Duplication of floral regulatory genes in the Lamiales

Duplication of some floral regulatory genes has occurred repeatedly in angiosperms, whereas others are thought to be single-copy in most lineages. We selected three genes that interact in a pathway regulating floral development conserved among higher tricolpates (LFY/FLO, UFO/FIM, and AP3/DEF) and screened for copy number among families of Lamiales that are closely related to the model species Antirrhinum majus. We show that two of three genes have duplicated at least twice in the Lamiales. Phylogenetic analyses of paralogs suggest that an ancient whole genome duplication shared among many families of Lamiales occurred after the ancestor of these families diverged from the lineage leading to Veronicaceae (including the single-copy species A. majus). Duplication is consistent with previous patterns among angiosperm lineages for AP3/DEF, but this is the first report of functional duplicate copies of LFY/FLO outside of tetraploid species. We propose Lamiales taxa will be good models for understanding mechanisms of duplicate gene preservation and how floral regulatory genes may contribute to morphological diversity.     

Photosynthesis and stomatal characteristics of Striga hermonthica in relation to its parasitic habit

Photosynthesis and stomatal characteristics of the angiosperm parasite Striga her-monthica (Del.) Benth. have been compared with those of Antirrhinum majus L. cv. Suttons Yellow Rust Resistant, a related, non-parasitic species. The concentration of photosynthetic pigments in S. hermonthica leaves was less than 40% of those of A. majus leaves. Light saturated CO₂ assimilation rate of S. hermonthica was less than 40% that of A. majus on a chlorophyll basis and under 20% than of A. majus on a leaf area basis.
Stomata of Striga showed only partial closure in darkness, remained open in water stressed leaves and showed little response to exogenously applied abscisic acid. Stomatal conductance and transpiration were considerably higher in Striga compared with Antirrhinum . The high transpiration and low photosynthetic rates of S. hermonthica resulted in a low water use efficiency. The water relations of Striga leaves, while seemingly inappropriate for growth in drought prone environments, do appear to maximise nutrient and water acquisition from the host and as such may be an adaptation to the parasitic habit.     

Molecular cytogenetic characterization of the Antirrhinum majus genome

As a model system in classical plant genetics, the genus Antirrhinum has been well studied, especially in gametophytic self-incompatibility, flower development biology, and transposon-induced mutation. In contrast to the advances in genetic and molecular studies, little is known about Antirrhinum cytogenetics. In this study, we isolated two tandem repetitive sequences, CentA1 and CentA2, from the centromeric regions of Antirrhinum chromosomes. A standard karyotype has been established by anchoring these centromeric repeats on meiotic pachytene chromosome using FISH. An ideogram based on the DAPI-staining pattern of pachytene chromosomes was developed to depict the distribution of heterochromatin in the Antirrhinum majus genome. To integrate the genetic and chromosomal maps, we selected one or two molecular markers from each linkage group to screen an Antirrhinum transformation-competent artificial chromosome (TAC) library. These genetically anchored TAC clones were labeled as FISH probes to hybridize to pachytene chromosomes of A. majus. As a result, the relationship between chromosomes and the linkage groups (LGs) in Antirrhinum has been established.     

Floral initiation and inflorescence architecture: a comparative view

BACKGROUND: A huge variety of plant forms can be found in nature. This is particularly noticeable for inflorescences, the region of the plant that contains the flowers. The architecture of the inflorescence depends on its branching pattern and on the relative position where flowers are formed. In model species such as Arabidopsis thaliana or Antirrhinum majus the key genes that regulate the initiation of flowers have been studied in detail and much is known about how they work. Studies being carried out in other species of higher plants indicate that the homologues of these genes are also key regulators of the development of their reproductive structures. Further, changes in these gene expression patterns and/or function play a crucial role in the generation of different plant architectures. SCOPE: In this review we aim to present a summarized view on what is known about floral initiation genes in different plants, particularly dicotyledonous species, and aim to emphasize their contribution to plant architecture.     

