Growth and cellular patterns in the petal epidermis of Antirrhinum majus: empirical studies.

BACKGROUND AND AIMS: Analysis of cellular patterns in plant organs provides information about the orientation of cell divisions and predominant growth directions. Such an approach was employed in the present study in order to characterize growth of the asymmetrical wild-type dorsal petal and the symmetrical dorsalized petal of the backpetals mutant in Antirrhinum majus. The aims were to determine how growth in an initially symmetrical petal primordium leads to the development of mature petals differing in their symmetry, and to determine how specific cellular patterns in the petal epidermis are formed. METHODS: Cellular patterns in the epidermis in both petal types over consecutive developmental stages were visualized and characterized quantitatively in terms of cell wall orientation and predominant types of four-cell packets. The data obtained were interpreted in terms of principal directions of growth (PDGs). KEY RESULTS: Both petal types grew predominantly along the proximo-distal axis. Anticlinal cell walls in the epidermis exhibited a characteristic fountain-like pattern that was only slightly modified in time. New cell walls were mostly perpendicular to PDG trajectories, but this alignment could change with wall age. CONCLUSIONS: The results indicate that the predominant orientation of cell division planes and the fountain-like cellular pattern observed in both petal types may be related to PDGs. The difference in symmetry between the two petal types arises because PDG trajectories in the field of growth rates (growth field) controlling petal growth undergo gradual redefinition. This redefinition probably takes place in both petal types but only in the wild-type does it eventually lead to asymmetry in the growth field. Two scenarios of how redefinition of PDGs may contribute to this asymmetry are considered.

A subcellular tug of war involving three MYB-like proteins underlies a molecular antagonism in Antirrhinum flower asymmetry.

The establishment of meristematic domains with different transcriptional activity is essential for many developmental processes. The asymmetry of the Antirrhinum majus flower is established by transcription factors with an asymmetric pattern of activity. To understand how this asymmetrical pattern is established, we studied the molecular mechanism through which the dorsal MYB protein RADIALIS (RAD) restricts the activity of the MYB transcription factor DIVARICATA (DIV) to the ventral region of the flower meristem. We show that RAD competes with DIV for binding with other MYB-like proteins, termed DRIF1 and DRIF2 (DIV- and-RAD-interacting-factors). DRIF1 and DIV interact to form a protein complex that binds to the DIV-DNA consensus region, suggesting that the DRIFs act as co-regulators of DIV transcriptional activity. In the presence of RAD, the interaction between DRIF1 and DIV bound to DNA is disrupted. Moreover, the DRIFs are sequestered in the cytoplasm by RAD, thus, preventing or reducing the formation of DRIF-DIV heterodimers in the nuclei. Our results suggest that in the dorsal region of the Antirrhinum flower meristem the dorsal protein RAD antagonises the activity of the ventral identity protein DIV in a subcellular competition for a DRIF protein promoting the establishment of the asymmetric pattern of gene activity in the Antirrhinum flower.

Generation of diverse biological forms through combinatorial interactions between tissue polarity and growth.

A major problem in biology is to understand how complex tissue shapes may arise through growth. In many cases this process involves preferential growth along particular orientations raising the question of how these orientations are specified. One view is that orientations are specified through stresses in the tissue (axiality-based system). Another possibility is that orientations can be specified independently of stresses through molecular signalling (polarity-based system). The axiality-based system has recently been explored through computational modelling. Here we develop and apply a polarity-based system which we call the Growing Polarised Tissue (GPT) framework. Tissue is treated as a continuous material within which regionally expressed factors under genetic control may interact and propagate. Polarity is established by signals that propagate through the tissue and is anchored in regions termed tissue polarity organisers that are also under genetic control. Rates of growth parallel or perpendicular to the local polarity may then be specified through a regulatory network. The resulting growth depends on how specified growth patterns interact within the constraints of mechanically connected tissue. This constraint leads to the emergence of features such as curvature that were not directly specified by the regulatory networks. Resultant growth feeds back to influence spatial arrangements and local orientations of tissue, allowing complex shapes to emerge from simple rules. Moreover, asymmetries may emerge through interactions between polarity fields. We illustrate the value of the GPT-framework for understanding morphogenesis by applying it to a growing Snapdragon flower and indicate how the underlying hypotheses may be tested by computational simulation. We propose that combinatorial intractions between orientations and rates of growth, which are a key feature of polarity-based systems, have been exploited during evolution to generate a range of observed biological shapes.

