Spatio-temporal expression of candidate genes for nectar spur development in Tropaeolum (Tropaeolaceae: Brassicales).

BACKGROUND AND AIMS: Tropaeolaceae (Brassicales) comprise ~100 species native to South and Central America. Tropaeolaceae flowers have a nectar spur, formed by a late expansion and evagination of the fused proximal region of the perianth (i.e. the floral tube). This spur is formed in the domain of the tube oriented towards the inflorescence axis, which corresponds to the adaxial floral region. However, little is known about the molecular mechanisms responsible for the evolution of spurs in Tropaeolaceae. METHODS: In this study, we examined the spatio-temporal expression of genes putatively responsible for differential patterns of cell division between the adaxial and abaxial floral regions in Tropaeolaceae. These genes include previously identified TCP and KNOX transcription factors and the cell division marker HISTONE H4 (HIS4). KEY RESULTS: We found a TCP4 homologue concomitantly expressed with spur initiation and elaboration. Tropaeolaceae possess two TCP4-like (TCP4L) copies, as a result of a Tropaeolaceae-specific duplication. The two copies (TCP4L1 and TCP4L2) in Tropaeolum longifolium show overlapping expression in the epidermis of reproductive apices (inflorescence meristems) and young floral buds, but only TlTCP4L2 shows differential expression in the floral tube at early stages of spur formation, restricted to the adaxial region. This adaxial expression of TlTCP4L2 overlaps with the expression of TlHIS4. Later in development, only TlTCP4L2 is expressed in the nectariferous tissue of the spur. CONCLUSIONS: Based on these results, we hypothesize that Tropaeolaceae TCP4L genes had a plesiomorphic role in epidermal development and that, after gene duplication, TCP4L2 acquired a new function in spur initiation and elaboration. To better understand spur evolution in Tropaeolaceae, it is critical to expand developmental genetic studies to their sister group, the Akaniaceae, which possess simultaneously an independent duplication of TCP4L genes and a spurless floral tube.

Comparative morphoanatomy and transcriptomic analyses reveal key factors controlling floral trichome development in Aristolochia (Aristolochiaceae).

Trichomes are specialized epidermal cells in aerial plant parts. Trichome development proceeds in three stages, determination of cell fate, specification, and morphogenesis. Most genes responsible for these processes have been identified in the unicellular branched leaf trichomes from the model Arabidopsis thaliana. Less is known about the molecular basis of multicellular trichome formation across flowering plants, especially those formed in floral organs of early diverging angiosperms. Here, we aim to identify the genetic regulatory network (GRN) underlying multicellular trichome development in the kettle-shaped trap flowers of Aristolochia (Aristolochiaceae). We selected two taxa for comparison, A. fimbriata, with trichomes inside the perianth, which play critical roles in pollination, and A. macrophylla, lacking specialized trichomes in the perianth. A detailed morphoanatomical characterization of floral epidermis is presented for the two species. We compared transcriptomic profiling at two different developmental stages in the different perianth portions (limb, tube, and utricle) of the two species. Moreover, we present a comprehensive expression map for positive regulators and repressors of trichome development, as well as cell cycle regulators. Our data point to extensive modifications in gene composition, expression, and putative roles in all functional categories when compared with model species. We also record novel differentially expressed genes (DEGs) linked to epidermis patterning and trichome development. We thus propose the first hypothetical genetic regulatory network (GRN) underlying floral multicellular trichome development in Aristolochia, and pinpoint key factors responsible for the presence and specialization of floral trichomes in phylogenetically distant species of the genus.

Expression and Functional Studies of Leaf, Floral, and Fruit Developmental Genes in Non-model Species.

Researchers working on evolutionary developmental plant biology are inclined to choose non-model taxa to address how specific features have been acquired during ontogeny and fixed during phylogeny. In this chapter we describe methods to extract RNA, to assemble de-novo transcriptomes, to isolate orthologous genes within gene families, and to evaluate expression and function of target genes. We have successfully optimized these protocols for non-model plant species including ferns, gymnosperms, and a large assortment of angiosperms. In the latter, we have ranged a large number of families including Aristolochiaceae, Apodanthaceae, Chloranthaceae, Orchidaceae, Papaveraceae, Rubiaceae, Solanaceae, and Tropaeolaceae.