Phylogenetic relationships of North American Antirrhinum (Veronicaceae)

Species of the genus Antirrhinum (Veronicaceae) provide excellent opportunities for research on plant evolution given their extensive morphological and ecological diversity. These opportunities are enhanced by genetic and developmental data from the model organism Antirrhinum majus. The genus Antirrhinum includes 15 New World species in section Saerorhinum and 21 Old World species in sections Antirrhinum and Orontium. Phylogenetic analyses of sequences of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA were conducted for 19 Antirrhinum species, including all species from the New World, and 13 related genera in the tribe Antirrhineae. These analyses confirm the monophyly of Antirrhinum given the inclusion of the small genus Mohavea and exclusion of A. cyathiferum. The New World species, all of which are tetraploid, form a clade that is weakly supported as sister to the Old World sect. Orontium. The Old World species in sect. Antirrhinum form a well-supported clade that is sister to the remainder of the genus. In addition, both molecular and morphological data are used in the most comprehensive effort to date focused on recovering the phylogenetic relationships among the extremely diverse species in section Saerorhinum.     

Evolution of novel morphological and reproductive traits in a clade containing Antirrhinum majus (Scrophulariaceae)

Phylogenetic analysis of DNA sequences of the chloroplast genes rcbL and ndhf revealed a highly supported clade composed of the families Plantaginaceae, Callitrichaceae, and Hippuridaceae in close association with the model organism Antirrhinum majus and other members of family Scrophulariaceae. Plantago has miniature actinomorphic wind-pollinated flowers that have evolved from zygomorphic animal-pollinated precursors. The aquatic Hippuridaceae have reduced windpollinated flowers with one reproductive organ per whorl, and three, rather than four, whorls. In monoecious aquatic Callitrichaceae, further reduction has occurred such that there is only one whorl per flower containing a single stamen or carpel. Optimization of character states showed that these families descended from an ancestor similar to Antirrhinum majus. Recent studies of plant developmental genetics have focused on distantly related species. Differences in the molecular mechanisms controlling floral development between model organisms are difficult to interpret due to phylogenetic distance. In order to understand evolutionary changes in floral morphology in terms of their underlying genetic processes, closely related species exhibiting morphological Variation should be examined. Studies of genes that regulate morphogenesis in the clade described here could aid in the elucidation of a general model tot such fundamental issues as how changes in floral symmetry, organ number, and whorl number are achieved, as well as providing insight on the evolution of dicliny and associated changes in pollination syndrome.     

The evolutionary history of Antirrhinum suggests that ancestral phenotype combinations survived repeated hybridizations

The model species Antirrhinum majus (the garden snapdragon) has over 20 close wild relatives that are morphologically diverse and adapted to different Mediterranean environments. Hybrids between Antirrhinum species have been used successfully to identify genes underlying their phenotypic differences, and to infer how selection acts on them. To better understand the genetic basis for this diversity, we have examined the evolutionary relationships between Antirrhinum species and how these relate to geography and patterns of phenotypic variation in the genus as a whole. Large population samples and both plastid and multilocus nuclear genotypes resolved the relationships between many species and provided some support for the traditional taxonomic division of the genus into morphological subsections. Morphometric analysis of plants grown in controlled conditions supported the phenotypic distinction of the two largest subsections, and the involvement of multiple underlying genes. Incongruence between nuclear and plastid genotypes further suggested that several species have arisen after hybridization between subsections, and that some species continue to hybridize. However, all potential hybrids appear to have retained a phenotype similar to one of their ancestors, suggesting that ancestral combinations of characters are maintained by selection at many different loci.     

Floral zygomorphy, the recurring evolution of a successful trait

The flowers of the primitive angiosperm plants were radially symmetrical (actinomorphic). Flowers with bilateral symmetry (zygomorphic) evolved in several clades independently as an adaptation to specialized methods of pollination and played an important role in the diversification of flowering plants. In the model species Antirrhinum majus (snapdragon), the related genes CYCLOIDEA (CYC) and DICHOTOMA (DICH) are key in the development of this trait. This raises the question of whether they played a role in the evolution of floral bilateral symmetry. To address this, the evolution of CYC in relation to the evolution of zygomorphy is being investigated. Phylogenetic and functional analyses of CYC-like genes are being carried out in groups either closely related to Antirhinum or in families where zygomorphy evolved as an independent event. In addition, the origin of zygomorphy is being studied by comparing the function of CYC-like genes in species with zygomorphic flowers with their function in species with radially symmetrical flowers.     

Origin of allelic diversity in antirrhinum S locus RNases.

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.     