Actomyosin mediates gravisensing and early transduction events in reoriented cut snapdragon spikes.

We investigated the involvement of the actomyosin network in the early events of the gravitropic response of cut snapdragon (Antirrhinum majus L.) spikes. The effects of the actin-modulating drug, cytochalasin D (CD) and/or the myosin inhibitor, 2,3-butanedione-2-monoxime (BDM) on amyloplast displacement, lateral auxin transport and consequently on stem bending were examined. The inhibitory effect on cytoskeleton integrity was studied by using indirect immunofluorescence double-labeling of actin and myosin. Our results demonstrate that no organizational changes in actin filaments occurred in cortical and endodermal cells of the stem bending zone during reorientation. These results suggest that actin depolymerization is not required for amyloplast sedimentation. Unlike the chloroplasts in the cortex, the amyloplasts in the endodermis were surrounded by actin and myosin, indicating that amyloplasts may be attached to the actin filaments via the motor protein, myosin. This suggests the involvement of myosin as part of the actomyosin complex in amyloplast movement in vertical as well as in reoriented stems. This suggestion was supported by the findings showing that: (a) BDM or CD disrupted the normal organization of actin either by altering characteristic distribution patterns of myosin-like protein in the cortex (BDM), or by causing actin fragmentation (CD); (b) both compounds inhibited the gravity-induced amyloplast displacement in the endodermis. Additionally, these compounds also inhibited lateral auxin transport across the stem and stem gravitropic bending. Our study suggests that during stem reorientation amyloplasts possibly remain attached to the actin filaments, using myosin as a motor protein. Thus, gravisensing and early transduction events in the gravitropic response of snapdragon spikes, manifested by amyloplast displacement and lateral auxin transport, are mediated by the actomyosin complex.

Determining the contribution of epidermal cell shape to petal wettability using isogenic Antirrhinum lines.

The petal epidermis acts not only as a barrier to the outside world but also as a point of interaction between the flower and potential pollinators. The presence of conical petal epidermal cells has previously been shown to influence the attractiveness of the flower to pollinating insects. Using Antirrhinum isogenic lines differing only in the presence of a single epidermal structure, conical cells, we were able to investigate how the structure of the epidermis influences petal wettability by measuring the surface contact angle of water drops. Conical cells have a significant impact on how water is retained on the flower surface, which may have indirect consequences for pollinator behaviour. We discuss how the petal epidermis is a highly multifunctional one and how a battery of methods, including the use of isogenic lines, is required to untangle the impacts of specific epidermal properties in an ecological context.

The molecular basis for venation patterning of pigmentation and its effect on pollinator attraction in flowers of Antirrhinum.

Pigment stripes associated with veins (venation) is a common flower colour pattern. The molecular genetics and function of venation were investigated in the genus Antirrhinum, in which venation is determined by Venosa (encoding an R2R3MYB transcription factor). Pollinator preferences were measured by field tests with Antirrhinum majus. Venosa function was examined using in situ hybridization and transient overexpression. The origin of the venation trait was examined by molecular phylogenetics. Venation and full-red flower colouration provide a comparable level of advantage for pollinator attraction relative to palely pigmented or white lines. Ectopic expression of Venosa confers pigmentation outside the veins. Venosa transcript is produced only in small areas of the corolla between the veins and the adaxial epidermis. Phylogenetic analyses suggest that venation patterning is an ancestral trait in Antirrhinum. Different accessions of three species with full-red pigmentation with or without venation patterning have been found. Epidermal-specific venation is defined through overlapping expression domains of the MYB (myoblastoma) and bHLH (basic Helix-Loop-Helix) co-regulators of anthocyanin biosynthesis, with the bHLH providing epidermal specificity and Venosa vein specificity. Venation may be the ancestral trait, with full-red pigmentation a derived, polyphyletic trait. Venation patterning is probably not fixed once species evolve full-red floral pigmentation.

