The Snapdragon Genomes Reveal the Evolutionary Dynamics of the S-Locus Supergene.

The genus Antirrhinum has been used as a model to study self-incompatibility extensively. The multi-allelic S-locus, carrying a pistil S-RNase and dozens of S-locus F-box (SLF) genes, underlies the genetic control of self-incompatibility (SI) in Antirrhinum hispanicum. However, there have been limited studies on the genomic organization of the S-locus supergene due to a lack of high-quality genomic data. Here, we present the chromosome-level reference and haplotype-resolved genome assemblies of a self-incompatible A. hispanicum line, AhS7S8. For the first time, 2 complete A. hispanicum S-haplotypes spanning ∼1.2 Mb and containing a total of 32 SLFs were reconstructed, whereas most of the SLFs derived from retroelement-mediated proximal or tandem duplication ∼122 Mya. Back then, the S-RNase gene and incipient SLFs came into linkage to form the pro-type of type-1 S-locus in the common ancestor of eudicots. Furthermore, we detected a pleiotropic cis-transcription factor (TF) associated with regulating the expression of SLFs, and two miRNAs may control the expression of this TF. Interspecific S-locus and intraspecific S-haplotype comparisons revealed the dynamic nature and polymorphism of the S-locus supergene mediated by continuous gene duplication, segmental translocation or loss, and TE-mediated transposition events. Our data provide an excellent resource for future research on the evolutionary studies of the S-RNase-based self-incompatibility system.

Painted flowers: Eluta generates pigment patterning in Antirrhinum.

In the early 1900s, Erwin Baur established Antirrhinum majus as a model system, identifying and characterising numerous flower colour variants. This included Picturatum/Eluta, which restricts the accumulation of magenta anthocyanin pigments, forming bullseye markings on the flower face. We identified the gene underlying the Eluta locus by transposon-tagging, using an Antirrhinum line that spontaneously lost the nonsuppressive el phenotype. A candidate MYB repressor gene at this locus contained a CACTA transposable element. We subsequently identified plants where this element excised, reverting to a suppressive Eluta phenotype. El alleles inhibit expression of anthocyanin biosynthetic genes, confirming it to be a regulatory locus. The modes of action of Eluta were investigated by generating stable transgenic tobacco lines, biolistic transformation of Antirrhinum petals and promoter activation/repression assays. Eluta competes with MYB activators for promoter cis-elements, and also by titrating essential cofactors (bHLH proteins) to reduce transcription of target genes. Eluta restricts the pigmentation established by the R2R3-MYB factors, Rosea and Venosa, with the greatest repression on those parts of the petals where Eluta is most highly expressed. Baur questioned the origin of heredity units determining flower colour variation in cultivated A. majus. Our findings support introgression from wild species into cultivated varieties.

CYCLOIDEA paralogs function partially redundantly to specify dorsal flower development in Mimulus lewisii.

PREMISE: Duplicated genes (paralogs) are abundant in plant genomes, and their retention may influence the function of genetic programs and contribute to evolutionary novelty. How gene duplication affects genetic modules and what forces contribute to paralog retention are outstanding questions. The CYCLOIDEA(CYC)-dependent flower symmetry program is a model for understanding the evolution of gene duplication, providing multiple examples of paralog partitioning and novelty. However, a novel CYC gene lineage duplication event near the origin of higher core Lamiales (HCL) has received little attention. METHODS: To understand the evolutionary fate of duplicated HCL CYC2 genes, we determined the effects on flower symmetry by suppressing MlCYC2A and MlCYC2B expression using RNA interference (RNAi). We determined the phenotypic effects on flower symmetry in single- and double-silenced backgrounds and coupled our functional analyses with expression surveys of MlCYC2A, MlCYC2B, and a putative downstream RADIALIS (MlRAD5) ortholog. RESULTS: MlCYC2A and MlCYC2B jointly contribute to bilateral flower symmetry. MlCYC2B exhibits a clear dorsal flower identity function and may additionally function in carpel development. MlCYC2A functions in establishing dorsal petal shape. Further, our results suggest an MlCYC2A-MlCYC2B regulatory interaction, which may affect pathway homeostasis. CONCLUSIONS: Our results suggest that CYC paralogs specific to higher core Lamiales may be selectively retained for their joint contribution to bilateral flower symmetry, similar to the independently derived CYC paralogs in the Lamiales model for bilateral flower symmetry research, Antirrhinum majus (snapdragon).