Polymorphic microsatellites in Antirrhinum (Scrophulariaceae), a genus with low levels of nuclear sequence variability

In Antirrhinum, reproductive systems range from self-compatible to self-incompatible, but the actual outcrossing rates of self-compatible populations are not known. Thus the extent to which levels of variability and inbreeding differ among Antirrhinum populations is not known. In order to address this issue we isolated nine Antirrhinum nuclear microsatellite loci. In contrast to several nuclear genes that show low levels of sequence variation, six of the microsatellite loci indicate high levels of variability within and between Antirrhinum species. The highly self-compatible Antirrhinum majus ssp. cirrhigerum population has high levels of variability and no significant deviation from Hardy-Weinberg equilibrium, suggesting substantial rates of outcrossing.     

Application of epidermal characters to the taxonomy of European species of Antirrhinum (Schrophulariaceae)

Epidermal characters provide additional features in the assessment of taxonomic relationships among the European species of Antirrhinum. Much of this study confirms the classification derived by Webb but important differences have been observed, viz. A. mollissimum may be considered as identical to A. molle whilst A. boissieri, A. ambiguum and A. rupestre may be assigned as distinct species. Evidence from epidermal characters supports Rothmaler's view that A. majus spp. litigiosum is a subspecies of A. majus and should not be amalgamated with A. barrelieri as proposed by Webb. A. × huteri is proposed to be a hybrid between A. hispanicum and A. boissieri. Thus it is proposed that the section Antirrhinum comprises 20 species with A. majus divided into five subspecies.     

Evolution of petaloid sepals independent of shifts in B-class MADS box gene expression

Attractive petals are an integral component of animal-pollinated flowers and in many flowering plant species are restricted to the second floral whorl. Interestingly, multiple times during angiosperm evolution, petaloid characteristics have expanded to adjacent floral whorls or to extra-floral organs. Here, we investigate developmental characteristics of petaloid sepals in Rhodochiton atrosanguineum, a close relative of the model species Antirrhinum majus (snapdragon). We undertook this in two ways, first using scanning electron microscopy we investigate the micromorphology of petals and sepals, followed by expression studies of genes usually responsible for the formation of petaloid structures. From our data, we conclude that R. atrosanguineum petaloid sepals lack micromorphological characteristics of petals and that petaloid sepals did not evolve through regulatory evolution of B-class MADS box genes, which have been shown to specify second whorl petal identity in a number of model flowering plant species including snapdragon. These data, in conjunction with other studies, suggests multiple convergent pathways for the evolution of showy sepals.     

LEAFY controls floral meristem identity in Arabidopsis.

The first step in flower development is the generation of a floral meristem by the inflorescence meristem. We have analyzed how this process is affected by mutant alleles of the Arabidopsis gene LEAFY. We show that LEAFY interacts with another floral control gene, APETALA1, to promote the transition from inflorescence to floral meristem. We have cloned the LEAFY gene, and, consistent with the mutant phenotype, we find that LEAFY RNA is expressed strongly in young flower primordia. LEAFY expression procedes expression of the homeotic genes AGAMOUS and APETALA3, which specify organ identify within the flower. Furthermore, we demonstrate that LEAFY is the Arabidopsis homolog of the FLORICAULA gene, which controls floral meristem identity in the distantly related species Antirrhinum majus.     

Isolation and Identification of Viable Embryo Sacs in Several Angiosperm Species

Since the enzymatic technique for isolating embryo sac (ES) has been established on fixed materials of several angiosperms as well as on fresh ovules of Antirrhinum majus in our lab, further works on isolation of viable ESs were carried on. Fresh ovules were macerated in a solution of enzymes, sucrose with or without potassium dextran sulphate by a microshaker at 28–30℃ for several hours. The enzymes included pectinase, cellulase, snailase and pectolyase Y-23, the combination and concentration of which varied with the plant species and the developmental stages of ESs. To date the mature ESs of Helianthus annuus, A. majus and Nicotiana tabacum and the ESs after fertilization with proembryo and endosperm cells in the two former species were well isolated. Nomarski interference contrast and Hoechst 33253 fluorescence microscopical observations revealed that the ESs retained their cell structure and were rich of ergastic substances. Fluorochromasia induced by fluorescein diacetate further proved that they were really viable.     

