A design principle for floral organ number and arrangement in flowers with bilateral symmetry.

The bilateral symmetry of flowers is a striking morphological achievement during floral evolution, providing high adaptation potential for pollinators. The symmetry can appear when floral organ primordia developmentally initiate. Primordia initiation at the ventral and dorsal sides of the floral bud is differentially regulated by several factors, including external organs of the flower and CYCLOIDEA (CYC) gene homologues, which are expressed asymmetrically on the dorso-ventral axis. It remains unclear how these factors control the diversity in the number and bilateral arrangement of floral organs. Here, we propose a mathematical model demonstrating that the relative strength of the dorsal-to-ventral inhibitions and the size of the floral stem cell region (meristem) determines the number and positions of the sepal and petal primordia. The simulations reproduced the diversity of monocots and eudicots, including snapdragon Antirrhinum majus and its cyc mutant, with respect to organ number, arrangement and initiation patterns, which were dependent on the inhibition strength. These theoretical results suggest that diversity in floral symmetry is primarily regulated by the dorso-ventral inhibitory field and meristem size during developmental evolution.

Genetics of Floral Development (By Christine Fleet).

Summaryplantcell;29/11/tpc.117.tt1117/FIG1F1fig1A basic model for floral organ identity has been developed using model systems such as Arabidopsis thaliana, snapdragon (Antirrhinum majus), and petunia (Petunia hybrida). In this model, different combinations of proteins known as ABCDE proteins, mostly MADS-domain transcription factors, activate the transcription of target genes to specify the identity of each whorl of floral organs. Changes in the regulation or activation of these target genes contribute to the wide variety of floral forms that we see within and across species. In addition, duplications and divergence of these genes in different groups of flowering plants have resulted in differences in gene function and expression patterns, contributing to differences in flower form across species. Posted December 8, 2017.Click HERE to access Teaching Tool Components.

Evolution of flower color pattern through selection on regulatory small RNAs.

Small RNAs (sRNAs) regulate genes in plants and animals. Here, we show that population-wide differences in color patterns in snapdragon flowers are caused by an inverted duplication that generates sRNAs. The complexity and size of the transcripts indicate that the duplication represents an intermediate on the pathway to microRNA evolution. The sRNAs repress a pigment biosynthesis gene, creating a yellow highlight at the site of pollinator entry. The inverted duplication exhibits steep clines in allele frequency in a natural hybrid zone, showing that the allele is under selection. Thus, regulatory interactions of evolutionarily recent sRNAs can be acted upon by selection and contribute to the evolution of phenotypic diversity.

Validation of Aintegumenta as a gene to modify floral size in ornamental plants.

The gene AINTEGUMENTA (AtANT) is an APETALA2 transcription factor in Arabidopsis activating growth downstream of auxin signalling. Lateral organ size is positively correlated with ANT expression in Arabidopsis. We tested the use of AtANT as a tool to modify floral size in two different plants used as model organisms and ornamental crops, Petunia × hybrida and Antirrhinum majus. Petunia plants expressing PhANT RNAi showed a decrease in PhANT expression correlated with smaller petal limbs. In contrast Petunia plants overexpressing AtANT had larger petal limbs. Petal tube length was less affected in down-regulation of PhANT or overexpression of AtANT. Overexpression of AtANT in Antirrhinum caused increased flower size via increased petal limb width and tube length. Down-regulation of PhANT showed an effect on cell size while overexpression of AtANT in Petunia and Antirrhinum caused significant increases in cell expansion that could explain the differences in floral organ size. The endogenous expression levels of PhANT and AmANT tended to be higher in the limb than in the tube in both Antirrhinum and Petunia. AtANT overexpression caused significant AmANT up-regulation in Antirrhinum limbs but not of PhANT in Petunia, indicating differences in the regulatory network. The differential effect of AtANT on limb and tube in Petunia and Antirrhinum correspond to phenotypic differences observed in natural variation in the corresponding genus indicating a relation between the phenotypic space of a genus and the effect of modified ANT levels, validating ANT as a gene to modify floral size.

Trends in flower symmetry evolution revealed through phylogenetic and developmental genetic advances.