Complete Mitogenomes of Two Aragoa Species and Phylogeny of Plantagineae (Plantaginaceae, Lamiales) Using Mitochondrial Genes and the Nuclear Ribosomal RNA Repeat.

Aragoa, comprising 19 high-altitude North Andean species, is one of three genera in the Plantagineae (Plantaginaceae, Lamiales), along with Littorella and Plantago. Based primarily on plastid data and nuclear ITS, Aragoa is sister to a clade of Littorella + Plantago, but Plantagineae relationships have yet to be assessed using multigene datasets from the nuclear and mitochondrial genomes. Here, complete mitogenomes were assembled for two species of Aragoa (A. abietina and A. cleefii). The mitogenomes of both species have a typical suite of genes for 34 proteins, 17 tRNAs, and three rRNAs. The A. abietina mitogenome assembled into a simple circular map, with no large repeats capable of producing alternative isoforms. The A. cleefii mitogenomic map was more complex, involving two circular maps bridged by a substoichiometric linear fragment. Phylogenetics of three mitochondrial genes or the nuclear rRNA repeat placed Aragoa as sister to Littorella + Plantago, consistent with previous studies. However, P. nubicola, the sole representative of subg. Bougueria, was nested within subg. Psyllium based on the mitochondrial and nuclear data, conflicting with plastid-based analyses. Phylogenetics of the nuclear rRNA repeat provided better resolution overall, whereas relationships from mitochondrial data were hindered by extensive substitution rate variation among lineages.

Molecular framework underlying floral bilateral symmetry and nectar spur development in Tropaeolum, an atypical member of the Brassicales.

PREMISE: Floral spurs are key innovations associated with elaborate pollination mechanisms that have evolved independently several times across angiosperms. Spur formation can shift the floral symmetry from radial to bilateral, as it is the case in Tropaeolum, the only member of the Brassicales with floral nectar spurs. The genetic mechanisms underlying both spur and bilateral symmetry in the family have not yet been investigated. METHODS: We studied flower development and morphoanatomy of Tropaeolum longifolium. We also generated a reference transcriptome and isolated all candidate genes involved in adaxial-abaxial differential growth during spur formation. Finally, we evaluated the evolution of the targeted genes across Brassicales and examined their expression in dissected floral parts. RESULTS: Five sepals initiate spirally, followed by five petals alternate to the sepals, five antesepalous stamens, three antepetalous stamens, and three carpels. Intercalary growth at the common base of sepals and petals forms a floral tube. The spur is an outgrowth from the adaxial region of the tube, lined up with the medial sepal. We identified Tropaeolum specific duplications in the TCP3/4L and STM gene lineages, which are critical for spur formation in other taxa. In addition, we found that TM6 (MADS-box), RL2 (RAD-like7), and KN2/6L2 and OSH6L (KNOX1 genes), have been lost in core Brassicales but retained in Tropaeolum. CONCLUSIONS: Three genes are pivotal during the extreme adaxial-abaxial asymmetry of the floral tube, namely, TlTCP4L2 restricted to the adaxial side where the spur is formed, and TlTCP12 and TlSTM1 to the abaxial side, lacking a spur.

Plastomes from tribe Plantagineae (Plantaginaceae) reveal infrageneric structural synapormorphies and localized hypermutation for Plantago and functional loss of ndh genes from Littorella.