The mutants compacta ähnlich, Nitida and Grandiflora define developmental compartments and a compensation mechanism in floral development in Antirrhinum majus.

In order to improve our understanding of floral size control we characterised three mutants of Antirrhinum majus with different macroscopic floral phenotypes. The recessive mutant compacta ähnlich has smaller flowers affected mainly in petal lobe expansion, the dominant mutant Grandiflora has overall larger organs, whilst the semidominant mutation Nitida exhibits smaller flowers in a dose-dependent manner. We developed a cell map in order to establish the cellular phenotypes of the mutants. Changes in organ size were both organ- and region-specific. Nitida and compacta ähnlich affected cell expansion in proximal and distal petal regions, respectively, suggesting differential regulation between petal lobe regions. Although petal size was smaller in compacta ähnlich than in wild type, conical cells were significantly bigger, suggesting a compensation mechanism involved in petal development. Grandiflora had larger cells in petals and increased cell division in stamens and styles, suggesting a relationship between genes controlling organ size and organ identity. The level of ploidy in petals of Grandiflora and coan was found to be equivalent to wild type petals and leaves, ruling out an excess of growth via endoreduplication. We discuss our results in terms of current models about control of lateral organ size.

Conservation and diversification of the symmetry developmental program among close relatives of snapdragon with divergent floral morphologies.

Multiple evolutionary shifts in floral symmetry and stamen number have occurred in the snapdragon (Antirrhinum majus) family Veronicaceae. In Mohavea, Veronica and Gratiola there have been independent evolutionary reductions in stamen number and modifications to corolla shape. It is hypothesized that changes in the regulation of homologs of snapdragon dorsal flower identity genes CYCLOIDEA (CYC) and RADIALIS (RAD) underlie these floral transitions. CYC-like and RAD-like genes from Veronica montana and Gratiola officinalis were cloned and sequenced, compared with homologs from other Veronicaceae species using phylogenetic analysis, and their expression was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. VmCYC1, GoCYC1, GoCYC2 and RAD-like genes are expressed exclusively in the dorsal region of floral meristems and developing flowers. Their expression patterns do not correlate with patterns of stamen arrest. VmCYC2 and GoCYC3 are expressed in both vegetative and floral tissues, with VmCYC2 being most abundant in all regions of the floral meristem and all petals. These results support conservation of the floral symmetry gene network for Veronicaceae RAD-like and some CYC-like paralogs, suggest regulatory evolution of other CYC-like genes following gene duplication and implicate different genetic mechanisms underlying dorsal versus ventral stamen abortion within Veronica and Gratiola.

FORMOSA controls cell division and expansion during floral development in Antirrhinum majus.

Control of organ size is the product of coordinated cell division and expansion. In plants where one of these pathways is perturbed, organ size is often unaffected as compensation mechanisms are brought into play. The number of founder cells in organ primordia, dividing cells, and the period of cell proliferation determine cell number in lateral organs. We have identified the Antirrhinum FORMOSA (FO) gene as a specific regulator of floral size. Analysis of cell size and number in the fo mutant, which has increased flower size, indicates that FO is an organ-specific inhibitor of cell division and activator of cell expansion. Increased cell number in fo floral organs correlated with upregulation of genes involved in the cell cycle. In Arabidopsis the AINTEGUMENTA (ANT) gene promotes cell division. In the fo mutant increased cell number also correlates with upregulation of an Antirrhinum ANT-like gene (Am-ANT) in inflorescences that is very closely related to ANT and shares a similar expression pattern, suggesting that they may be functional equivalents. Increased cell proliferation is thought to be compensated for by reduced cell expansion to maintain organ size. In Arabidopsis petal cell expansion is inhibited by the BIGPETAL (BPE) gene, and in the fo mutant reduced cell size corresponded to upregulation of an Antirrhinum BPE-like gene (Am-BPE). Our data suggest that FO inhibits cell proliferation by negatively regulating Am-ANT, and acts upstream of Am-BPE to coordinate floral organ size. This demonstrates that organ size is modulated by the organ-specific control of both general and local gene networks.

Heterochromatic and genetic features are consistent with recombination suppression of the self-incompatibility locus in Antirrhinum.