A striking aspect of flowering plant (angiosperm) diversity is variation in flower symmetry. From an ancestral form of radial symmetry (polysymmetry, actinomorphy), multiple evolutionary transitions have contributed to instances of non-radial forms, including bilateral symmetry (monosymmetry, zygomorphy) and asymmetry. Advances in flowering plant molecular phylogenetic research and studies of character evolution as well as detailed flower developmental genetic studies in a few model species (e.g. Antirrhinum majus, snapdragon) have provided a foundation for deep insights into flower symmetry evolution. From phylogenetic studies, we have a better understanding of where during flowering plant diversification transitions from radial to bilateral flower symmetry (and back to radial symmetry) have occurred. From developmental studies, we know that a genetic programme largely dependent on the functional action of the CYCLOIDEA gene is necessary for differentiation along the snapdragon dorsoventral flower axis. Bringing these two lines of inquiry together has provided surprising insights into both the parallel recruitment of a CYC-dependent developmental programme during independent transitions to bilateral flower symmetry, and the modifications to this programme in transitions back to radial flower symmetry, during flowering plant evolution.

The influence of pigmentation patterning on bumblebee foraging from flowers of Antirrhinum majus.

Patterns of pigmentation overlying the petal vasculature are common in flowering plants and have been postulated to play a role in pollinator attraction. Previous studies report that such venation patterning is significantly more attractive to bee foragers in the field than ivory or white flowers without veins. To dissect the ways in which venation patterning of pigment can influence bumblebee behaviour, we investigated the response of flower-naïve individuals of Bombus terrestris to veined, ivory and red near-isogenic lines of Antirrhinum majus. We find that red venation shifts flower colour slightly, although the ivory background is the dominant colour. Bees were readily able to discriminate between ivory and veined flowers under differential conditioning but showed no innate preference when presented with a free choice of rewarding ivory and veined flowers. In contrast, both ivory and veined flowers were selected significantly more often than were red flowers. We conclude that advantages conferred by venation patterning might stem from bees learning of their use as nectar guides, rather than from any innate preference for striped flowers.

Quantitative levels of Deficiens and Globosa during late petal development show a complex transcriptional network topology of B function.

The transcriptional network topology of B function in Antirrhinum, required for petal and stamen development, is thought to rely on initial activation of transcription of DEFICIENS (DEF) and GLOBOSA (GLO), followed by a positive autoregulatory loop maintaining gene expression levels. Here, we show that the mutant compacta (co), whose vegetative growth and petal size are affected, plays a role in B function. Late events in petal morphogenesis such as development of conical cell area and scent emissions were reduced in co and def (nicotianoides) (def (nic) ), and absent in co def (nic) double mutants, suggesting a role for CO in petal identity. Expression of DEF was down-regulated in co but surprisingly GLO was not affected. We investigated the levels of DEF and GLO at late stages of petal development in the co, def (nic) and glo-1 mutants, and established a reliable transformation protocol that yielded RNAi-DEF lines. We show that the threshold levels of DEF or GLO required to obtain petal tissue are approximately 11% of wild-type. The relationship between DEF and GLO transcripts is not equal or constant and changes during development. Furthermore, down-regulation of DEF or GLO does not cause parallel down-regulation of the partner. Our results demonstrate that, at late stages of petal development, the B function transcriptional network topology is not based on positive autoregulation, and has additional components of transcriptional maintenance. Our results suggest changes in network topology that may allow changes in protein complexes that would explain the fact that not all petal traits appear early in development.

Generation of leaf shape through early patterns of growth and tissue polarity.

A major challenge in biology is to understand how buds comprising a few cells can give rise to complex plant and animal appendages like leaves or limbs. We address this problem through a combination of time-lapse imaging, clonal analysis, and computational modeling. We arrive at a model that shows how leaf shape can arise through feedback between early patterns of oriented growth and tissue deformation. Experimental tests through partial leaf ablation support this model and allow reevaluation of previous experimental studies. Our model allows a range of observed leaf shapes to be generated and predicts observed clone patterns in different species. Thus, our experimentally validated model may underlie the development and evolution of diverse organ shapes.

Patterns of floral colour neighbourhood and their effects on female reproductive success in an Antirrhinum hybrid zone.