Antirrhinum majus microspore maturation and transient transformation in vitro

The male gametophyte of higher plants represents an excellent system to study gene regulation, cell fate determination and cellular differentiation in plants because of its relative simplicity compared to the sporophyte and its accessibility for cytological and molecular analysis. Unicellular plant microspores are single haploid cells, which can be isolated in large amounts at a defined developmental stage. Microspores cultured in vitro in a rich medium develop into mature pollen grains, which are fertile upon pollination in vivo. It is reported here that isolated Antirrhinum majus microspores when cultured in an optimal medium develop to form mature, fertile pollen. Their development closely resembled that of pollen formed in vivo. Isolated microspores were bombarded with Aquorea victoria Green Fluorescent Protein (GFP), Discosoma Red Fluorescent Protein (dsRFP) and beta-glucuronidase (GUS) reporter genes under the control of various promoters and transient expression was observed throughout pollen development in vitro. Bombarded and not bombarded in vitro-matured pollen grains were able to germinate both in vitro and on receptive stigmas and to set seed. The protocol of maturation, transient transformation and germination of Antirrhinum majus pollen in vitro described here provides a valuable tool for basic and applied research.     

Functional conservation of PISTILLATA activity in a pea homolog lacking the PI motif

Current understanding of floral development is mainly based on what we know from Arabidopsis (Arabidopsis thaliana) and Antirrhinum majus. However, we can learn more by comparing developmental mechanisms that may explain morphological differences between species. A good example comes from the analysis of genes controlling flower development in pea (Pisum sativum), a plant with more complex leaves and inflorescences than Arabidopsis and Antirrhinum, and a different floral ontogeny. The analysis of UNIFOLIATA (UNI) and STAMINA PISTILLOIDA (STP), the pea orthologs of LEAFY and UNUSUAL FLORAL ORGANS, has revealed a common link in the regulation of flower and leaf development not apparent in Arabidopsis. While the Arabidopsis genes mainly behave as key regulators of flower development, where they control the expression of B-function genes, UNI and STP also contribute to the development of the pea compound leaf. Here, we describe the characterization of P. sativum PISTILLATA (PsPI), a pea MADS-box gene homologous to B-function genes like PI and GLOBOSA (GLO), from Arabidopsis and Antirrhinum, respectively. PsPI encodes for an atypical PI-type polypeptide that lacks the highly conserved C-terminal PI motif. Nevertheless, constitutive expression of PsPI in tobacco (Nicotiana tabacum) and Arabidopsis shows that it can specifically replace the function of PI, being able to complement the strong pi-1 mutant. Accordingly, PsPI expression in pea flowers, which is dependent on STP, is identical to PI and GLO. Interestingly, PsPI is also transiently expressed in young leaves, suggesting a role of PsPI in pea leaf development, a possibility that fits with the established role of UNI and STP in the control of this process.     

Locally asymmetric introgressions between subspecies suggest circular range expansion at the Antirrhinum majus global scale

Assessing processes of geographic expansion in contact zones is a crucial step towards an accurate prediction of the evolution of species genetic diversity. The geographic distribution of cytonuclear discordance often reflects genetic introgression patterns across a species geographic range. Antirrhinum majus pseudomajus and A. m. striatum are two interfertile subspecies that occupy nonoverlapping areas but enter in contact in many locations at the margin of their geographic distribution. We found that genetic introgression between both subspecies was asymmetric at the local scale and geographically oriented in opposite directions at both ends of their contact zone perimeter in the Pyrenees. Our results suggest that the geographic expansion of A. majus subspecies was circular around the perimeter of their contact zone and pinpoint the need to integrate different spatial scales to unravel complex patterns of species geographic expansion.     

Ecology predicts parapatric distributions in two closely related Antirrhinum majus subspecies

Using a species distribution model, we reconstructed the environmental niches of Antirrhinum majus pseudomajus and Antirrhinum majus striatum, two closely related species with parapatric distributions. We tested whether retention of ancestral environmental niche (i.e. niche conservatism) or adaptation to different ecological conditions (i.e. niche divergence) could explain the maintenance of their non-overlapping geographic ranges. We found that the environmental niche of A. m. pseudomajus is almost twice as large as that of A. m. striatum, with substantial overlap indicating that A. m. pseudomajus and A. m. striatum should co-occur frequently within the geographic range of A. m. striatum. By analysing contact zones where both subspecies are geographically close, we found that the presence of one subspecies instead of the other was significantly influenced by particular combinations of climatic factors. Since independent genetic evidence indicates that the two subspecies have experienced phases of range overlap at or near contact zones over the course of their evolutionary history, we propose that ecological niche displacement might be an important factor in explaining the absence of current range overlap between A. majus subspecies.