Tribe Plantagineae (Plantaginaceae) comprises ~ 270 species in three currently recognized genera (Aragoa, Littorella, Plantago), of which Plantago is most speciose. Plantago plastomes exhibit several atypical features including large inversions, expansions of the inverted repeat, increased repetitiveness, intron losses, and gene-specific increases in substitution rate, but the prevalence of these plastid features among species and subgenera is unknown. To assess phylogenetic relationships and plastomic evolutionary dynamics among Plantagineae genera and Plantago subgenera, we generated 25 complete plastome sequences and compared them with existing plastome sequences from Plantaginaceae. Using whole plastome and partitioned alignments, our phylogenomic analyses provided strong support for relationships among major Plantagineae lineages. General plastid features-including size, GC content, intron content, and indels-provided additional support that reinforced major Plantagineae subdivisions. Plastomes from Plantago subgenera Plantago and Coronopus have synapomorphic expansions and inversions affecting the size and gene order of the inverted repeats, and particular genes near the inversion breakpoints exhibit accelerated nucleotide substitution rates, suggesting localized hypermutation associated with rearrangements. The Littorella plastome lacks functional copies of ndh genes, which may be related to an amphibious lifestyle and partial reliance on CAM photosynthesis.

Evolution of the Subgroup 6 R2R3-MYB Genes and Their Contribution to Floral Color in the Perianth-Bearing Piperales.

Flavonoids, carotenoids, betalains, and chlorophylls are the plant pigments responsible for floral color. Anthocyanins, a class of flavonoids, are largely responsible for the red, purple, pink, and blue colors. R2R3-MYB genes belonging to subgroup 6 (SG6) are the upstream regulatory factors of the anthocyanin biosynthetic pathway. The canonical members of these genes in Arabidopsis include AtMYB75, AtMYB90, AtMYB113, and AtMYB114. The Aristolochiaceae is an angiosperm lineage with diverse floral groundplans and perianth colors. Saruma henryi exhibits a biseriate perianth with green sepals and yellow petals. All other genera have sepals only, with colors ranging from green (in Lactoris) to a plethora of yellow to red and purple mixtures. Here, we isolated and reconstructed the SG6 R2R3-MYB gene lineage evolution in angiosperms with sampling emphasis in Aristolochiaceae. We found numerous species-specific duplications of this gene lineage in core eudicots and local duplications in Aristolochiaceae for Saruma and Asarum. Expression of SG6 R2R3-MYB genes examined in different developmental stages and plant organs of four Aristolochiaceae species, largely overlaps with red and purple pigments, suggesting a role in anthocyanin and flavonoid synthesis and accumulation. A directed RNA-seq analysis corroborated our RT-PCR analyses, by showing that these structural enzymes activate during perianth development in Aristolochia fimbriata and that the regulatory genes are expressed in correlation with color phenotype. Finally, the reconstruction of the flavonoid and anthocyanin metabolic pathways using predicted peptides from transcriptomic data show that all pivotal enzymes are present in the analyzed species. We conclude that the regulatory genes as well as the biosynthetic pathway are largely conserved across angiosperms. In addition, the Aristolochiaceae emerges as a remarkable group to study the genetic regulatory network for floral color, as their members exhibit an outstanding floral diversity with elaborate color patterns and the genetic complement for SG6 R2R3-MYB genes is simpler than in core eudicot model species.

Gene expression underlying floral epidermal specialization in Aristolochia fimbriata (Aristolochiaceae).

BACKGROUND AND AIMS: The epidermis constitutes the outermost tissue of the plant body. Although it plays major structural, physiological and ecological roles in embryophytes, the molecular mechanisms controlling epidermal cell fate, differentiation and trichome development have been scarcely studied across angiosperms, and remain almost unexplored in floral organs. METHODS: In this study, we assess the spatio-temporal expression patterns of GL2, GL3, TTG1, TRY, MYB5, MYB6, HDG2, MYB106-like, WIN1 and RAV1-like homologues in the magnoliid Aristolochia fimbriata (Aristolochiaceae) by using comparative RNA-sequencing and in situ hybridization assays. KEY RESULTS: Genes involved in Aristolochia fimbriata trichome development vary depending on the organ where they are formed. Stem, leaf and pedicel trichomes recruit most of the transcription factors (TFs) described above. Conversely, floral trichomes only use a small subset of genes including AfimGL2, AfimRAV1-like, AfimWIN1, AfimMYB106-like and AfimHDG2. The remaining TFs, AfimTTG1, AfimGL3, AfimTRY, AfimMYB5 and AfimMYB6, are restricted to the abaxial (outer) and the adaxial (inner) pavement epidermal cells. CONCLUSIONS: We re-evaluate the core genetic network shaping trichome fate in flowers of an early-divergent angiosperm lineage and show a morphologically diverse output with a simpler genetic mechanism in place when compared to the models Arabidopsis thaliana and Cucumis sativus. In turn, our results strongly suggest that the canonical trichome gene expression appears to be more conserved in vegetative than in floral tissues across angiosperms.