Self-incompatibility (SI) is a genetic mechanism to prevent self-fertilization that is found in many species of flowering plants. Molecular studies have demonstrated that the S-RNase and SLF/SFB genes encoded by the single polymorphic S locus, which control the pollen and pistil functions of SI in three distantly related families, the Solanaceae, Scrophulariaceae and Rosaceae, are organized in a haplotype-specific manner. Previous work suggested that the haplotype structure of the two genes is probably maintained by recombination suppression at the S locus. To examine features associated with this suppression, we first mapped the S locus of Antirrhinum hispanicum, a member of the Scrophulariaceae, to a highly heterochromatic region close to the distal end of the short arm of chromosome 8. Both leptotene chromosome and DNA fiber fluorescence in situ hybridization analyses showed an obvious haplotype specificity of the Antirrhinum S locus that is consistent with its haplotype structure. A chromosome inversion was also detected around this region between A. majus and A. hispanicum. These results revealed that DNA sequence polymorphism and a heterochromatic location are associated with the S locus. Possible roles of these features in maintenance of the haplotype specificity involved in both self and non-self recognition are discussed.

A truncated MYB transcription factor from Antirrhinum majus regulates epidermal cell outgrowth.

Plant MYB genes can be divided into subgroups on the basis of additional conserved regions of sequence. In some cases, genes within a subgroup share similarities in function, as well as sequence. The functions of three proteins in subgroup 9 have been described, with AmMYBMX regulating the differentiation of conical-papillate petal epidermal cells, PhMYB1 involved in extending the growth of these cells, and AmMYBML1 involved in differentiation of several petal epidermal cell types. Here, the isolation of a gene encoding a new member of MYB subgroup 9, AmMYBML3 (Antirrhinum majus MYB MIXTA-LIKE 3) is described, which contains the defining regions of conserved sequence but is lacking the majority of the C-terminus, including the amphipathic alpha-helix presumed necessary for transcriptional activation. AmMYBML3 is expressed in all aerial organs, but its expression is restricted to outgrowing epidermal cells, including trichomes, stigmatic papillae, and petal conical-papillate cells. Ectopic expression of AmMYBML3 in tobacco results in the formation of conical-papillate cells in the usually flat carpel epidermis. These data suggest that this protein is capable of altering epidermal development, thus resulting in cellular outgrowth, despite the missing C-terminus, and may act in conjunction with other transcriptional activators to enhance cellular outgrowth from the epidermis of all aerial organs.

Control of cell and petal morphogenesis by R2R3 MYB transcription factors.

Petals of animal-pollinated angiosperms have adapted to attract pollinators. Factors influencing pollinator attention include colour and overall size of flowers. Colour is determined by the nature of the pigments, their environment and by the morphology of the petal epidermal cells. Most angiosperms have conical epidermal cells, which enhance the colour intensity and brightness of petal surfaces. The MYB-related transcription factor MIXTA controls the development of conical epidermal cells in petals of Antirrhinum majus. Another gene encoding an R2R3 MYB factor very closely related to MIXTA, AmMYBML2, is also expressed in flowers of A. majus. We have analysed the roles of AmMYBML2 and two MIXTA-related genes, PhMYB1 from Petunia hybrida and AtMYB16 from Arabidopsis thaliana, in petal development. The structural similarity between these genes, their comparable expression patterns and the similarity of the phenotypes they induce when ectopically expressed in tobacco, suggest they share homologous functions closely related to, but distinct from, that of MIXTA. Detailed phenotypic analysis of a phmyb1 mutant confirmed the role of PhMYB1 in the control of cell morphogenesis in the petal epidermis. The phmyb1 mutant showed that epidermal cell shape affects petal presentation, a phenotypic trait also observed following re-examination of mixta mutants. This suggests that the activity of MIXTA-like genes also contributes to petal form, another important factor influencing pollinator attraction.

DNA degradation and nuclear degeneration during programmed cell death in petals of Antirrhinum, Argyranthemum, and Petunia.

