inflorescence of Antirrhinum to terminate in a flower. We show that centroradialis is expressed in the inflorescence apex a few days after floral induction, and interacts with the floral-meristem-identity gene floricaula to regulate flower position and morphology. The protein CEN is similar to animal proteins that associate with lipids and GTP-binding proteins. We propose a model for how different inflorescence structures may arise through the action and evolution of centroradialis.

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 activation 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.

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 floricaula and the organ identity gene plena.

One of the unifying theories of plant biology is that the variety of plant forms are simply different modifications of a common growth plan. Different permutations of a few key features of plant growth can generate a bewildering array of seemingly distinct forms. There is perhaps no better illustration of this than the comparison of a flower and a shoot. The idea that these two apparently different structures might be fundamentally equivalent goes back to Goethe's treatise on metamorphosis, published in 1790. He concluded, "Flowers which develop from lateral buds are to be regarded as entire plants, which are set in the mother plant, as the mother plant is set in the earth" (Goethe, 1790). In equating flowers and shoots, four key assertions need to be made. First, the different parts of the flower (sepals, petals, stamens, and carpels) are equivalent to the leaves of a shoot. Second, the organs of both shoot and flower are separated by internodes, but in the case of the flower these are so short as to be barely visible. Third, the organs of shoot and flower usually have a distinct phyllotaxy, or arrangement around the central axis. Finally, the indeterminate growth that so characterizes a shoot is suppressed in the case of a flower, both apically, because it eventually stops producing organs around the central axis, and laterally, because branches do not normally arise in the axils of floral organs. The comparison of flower and shoot therefore highlights four key variables: organ identity, internode length, phyllotaxy, and determinacy. The numerous forms and habits of plants simply reflect different variations and permutations of these four fundamental aspects of growth. What is their developmental basis?

The delila (del) gene regulates the pattern of red anthocyanin pigmentation in Antirrhinum majus plants. We describe the cloning of the del locus by transposon tagging and show that it encodes a protein with extensive homology to products of the R gene family, which regulates pigmentation in maize. This shows that in spite of the many differences in morphology and coloration between maize and Antirrhinum, the control of pigmentation pattern is mediated by a common regulator. The del and R products contain a region similar to the conserved domain of the helix-loop-helix family of transcription factors. In situ hybridization and RNA analysis show that the expression of del correlates with the distribution of anthocyanins in the flowers. We discuss the implications of these findings for the evolution of regulatory networks.

Self-incompatibility (SI) systems have been reported in almost half of the families of all flowering plants (de Nettancourt, 1977). Most commonly, SI is regulated by a single multiallelic locus, with the compatibility of the pollen with respect to the stigma being controlled by the haploid nucleus (East, 1940). The genus Antirrhinum possesses such a gametophytically-regulated monofactorial SI system and, when pollen and pistil share SI (S) alleles, tube growth is arrested in the style. The significance of SI systems to plant breeders, combined with the fact that they represent some of the best defined systems of intercellular interaction in plants has resulted in their being intensively studied (Anderson et al., 1986; Nasrallah et al., 1985). For example, molecular and biochemical research has identified the female S-allele products of members of the Solanaceae as being highly charged glycoproteins with molecular weights of between 20–35 kDa (for review, see Ebert et al., 1989). Many putative S-alleles have been cloned and sequenced, and it has been recently discovered that all of the S-glycoproteins in the Solanaceae have ribonuclease activity (McClure et al., 1990), leading to the proposal that this type of SI may involve a step in which pollen tube RNA is selectively degraded. Although progress has been made in our understanding of the S-allele products of the pistil in gametophytically-controlled SI (GSI), and also in the sporophytically-controlled systems where pollen compatibility is regulated by two alleles (Bateman, 1952; Nasrallah et al., 1970), little is known of the organisation of the S-locus and of S-allele expression in the pollen.

Homeotic mutants have been used to define the genetic interactions controlling flowering in Antirrhinum. Three categories of homeotic genes were identified by transposon mutagenesis. The first includes floricaula (flo), which is required to switch inflorescence meristems to floral. This gene has been isolated and shown to be expressed transiently in bract, sepal, petal and carpel primordia. The second group of genes controls the identity (and sometimes the number) of organs in a whorl. These genes affect overlapping whorls and their mutant phenotypes suggest a combinatorial model for gene action in determining the fate of floral primordia. Genes of the third category determine the identity of organs within one whorl and thus affect the symmetry of the flower. We propose that the interactions of these homeotic genes not only control the basic patterns of inflorescence and flower development in Antirrhinum, but possibly in a diverse range of plant species.