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.

Recent parallel speciation in Antirrhinum involved complex haplotypes and multiple adaptive characters.

A role of ecological adaptation in speciation can be obscured by stochastic processes and differences that species accumulate after genetic isolation. One way to identify adaptive characters and their underlying genes is to study cases of speciation involving parallel adaptations. Recently resolved phylogenies reveal that alpine morphology has evolved in parallel in the genus Antirrhinum (snapdragons): first in an early split of an alpine from a lowland lineage and, more recently, from within the lowland lineage to produce closely related sympatric species with contrasting alpine and lowland forms. Here, we find that two of these later diverged sympatric species are differentiated by only around 2% of nuclear loci. Though showing evidence of recent gene flow, the species remain distinct for a suite of morphological characters typical of earlier-diverged alpine or lowland lineages and their morphologies correlate with features of the local landscape, as expected of ecological adaptations. Morphological differences between the two species involve multiple, unlinked genes so that parental character combinations are readily broken up by recombination in hybrids. We detect little evidence for post-pollination barriers to gene flow or recombination, suggesting that genetic isolation related to ecological adaptation is important in maintaining character combinations and might have contributed to parallel speciation. We also find evidence that genes involved in the earlier alpine-lowland split were reused in parallel evolution of alpine species, consistent with introgressive hybridisation, and speculate that many non-ecological barriers to gene flow might have been purged during the process.

Cloning and functional analysis of RADIALIS-like 1 gene from Antirrhinum majus.

MYB is one of the largest transcription factor families in plants. Among them, the R3-MYB transcription factor RADIALIS (RAD) plays a very important role in the flowers development in Antirrhinum majus. In this study, a R3-MYB gene similar to RAD was found by analyzing the genome of A. majus, which was named AmRADIALIS-like 1 (AmRADL1). The gene function was predicted through bioinformatics. The relative expression levels in different tissues and organs of wild-type A. majus were analyzed by qRT-PCR. AmRADL1 was overexpressed in A. majus, and the transgenic plants were analyzed by morphological observation and histological staining. The results showed that the open reading frame (ORF) of AmRADL1 gene was 306 bp in length, encoding 101 amino acids. It has typical SANT domain, and the C-terminal contains a CREB motif, which was highly homologous to tomato SlFSM1. The results of qRT-PCR showed that AmRADL1 was expressed in roots, stems, leaves and flowers, and the expression level was higher in flowers. Further analysis of its expression in different floral organs showed that AmRADL1 had the highest expression in carpel. The results of histological staining analysis of the transgenic plants showed that compared with the wild type, although the size of the carpel cells of the transgenic plants did not change significantly, the placenta area in the carpel became smaller and the number of cell decreased. In summary, AmRADL1 may be involved in the regulation of carpel development, but the specific mechanism of action in carpel remains to be further studied.

A phylogeny of Antirrhinum reveals parallel evolution of alpine morphology.

Parallel evolution of similar morphologies in closely related lineages provides insight into the repeatability and predictability of evolution. In the genus Antirrhinum (snapdragons), as in other plants, a suite of morphological characters are associated with adaptation to alpine environments. We tested for parallel trait evolution in Antirrhinum by investigating phylogenetic relationships using restriction-site associated DNA (RAD) sequencing. We then associated phenotypic information to our phylogeny to reconstruct the patterns of morphological evolution and related this to evidence for hybridisation between emergent lineages. Phylogenetic analyses showed that the alpine character syndrome is present in multiple groups, suggesting that Antirrhinum has repeatedly colonised alpine habitats. Dispersal to novel environments happened in the presence of intraspecific and interspecific gene flow. We found support for a model of parallel evolution in Antirrhinum. Hybridisation in natural populations, and a complex genetic architecture underlying the alpine morphology syndrome, support an important role of natural selection in maintaining species divergence in the face of gene flow.

Non-reproducible signals of adaptation to elevation between open and understorey microhabitats in snapdragon plants.