Programmed cell death (PCD) was studied in the petals of Antirrhinum majus, Argyranthemum frutescens, and Petunia hybrida, using DNA degradation and changes in nuclear morphology as parameters. The petals exhibit loss of turgor (wilting) as a visible symptom of PCD. DNA degradation, as shown on agarose gels, occurred in all species studied, prior to visible wilting. The number of DNA masses in all the petals of a flower, determined by flow cytometry, markedly increased in Argyranthemum and Petunia, but decreased in Antirrhinum. Many small DNA masses were observed in Argyranthemum and Petunia. The surface of each small DNA mass stained with the lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide (DiOC6), indicating that these masses were surrounded by a membrane. In Antirrhinum, in contrast, the chromatin fragmented into several small spherical clumps that remained inside a large membranous structure. Nuclear fragmentation, therefore, did not occur in Antirrhinum, whereas nuclear fragmentation possibly was a cause of the small DNA masses in Argyranthemum and Petunia. It is concluded that at least two contrasting nuclear morphologies exist during PCD. In the first, the chromatin fragments inside the nucleus, not accompanied--or followed--by nuclear fragmentation. In the second, a large number of DNA masses were observed each enveloped by a membrane. The second type was probably due, at least partially, to nuclear fragmentation.

Analysis of the transcription factor WUSCHEL and its functional homologue in Antirrhinum reveals a potential mechanism for their roles in meristem maintenance.

One of the most significant features of plant development is the way in which it can be elaborated and modulated throughout the life of the plant, an ability that is conferred by meristems. The Arabidopsis thaliana WUSCHEL gene (WUS), which encodes a homeodomain transcription factor, is required to maintain the stem cells in the shoot apical meristem in an undifferentiated state. The mechanism by which WUS prevents the differentiation of stem cells is unknown. We have characterized a meristem maintenance mutant in Antirrhinum majus and shown that it arises from a defect in the WUS orthologue ROSULATA (ROA). Detailed characterization of a semidominant roa allele revealed an essential role for the conserved C-terminal domain. Expression of either ROA or WUS lacking this domain causes a failure of meristem maintenance. The conserved domain mediates an interaction between WUS and two members of a small family of corepressor-like proteins in Arabidopsis. Our results suggest that WUS functions by recruiting transcriptional corepressors to repress target genes that promote differentiation, thereby ensuring stem cell maintenance.

The pollen-specific DEFH125 promoter from Antirrhinum is bound in vivo by the MADS-box proteins DEFICIENS and GLOBOSA.

The Antirrhinum DEFH125 MADS-box protein is expressed in maturing pollen and thus likely participates in the regulation of pollen development. Here, we describe the characterization of a 2.5 kbp promoter fragment conferring pollen-specific GUS expression in Antirrhinum, as well as in the distantly related species Arabidopsis. Taking advantage of the higher sensitivity of the diphtheria toxin A-chain (DTA) reporter gene assay, onset of DEFH125 promoter activity could be defined to start at the late unicellular microspore stage. Stamen development in Antirrhinum is governed by the class B MADS-box genes DEFICIENS (DEF) and GLOBOSA (GLO). The respective proteins form a heterodimer and are expressed throughout stamens, except for microspores. Complementary expression patterns of DEFH125 and DEF/GLO during later stamen development tempted us to investigate whether the DEF/GLO heterodimer might bind the DEFH125 promoter and could thus be involved in repressing the DEFH125 expression. The ChIP technique was applied to investigate protein/DNA interactions occurring in vivo. We report the identification of a 200 bp DEFH125 promoter fragment that is in vivo bound by DEF and GLO proteins. This fragment contains a CArG-box motif, known to mediate DNA binding of MADS-box proteins. Implications for a likely function of DEF and GLO in the transcriptional control of DEFH125 are discussed.

A computational method for inferring growth parameters and shape changes during development based on clonal analysis.

We describe a method for estimating growth parameters in various regions of a developing organ undergoing cell divisions, along with the corresponding changes in organ shape. Growth parameters are computed by coupling clonal analysis with a growth model, allowing a wide range of developmental stages to be covered. The method was applied to the development of dorsal petal lobes of Antirrhinum majus. The resulting description of growth patterns and shape changes is consistent with direct observations using scanning electron microscopy. This method can potentially be applied to other organs, and opens the way to comparative studies of growth and gene expression patterns.

Regulation of developmental pathways in cultured microspores of tobacco and snapdragon by medium pH.