The maintenance of genetic integrity of parental populations is often explained by selection against hybrids. However, the selection agents are usually unknown. The role of environmental biotic interactions is often suspected but has rarely been demonstrated. In plants for instance, mutualism with pollinators may be involved. After verification that pollen deposition is a limiting factor for fruit set, we used an individual-based study and a representation of pollinator colour perception to test the effects of local plant density and floral colour neighbourhood on female reproductive success in an Antirrhinum hybrid zone. In addition to flower colour and density effects, the composition of the floral neighbourhood was found to influence fruit set, suggesting that most plants were usually better fertilized when similar to their neighbours. However, the plants of one particular type were sometimes favoured when very different from their neighbours. The implications for hybrid zone dynamics are discussed.

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.

Evolution of allometry in antirrhinum.

Correlated variation in shape and size (allometry) is a major component of natural diversity. We examined the evolutionary and genetic basis for allometry using leaves and flower petals of snapdragon species (Antirrhinum). A computational method was developed to capture shape and size variation in both types of organ within the Antirrhinum species group. The results show that the major component of variation between species involves positively correlated changes in leaf and petal size. The correlation was maintained in an F2 population derived from crossing two species with organs of different sizes, suggesting that developmental constraints were involved. Identification of the underlying genes as quantitative trait loci revealed that the larger species carried alleles that increased organ size at all loci. Although this was initially taken as evidence that directional selection has driven diversity in both leaf and petal size, simulations revealed that evolution without consistent directional selection, an undirected walk, could also account for the parental distribution of organ size alleles.

An expanded evolutionary role for flower symmetry genes.

CYCLOIDEA (CYC)-like TCP genes are critical for flower developmental patterning. Exciting recent breakthroughs, including a study by Song et al. published in BMC Evolutionary Biology, demonstrate that CYC-like genes have also had an important role in the evolution of flower form.

LeafAnalyser: a computational method for rapid and large-scale analyses of leaf shape variation.

A comprehensive understanding of leaf shape is important in many investigations in plant biology. Techniques to assess variation in leaf shape are often time-consuming, labour-intensive and prohibited by complex calculation of large data sets. We have developed LeafAnalyser, software that uses image-processing techniques to greatly simplify the measurement of leaf shape variation. LeafAnalyser places a large number of evenly distributed landmarks along leaf margins and records the position of each automatically. We used LeafAnalyser to analyse the variation in 3000 leaves from 400 plants of Antirrhinum majus. We were able to summarise the major trends in leaf shape variation using a principal components (PC) analysis and assess the changes in size, width and tip-to-base asymmetry within our leaf library. We demonstrate how this information can be used to develop a model that describes the range and variation of leaf shape within standard wild-type lines, and illustrate the shape transformations that occur between leaf nodes. We also show that information from LeafAnalyser can be used to identify novel trends in shape variation, as low-variance PCs that only affect a subset of position landmarks. These results provide a high-throughput method to calculate leaf shape variation that allows a large number of leaves to be visualised in higher-dimensional phenotypic space. To illustrate the applicability of LeafAnalyser we also calculated the leaf shape variation in 300 leaves from Arabidopsis thaliana.

Phylogenetic analysis of the "ECE" (CYC/TB1) clade reveals duplications predating the core eudicots.

Flower symmetry is of special interest in understanding angiosperm evolution and ecology. Evidence from the Antirrhineae (snapdragon and relatives) indicates that several TCP gene-family transcription factors, especially CYCLOIDEA (CYC) and DICHOTOMA (DICH), play a role in specifying dorsal identity in the corolla and androecium of monosymmetric (bilateral) flowers. Studies of rosid and asterid angiosperms suggest that orthologous TCP genes may be important in dorsal identity, but there has been no broad phylogenetic context to determine copy number or orthology. Here, we compare published data from rosids and asterids with newly collected data from ranunculids, caryophyllids, Saxifragales, and Asterales to ascertain the phylogenetic placement of major duplications in the "ECE" (CYC/TB1) clade of TCP transcription factors. Bayesian analyses indicate that there are three major copies of "CYC" in the ECE clade, and that duplications leading to these copies predate the core eudicots. CYC1 contains no subsequent duplications and may not be expressed in floral tissue. CYC3 exhibits similar patterns of duplication to CYC2 in several groups. Using RT-PCR, we show that, in flowers of Lonicera morrowii (Caprifoliaceae), DipsCYC2B is expressed in the four dorsal petals and not in the ventral petal. DipsCYC3B is expressed in flower and petal primordia, possibly most strongly in the ventral petal.