Experimental studies on local adaptation rarely investigate how different environmental variables might modify signals of adaptation or maladaptation. In plant common garden experiments, signals of adaptation or maladaptation to elevation are usually investigated in open habitats under full light. However, most plants inhabit heterogeneous habitats where environmental conditions differ. Understorey microhabitats are common and differ in terms of tree shade, temperature, water availability, microbiota, allelochemicals etc. Germination is a fitness-related trait of major importance for the adaptation of plants to contrasted climate conditions. It is affected by shade in snapdragon plants (Antirrhinum majus) and many other plant species. Here, we tested for the reproducibility of signals extrapolated from germination results between open and understorey microhabitats in two parapatric snapdragon plant subspecies (A. m. striatum and A. m. pseudomajus) characterized by a similar elevation range by using common garden experiments at different elevations. Signals observed under one microhabitat systematically differed in the other. Most scenarios could be inferred, with signals either shifting, appearing or disappearing between different environments. Our findings imply that caution should be taken when extrapolating the evolutionary significance of these types of experimental signals because they are not stable from one local environmental condition to the next. Forecasting the ability of plants to adapt to environmental changes based on common garden and reciprocal transplant experiments must account for the multivariate nature of the environment.

Discrete bHLH transcription factors play functionally overlapping roles in pigmentation patterning in flowers of Antirrhinum majus.

Floral pigmentation patterning is important for pollinator attraction as well as aesthetic appeal. Patterning of anthocyanin accumulation is frequently associated with variation in activity of the Myb, bHLH and WDR transcription factor complex (MBW) that regulates anthocyanin biosynthesis. Investigation of two classic mutants in Antirrhinum majus, mutabilis and incolorata I, showed they affect a gene encoding a bHLH protein belonging to subclade bHLH-2. The previously characterised gene, Delila, which encodes a bHLH-1 protein, has a bicoloured mutant phenotype, with residual lobe-specific pigmentation conferred by Incolorata I. Both Incolorata I and Delila induce expression of the anthocyanin biosynthetic gene DFR. Rosea 1 (Myb) and WDR1 proteins compete for interaction with Delila, but interact positively to promote Incolorata I activity. Delila positively regulates Incolorata I and WDR1 expression. Hierarchical regulation can explain the bicoloured patterning of delila mutants, through effects on both regulatory gene expression and the activity of promoters of biosynthetic genes like DFR that mediate MBW regulation. bHLH-1 and bHLH-2 proteins contribute to establishing patterns of pigment distribution in A. majus flowers in two ways: through functional redundancy in regulating anthocyanin biosynthetic gene expression, and through differences between the proteins in their ability to regulate genes encoding transcription factors.

Bumblebee electric charge stimulates floral volatile emissions in Petunia integrifolia but not in Antirrhinum majus.

The timing of volatile organic compound (VOC) emission by flowering plants often coincides with pollinator foraging activity. Volatile emission is often considered to be paced by environmental variables, such as light intensity, and/or by circadian rhythmicity. The question arises as to what extent pollinators themselves provide information about their presence, in keeping with their long co-evolution with flowering plants. Bumblebees are electrically charged and provide electrical stimulation when visiting plants, as measured via the depolarisation of electric potential in the stem of flowers. Here we test the hypothesis that the electric charge of foraging bumblebees increases the floral volatile emissions of bee pollinated plants. We investigate the change in VOC emissions of two bee-pollinated plants (Petunia integrifolia and Antirrhinum majus) exposed to the electric charge typical of foraging bumblebees. P. integrifolia slightly increases its emissions of a behaviorally and physiologically active compound in response to visits by foraging bumblebees, presenting on average 121 pC of electric charge. We show that for P. integrifolia, strong electrical stimulation (600-700 pC) promotes increased volatile emissions, but this is not found when using weaker electrical charges more representative of flying pollinators (100 pC). Floral volatile emissions of A. majus were not affected by either strong (600-700 pC) or weak electric charges (100 pC). This study opens a new area of research whereby the electrical charge of flying insects may provide information to plants on the presence and phenology of their pollinators. As a form of electroreception, this sensory process would bear adaptive value, enabling plants to better ensure that their attractive chemical messages are released when a potential recipient is present.

Selection and gene flow shape genomic islands that control floral guides.

Genomes of closely-related species or populations often display localized regions of enhanced relative sequence divergence, termed genomic islands. It has been proposed that these islands arise through selective sweeps and/or barriers to gene flow. Here, we genetically dissect a genomic island that controls flower color pattern differences between two subspecies of Antirrhinum majus, A.m.striatum and A.m.pseudomajus, and relate it to clinal variation across a natural hybrid zone. We show that selective sweeps likely raised relative divergence at two tightly-linked MYB-like transcription factors, leading to distinct flower patterns in the two subspecies. The two patterns provide alternate floral guides and create a strong barrier to gene flow where populations come into contact. This barrier affects the selected flower color genes and tightly-linked loci, but does not extend outside of this domain, allowing gene flow to lower relative divergence for the rest of the chromosome. Thus, both selective sweeps and barriers to gene flow play a role in shaping genomic islands: sweeps cause elevation in relative divergence, while heterogeneous gene flow flattens the surrounding "sea," making the island of divergence stand out. By showing how selective sweeps establish alternative adaptive phenotypes that lead to barriers to gene flow, our study sheds light on possible mechanisms leading to reproductive isolation and speciation.