The regulation of developmental pathways in cultured microspores of tobacco ( Nicotiana tabacum L) and snapdragon ( Antirrhinum majus L) by medium pH is described for the first time. Unicellular tobacco and snapdragon microspores developed into normal, fertile pollen when cultured in media T1 and AT3 at pH 7.0 and 25 degrees C for 6 and 8 days, respectively. First, pollen mitosis was asymmetric and mature pollen grains were filled with starch granules and germinated upon transfer to a germination medium. However, when tobacco and snapdragon microspores were cultured in media T1 and AT3, respectively, at pH 8.0-8.5 for 4-6 days at 25 degrees C, the frequency of symmetric division increased significantly with the formation two nuclei of equal size, and the gametophytic pathway was blocked, as seen by the lack of starch accumulation and the inhibition of pollen germination. The transfer of these microspores to embryogenesis medium AT3 at pH 6.5 resulted in the formation of multicellular structures in both species and, in tobacco, in the formation of embryos and plants. In order to understand the possible mechanisms of the action of high pH, sucrose metabolism was analysed in isolated microspores of tobacco cultured at various pH values. Invertase (EC 3.2.1.26) activity in microspores was maximal at pH 5.0 and strongly decreased at higher pH, leading to a slow-down of sucrose cleavage. At the same time the incorporation of (14)C-labelled sucrose from the medium into microspores was drastically reduced at high pH. These data suggest that isolated microspores are not able to metabolise carbohydrates at high pH and thus undergo starvation stress, which was shown earlier to block the gametophytic pathway and trigger sporophytic development.

Allozymic differentiation of the Antirrhinum graniticum and the Antirrhinum meonanthum species groups.

Forty populations from eight taxa belonging to six species of Antirrhinum were studied for electrophoretic differentiation at 14 allozyme loci. All the studied species show that marker alleles and genetic distances between conspecific populations are lower than for other species of the genus. Results permit the recognition of A. onubensis and A. boissieri at specific rank. The low level of genetic distance together with morphological characters and sympatric range, support the recognition of A. ambiguum as a new subspecies of A. graniticum, as A. graniticum ssp. ambiguum Mateu & Segarra stat. nov. The results of the study support separation of the studied taxa into two groups coinciding with subsections Streptosepalum and Antirrhinum, but disagreeing with the arrangement of species into them. According to the results of the study these groups consist of A. meonanthum and A. braun-blanquetii (Streptosepalum) and A. graniticum s.l., A. onubensis and A. boissieri (Antirrhinum). The status of A. meonanthum var. rothmaleri requires further clarification.

The role of actin filaments in the gravitropic response of snapdragon flowering shoots.

The involvement of the actin and the microtubule cytoskeleton networks in the gravitropic response of snapdragon ( Antirrhinum majus L.) flowering shoots was studied using various specific cytoskeleton modulators. The microtubule-depolymerizing drugs tested had no effect on gravitropic bending. In contrast, the actin-modulating drugs, cytochalasin D (CD), cytochalasin B (CB) and latrunculin B (Lat B) significantly inhibited the gravitropic response. CB completely inhibited shoot bending via inhibiting general growth, whereas CD completely inhibited bending via specific inhibition of the differential flank growth in the shoot bending zone. Surprisingly, Lat B had only a partial inhibitory effect on shoot bending as compared to CD. This probably resulted from the different effects of these two drugs on the actin cytoskeleton, as was seen in cortical cells. CD caused fragmentation of the actin cytoskeleton and delayed amyloplast displacement following gravistimulation. In contrast, Lat B caused a complete depolymerization of the actin filaments in the shoot bending zone, but only slightly reduced the amyloplast sedimentation rate following gravistimulation. Taken together, our results suggest that the actin cytoskeleton is involved in the gravitropic response of snapdragon shoots. The actin cytoskeleton within the shoot cells is necessary for normal amyloplast displacement upon gravistimulation, which leads to the gravitropic bending.

Regulating shapes and sizes.

Mutations at many loci lead to altered shapes and sizes, suggesting complex regulation of the overall morphology of an organism. Two recent studies present data on how orientation of growth axes and perception of maturation signals might regulate growth processes.