Most null alleles at the nivea (niv) locus are recessive to Niv+ and, when homozygous, give white flowers rather than the red of the wild type. In contrast, the niv-571 allele is semidominant; although it gives white flowers when homozygous, very pale flowers result when this allele is heterozygous with NIV+. We showed that in heterozygotes, niv-571 acts in trans to inhibit expression of its Niv+ homology 25-fold to 50-fold. The inhibition is reversible after meiosis and partially reversible somatically. The niv-571 allele carries a transposable element Tam3 insertion and three truncated copies of the niv gene, one copy being in inverse orientation. Analysis of two further niv alleles, niv-572 and niv-527, showed that excision of Tam3 from niv-571 does not affect the ability of the allele to repress Niv+ and that one truncated niv copy alone is insufficient to confer semidominance. The detailed structures of various semidominant niv alleles suggest that their effects in trans are not readily explained by production of antisense RNA but are more easily reconciled with a direct recognition/interaction between homologous genes, reminiscent of cosuppression and transvection phenomena described in other systems.

Many homoeotic genes affecting flower morphogenesis have been described in diverse species, although there have been few attempts to relate these in a systematic way to the mechanism of floral development (Meyerowitz et al., 1989). In 1742 it was the peloric form of Linaria vulgaris that made Linnaeus change his mind about the fixity of species (Linnaeus, 1744) and over a century later Darwin described, and was intrigued by, the behaviour of the peloric Antirrhinum (Darwin, 1868). We have used transposon-mutagenesis to generate floral homoeotic mutations in Antirrhinum with a view to studying and isolating the genes involved. One advantage of this approach is that transposon integration can be used to identify genes and subsequent excision can be used to prove that the correct gene has been isolated (Martin et al., 1985). In addition, imprecise excision can generate alleles with altered gene expression (Almeida et al., 1989).

The chronic blockade of neuromuscular activity is known to promote the survival of developing motoneurons in vivo in the chick, mouse and rat embryo. Increased survival in this situation may reflect an activity-dependent mechanism for the regulation of trophic factor production by target cells. To test this notion, we have examined motoneuron survival in vivo and choline acetyltransferase (ChAT) development in vitro following treatment of chick embryos and rat spinal cord cultures with partially purified skeletal muscle extracts derived from normally active, chronically paralyzed and aneural embryos, and from denervated postnatal chickens. Extracts from active and paralyzed chick embryos were equally effective in promoting motoneuron survival and ChAT activity. Aneural embryonic muscle extracts were slightly less effective in promoting motoneuron survival in vivo , but were not significantly different from control extracts in the in vitro ChAT assay. Denervated postnatal muscle extracts, however, were more effective in enhancing both motoneuron survival and ChAT activity. These data indicate that: (1) the promotion of motoneuron survival in vivo by activity blockade may not be mediated by an up-regulation of trophic factor synthesis in target cells; (2) postnatal innervation may regulate the production of putative muscle-derived neurotrophic factors; and (3) the synthesis or availability of trophic agents may be regulated differently in embryonic and postnatal muscle.

Plants carrying the floricaula (flo) mutation cannot make the transition from inflorescence to floral meristems and have indeterminate shoots in place of flowers. The flo-613 allele carries a Tam3 transposon insertion, which allowed the isolation of the flo locus. The flo gene encodes a putative protein (FLO) containing a proline-rich N-terminus and a highly acidic region. In situ hybridization shows that the flo gene is transiently expressed in the very early stages of flower development. The earliest expression seen is in bract primordia, followed by sepal, petal, and carpel primordia, but no expression is detected in stamen primordia. This pattern of expression has implications for how flo affects phyllotaxis, organ identity, and determinacy. We propose that flo interacts in a sequential manner with other homeotic genes affecting floral organ identity.

In many plant species, self-incompatibility (SI) is genetically controlled by a single multiallelic S locus. Previous analysis of S alleles in the Solanaceae, in which S locus ribonucleases (S RNases) are responsible for stylar expression of SI, has demonstrated that allelic diversity predated speciation within this family. To understand how allelic diversity has evolved, we investigated the molecular basis of gametophytic SI in Antirrhinum, a member of the Scrophulariaceae, which is closely related to the Solanaceae. We have characterized three Antirrhinum cDNAs encoding polypeptides homologous to S RNases and shown that they are encoded by genes at the S locus. RNA in situ hybridization revealed that the Antirrhinum S RNase are primarily expressed in the stylar transmitting tissue. This expression is consistent with their proposed role in arresting the growth of self-pollen tubes. S alleles from the Scrophulariaceae form a separate group from those of the Solanaceae, indicating that new S alleles have been generated since these families separated (approximately 40 million years). We propose that the recruitment of an ancestral RNase gene into SI occurred during an early stage of angiosperm evolution and that, since that time, new alleles subsequently have arisen at a low rate.

The flower meristem identity genes floricaula (flo) and squamosa (squa) promote a change in phyllotaxy from spiral to whorled in Antirrhinum. To determine how this might be achieved, we have performed a combination of morphological, genetic, and expression analyses. Comparison of the phenotypes and RNA expression patterns of single and double mutants with the wild type showed that flo and squa act together to promote flower development but that flo is epistatic to squa with respect to early effects on phyllotaxy. We propose that a common process underlies the phyllotaxy of wildtype, flo, and squa meristem development but that the relative timing of primordium initiation or growth is altered. This process depends on two separable events: setting aside zones for potential primordium initiation and partitioning these zones into discrete primordia. Failure of the second event can lead to the formation of continuous double spirals, which are occasionally seen in flo mutants.