Potential adaptive divergence between subspecies and populations of snapdragon plants inferred from QST -FST comparisons.

Phenotypic divergence among natural populations can be explained by natural selection or by neutral processes such as drift. Many examples in the literature compare putatively neutral (FST ) and quantitative genetic (QST ) differentiation in multiple populations to assess their evolutionary signature and identify candidate traits involved with local adaptation. Investigating these signatures in closely related or recently diversified species has the potential to shed light on the divergence processes acting at the interspecific level. Here, we conducted this comparison in two subspecies of snapdragon plants (eight populations of Antirrhinum majus pseudomajus and five populations of A. m. striatum) in a common garden experiment. We also tested whether altitude was involved with population phenotypic divergence. Our results identified candidate phenological and morphological traits involved with local adaptation. Most of these traits were identified in one subspecies but not the other. Phenotypic divergence increased with altitude for a few biomass-related traits, but only in A. m. striatum. These traits therefore potentially reflect A. m. striatum adaptation to altitude. Our findings imply that adaptive processes potentially differ at the scale of A. majus subspecies.

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.

Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species.

Flavonoid metabolons (weakly-bound multi-enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein-protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS); FNSII/chalcone isomerase (CHI); FNSII/dihydroflavonol 4-reductase (DFR); CHS/CHI; CHI/DFR; and flavonoid 3'-hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage-dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co-suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long-suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.

Regulation of flowering by green light depends on its photon flux density and involves cryptochromes.

Photoperiodic lighting can promote flowering of long-day plants (LDPs) and inhibit flowering of short-day plants (SDPs). Red (R) and far-red (FR) light regulate flowering through phytochromes, whereas blue light does so primarily through cryptochromes. In contrast, the role of green light in photoperiodic regulation of flowering has been inconsistent in previous studies. We grew four LDP species (two petunia cultivars, ageratum, snapdragon and Arabidopsis) and two SDP species (three chrysanthemum cultivars and marigold) in a greenhouse under truncated 9-h short days with or without 7-h day-extension lighting from green light (peak = 521 nm) at 0, 2, 13 or 25 µmol m-2  s-1 or R + white (W) + FR light at 2 µmol m-2  s-1 . Increasing the green photon flux density from 0 to 25 µmol m-2  s-1 accelerated flowering of all LDPs and delayed flowering of all SDPs. Petunia flowered similarly fast under R + W + FR light and moderate green light but was shorter and developed more branches under green light. To be as effective as R + W + FR light, saturation green photon flux densities were 2 µmol m-2  s-1 for LDP ageratum and SDP marigold and 13 µmol m-2  s-1 for LDP petunia. Snapdragon was the least sensitive to green light. In Arabidopsis, cryptochrome 2 mediated promotion of flowering under moderate green light, whereas both phytochrome B and cryptochrome 2 mediated that under R + W + FR light. We conclude that 7-h day-extension lighting from green light-emitting diodes can control flowering of photoperiodic ornamentals and that in Arabidopsis, cryptochrome 2 mediates promotion of flowering under green light.

Inverted stereocontrol of iridoid synthase in snapdragon.

The natural product class of iridoids, found in various species of flowering plants, harbors astonishing chemical complexity. The discovery of iridoid biosynthetic genes in the medicinal plant Catharanthus roseus has provided insight into the biosynthetic origins of this class of natural product. However, not all iridoids share the exact five- to six-bicyclic ring scaffold of the Catharanthus iridoids. For instance, iridoids in the ornamental flower snapdragon (Antirrhinum majus, Plantaginaceae family) are derived from the C7 epimer of this scaffold. Here we have cloned and characterized the iridoid synthase enzyme from A. majus (AmISY), the enzyme that is responsible for converting 8-oxogeranial into the bicyclic iridoid scaffold in a two-step reduction-cyclization sequence. Chiral analysis of the reaction products reveals that AmISY reduces C7 to generate the opposite stereoconfiguration in comparison with the Catharanthus homologue CrISY. The catalytic activity of AmISY thus explains the biosynthesis of 7-epi-iridoids in Antirrhinum and related genera. However, although the stereoselectivity of the reduction step catalyzed by AmISY is clear, in both AmISY and CrISY, the cyclization step produces a diastereomeric mixture. Although the reduction of 8-oxogeranial is clearly enzymatically catalyzed, the cyclization step appears to be subject to less stringent enzyme control.