The temperature-dependent change in methylation of the Antirrhinum transposon Tam3 is controlled by the activity of its transposase.

The Antirrhinum majus transposon Tam3 undergoes low temperature-dependent transposition (LTDT). Growth at 15 degrees C permits transposition, whereas growth at 25 degrees C strongly suppresses it. The degree of Tam3 DNA methylation is altered somatically and positively correlated with growth temperature, an exceptional epigenetic system in plants. Using a Tam3-inactive line, we show that methylation change depends on Tam3 activity. Random binding site selection analysis and electrophoretic mobility shift assays revealed that the Tam3 transposase (TPase) binds to the major repeat in the subterminal regions of Tam3, the site showing the biggest temperature-dependent change in methylation state. Methylcytosines in the motif impair the binding ability of the TPase. Proteins in a nuclear extract from plants grown at 15 degrees C but not 25 degrees C bind to this motif in Tam3. The decrease in Tam3 DNA methylation at low temperature also requires cell division. Thus, TPase binding to Tam3 occurs only during growth at low temperature and immediately after DNA replication, resulting in a Tam3-specific decrease in methylation of transposon DNA. Consequently, the Tam3 methylation level in LTDT is regulated by Tam3 activity, which is dependent on the ability of its TPase to bind DNA and affected by growth temperature. Thus, the methylation/demethylation of Tam3 is the consequence, not the cause, of LTDT.

Flower symmetry and shape in Antirrhinum.

According to their symmetry, flowers are classified as radially symmetrical or bilaterally symmetrical. Bilateral symmetry, which is thought to have evolved from radial symmetry, results from establishment of asymmetry relative to a dorsoventral axis of flowers. Here we consider developmental genetic mechanisms underlying the generation of this asymmetry and how they relate to controls of petal shape and growth in Antirrhinum. Two genes, CYC and DICH, are expressed in dorsal domains of the Antirrhinum flower and determine its overall dorsoventral asymmetry and the asymmetries and shapes of individual floral organs, by influencing regional growth. Another gene, DIV, influences regional asymmetries and shapes in ventral regions of the flower through a quantitative effect on growth. However, DIV is not involved in determining the overall dorsoventral asymmetry of the flower and its effects on regional asymmetries depend on interactions with CYC/DICH. These interactions illustrate how gene activity, symmetry, shape and growth may be related.

Evolution through genetically controlled allometry space.

Understanding evolutionary change requires phenotypic differences between organisms to be placed in a genetic context. However, there are few cases where it has been possible to define an appropriate genotypic space for a range of species. Here we address this problem by defining a genetically controlled space that captures variation in shape and size between closely related species of Antirrhinum. The axes of the space are based on an allometric model of leaves from an F2 of an interspecific cross between Antirrhinum majus and Antirrhinum charidemi. Three principal components were found to capture most of the genetic variation in shape and size, allowing a three-dimensional allometric space to be defined. The contribution of individual genetic loci was determined from QTL analysis, allowing each locus to be represented as a vector in the allometric space. Leaf shapes and sizes of 18 different Antirrhinum taxa, encompassing a broad range of leaf morphologies, could be accurately represented as clouds within the space. Most taxa overlapped with, or were near to, at least one other species in the space, so that together they defined a largely interconnected domain of viable forms. It is likely that the pattern of evolution within this domain reflects a combination of directional selection and evolutionary tradeoffs within a high dimensional space.

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.

Growth dynamics underlying petal shape and asymmetry.

Development commonly involves the generation of complex shapes from simpler ones. One way of following this process is to use landmarks to track the fate of particular points in a developing organ, but this is limited by the time over which it can be monitored. Here we use an alternative method, clonal analysis, whereby dividing cells are genetically marked and their descendants identified visually, to observe the development of Antirrhinum (snapdragon) petals. Clonal analysis has previously been used to estimate growth parameters of leaves and Drosophila wings but these results were not integrated within a dynamic growth model. Here we develop such a model and use it to show that a key aspect of shape--petal asymmetry--in the petal lobe of Antirrhinum depends on the direction of growth rather than regional differences in growth rate. The direction of growth is maintained parallel to the proximodistal axis of the flower, irrespective of changes in shape, implying that long-range signals orient growth along the petal as a whole. Such signals may provide a general mechanism for orienting growth in other growing structures.