Several unstable mutations in Antirrhinum majus are caused by the insertion of transposable elements into genes. Excision and rearrangements of these elements can give rise to quantitative genetic variation and altered spatial patterns of gene expression. Element transposition is controlled by genetic and environmental factors and may make an important contribution to natural variation.

The 3.5 kb transposable element, Tam3, has been shown to cause somatic and germinal instability at the nivea locus, which encodes chalcone synthase, of Antirrhinum majus. Molecular cloning and sequence analysis of the niv-98::Tam3 allele revealed that the termini of Tam3 consist of 12 bp perfect inverted repeats. Tam3 is integrated in the promoter region of the chalcone synthase gene and generates an 8 bp duplication of target sequences upon integration. DNA sequencing of a niv +x revertant, niv-164, revealed a new type of sequence alteration upon excision: the duplications are displaced by ten nucleotides generated from adjacent sequences. Structural similarities of Tam3 and the maize elements Ac/Ds suggest that these elements belong to a common family.

Diverse spatial patterns of flower color in Antirrhinum can be produced by a series of alleles of pallida, a gene encoding an enzyme required for pigment biosynthesis. The alleles arose by imprecise excision of a transposable element, Tam3, and we show that they carry a series of deletions involving progressive removal of sequences adjacent to the excision site. This has enabled us to define three cis-acting upstream regions, A, B, and C, which differentially affect the level of pallida expression in distinct areas of the flower. We show further that an unlinked locus, delila, regulates the spatial distribution of pallida transcript. Deletion of regions ABC at the pallida locus uncouples pallida from regulation by delila, whereas deletion of A or AB brings pallida under regulation by delila in a new area of the flower. These results suggest that diverse patterns of pallida expression reflect the different ways in which alleles interact with a prepattern of both common and spatially specific genetic signals in the flower.

SUMMARY The transposable element, Tam 3, gives rise to large-scale chromosomal rearrangements at a low frequency, when it is inserted at the nivea locus of Antirrhinum majus. Some deletions that result from imprecise excision of Tam 3 provide an allelic series for functional analysis of the nivea gene promoter. Rearrangements involving deletion, dispersion and inverted duplication of flanking sequences, where Tam 3 remains in situ, have also been identified. These rearrangements have been mapped at the molecular level, and the behaviour of Tam 3 following rearrangement has been observed. It is clear that Tam 3 has enormous potential to restructure chromosomes through successive rounds of large-scale rearrangements.

The nivea locus of Antirrhinum majus encodes the enzyme chalcone synthase required for the synthesis of red anthocyanin pigment. The stable allele niv-44 contains an insertion in the nivea gene (Tam2) which has all the structural features of a transposable element. We have shown that this insertion can excise from the nivea locus when niv-44 is combined with another allele (niv-99) in a heterozygote. Activation of Tam2 excision is caused by a factor tightly linked to the niv-99 allele and may be due to complementation between Tam2 and a related element, Tam1. Factors which repress the excision of Tam2 and Tam1 are also described. Repression is not inherited in a simple mendelian way. Many stable mutations may be due to the insertion of transposable elements. Our data suggest that their stability may be due to the absence in the genome of activating factors and to the presence of repressors.

The frequent somatic change pallida—recurrens (pal—rec) → Pallida (Pal) in Antirrhinum majus to give full or spotted pigmentation varies inversely with temperature and is regulated by a stabiliser (st—St) system. Some Pal/Pal lines derived from highly unstable pal—rec/pal—rec had many pale sites indicating a resurgence of genetic instability. F1s between these resurgent (R) lines and lines homozygous recessive for different pigment-controlling genes showed numerous acyanic and occasional unstable rec areas. The frequency of these sites on the flowers also varied inversely with temperature and with the pal—rec stabiliser constitution. The application of 0.25% caffeine quadrupled the frequency of pale sites in R/R plants. Unstable somatic rec-type sites occurred de novo in crosses of R/R/ with 4 acyanic (or partially acyanic) genotypes. The administration of chemically defined precursors to the sites identified the genes involved somatically. Two contrasting types of instability in A. majus are described and compared. In the first, a blocked pigment-producing gene resumes its function (e.g. pal—rec → Pal) to give pigmented sites against an acyanic background. In the second, acyanic (or pale) sites are seen against an otherwise fully pigmented background. These occurrences together with the occasional appearance of new rec-type sites in crosses with the resurgent R/R lines are considered in terms of transposition of controlling elements.

Many aberrant somatic sectors on the corollas of heterozygous Antirrhinum majus can be related to a number of genetic causes while twin-spots are attributed to somatic crossing-over, hitherto, considered a relatively rare occurrence in higher plants. The administration of caffeine increases the frequency of single aberrant sites and also of twin-spots. The inclusion of the unstable genes nivea-recurrens and pallida-recurrens has no effect on the frequency of twin-spots. Homozygosity following somatic crossing-over is presumed for several loci and some evidence is presented for deficiency aberrations. The use of precise precursors in the biosynthetic pathway to anthocyanidin formation provided confirmatory evidence for the identification of the genes involved in the somatic aberrations.