Changes in cis-regulatory elements of a key floral regulator are associated with divergence of inflorescence architectures.

Higher plant species diverged extensively with regard to the moment (flowering time) and position (inflorescence architecture) at which flowers are formed. This seems largely caused by variation in the expression patterns of conserved genes that specify floral meristem identity (FMI), rather than changes in the encoded proteins. Here, we report a functional comparison of the promoters of homologous FMI genes from Arabidopsis, petunia, tomato and Antirrhinum. Analysis of promoter-reporter constructs in petunia and Arabidopsis, as well as complementation experiments, showed that the divergent expression of leafy (LFY) and the petunia homolog aberrant leaf and flower (ALF) results from alterations in the upstream regulatory network rather than cis-regulatory changes. The divergent expression of unusual floral organs (UFO) from Arabidopsis, and the petunia homolog double top (DOT), however, is caused by the loss or gain of cis-regulatory promoter elements, which respond to trans-acting factors that are expressed in similar patterns in both species. Introduction of pUFO:UFO causes no obvious defects in Arabidopsis, but in petunia it causes the precocious and ectopic formation of flowers. This provides an example of how a change in a cis-regulatory region can account for a change in the plant body plan.

Detainment of Tam3 Transposase at Plasma Membrane by Its BED-Zinc Finger Domain.

Transposable elements (TEs) are considered to be parasites of host genomes because they act as powerful mutagens. If not kept in check, they can cause gene disruption, genome rearrangement, and genomic takeover. Hence, activities of TEs are under the rigid control of hosts. To date, all identified TE regulations have been epigenetic dependent, with the exception of the DNA transposon Tam3. Blocking nuclear translocation of Tam3 transposase (TPase) is consistent with the suppression of Tam3 in Antirrhinum majus In this article, we discovered that epigenetic-independent regulation of Tam3 is mediated by the BED-zinc finger (Znf-BED) domain of Tam3 TPase. The host targets the N terminus of the Znf-BED domain, which contains two highly conserved aromatic amino acids, to detain Tam3 TPase at the plasma membrane and to silence Tam3. Zinc finger proteins perform broader functions in transcriptional regulation through their DNA binding ability. Our data revealed that the posttranslational epigenetic-independent silencing against TEs was a result of the protein binding ability of the Znf-BED domain.

Control of Floret Symmetry by RAY3, SvDIV1B, and SvRAD in the Capitulum of Senecio vulgaris

All members of Asteraceae, the largest flowering family, have a unique compressed inflorescence known as a capitulum, which resembles a solitary flower. The capitulum often consists of bilateral (zygomorphic) ray florets and radial (actinomorphic) disc florets. In Antirrhinum majus, floral zygomorphy is established by the interplay between dorsal petal identity genes, CYCLOIDEA (CYC) and RADIALIS (RAD), and a ventral gene DIVARICATA (DIV). To investigate the role of CYC, RAD, and DIV in the development of ray and disc florets within a capitulum, we isolated homologs of these genes from an Asteraceae species, Senecio vulgaris (common groundsel). After initial uniform expression of RAY3 (CYC), SvRAD, and SvDIV1B in ray florets only, RAY3 and SvRAD were exclusively expressed in the ventral petals of the ray florets. Our functional analysis further showed that RAY3 promotes and SvDIV1B represses petal growth, confirming their roles in floral zygomorphy. Our results highlight that while floral symmetry genes such as RAY3 and SvDIV1B appear to have a conserved role in petal growth in both Senecio and Antirrhinum, the regulatory relationships and expression domains are divergent, allowing ventral petal elongation in Senecio versus dorsal petal elongation in Antirrhinum In S vulgaris, diversification of CYC genes has led to novel interactions; SvDIV1B inhibits RAY3 and SvRAD, and may activate RAY2 This highlights how recruitment of floral symmetry regulators into dynamic networks was crucial for creating a complex and elaborate structure such as the capitulum.