Evolution of Class II TCP genes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia.

Controlled spatiotemporal cell division and expansion are responsible for floral bilateral symmetry. Genetic studies have pointed to class II TCP genes as major regulators of cell division and floral patterning in model core eudicots. Here we study their evolution in perianth-bearing Piperales and their expression in Aristolochia, a rare occurrence of bilateral perianth outside eudicots and monocots. The evolution of class II TCP genes reveals single-copy CYCLOIDEA-like genes and three paralogs of CINCINNATA (CIN) in early diverging angiosperms. All class II TCP genes have independently duplicated in Aristolochia subgenus Siphisia. Also CIN2 genes duplicated before the diversification of Saruma and Asarum. Sequence analysis shows that CIN1 and CIN3 share motifs with Cyclin proteins and CIN2 genes have lost the miRNA319a binding site. Expression analyses of all paralogs of class II TCP genes in Aristolochia fimbriata point to a role of CYC and CIN genes in maintaining differential perianth expansion during mid- and late flower developmental stages by promoting cell division in the distal and ventral portion of the limb. It is likely that class II TCP genes also contribute to cell division in the leaf, the gynoecium and the ovules in A. fimbriata.

Expression of gynoecium patterning transcription factors in Aristolochia fimbriata (Aristolochiaceae) and their contribution to gynostemium development.

BACKGROUND: In Aristolochia (Aristolochiaceae) flowers, the congenital fusion of the anthers and the commissural, stigmatic lobes forms a gynostemium. Although the molecular bases associated to the apical-basal gynoecium patterning have been described in eudicots, comparative expression studies of the style and stigma regulatory genes have never been performed in early divergent angiosperms possessing a gynostemium. RESULTS: In this study, we assess the expression of five genes typically involved in gynoecium development in Aristolochia fimbriata. We found that all five genes (AfimCRC, AfimSPT, AfimNGA, AfimHEC1 and AfimHEC3) are expressed in the ovary, the placenta, the ovules and the transmitting tract. In addition, only AfimHEC3, AfimNGA and AfimSPT are temporarily expressed during the initiation of the stigma, while none of the genes studied is maintained during the elaboration of the stigmatic surfaces in the gynostemium. CONCLUSIONS: Expression patterns suggest that CRC, HEC, NGA and SPT homologs establish ovary and style identity in Aristolochia fimbriata. Only NGA,HEC3 and SPT genes may play a role in the early differentiation of the stigmatic lobes, but none of the genes studied seems to control late stigma differentiation in the gynostemium. The data gathered so far raises the possibility that such transient expression early on provides sufficient signal for late stigma differentiation or that unidentified late identity genes are controlling stigma development in the gynostemium. Our data does not rule out the possibility that stigmas could correspond to staminal filaments with convergent pollen-receptive surfaces.

Evolution of RADIALIS and DIVARICATA gene lineages in flowering plants with an expanded sampling in non-core eudicots.

PREMISE OF THE STUDY: Bilateral symmetry in core eudicot flowers is established by the differential expression of CYCLOIDEA (CYC), DICHOTOMA (DICH), and RADIALIS (RAD), which are restricted to the dorsal portion of the flower, and DIVARICATA (DIV), restricted to the ventral and lateral petals. Little is known regarding the evolution of these gene lineages in non-core eudicots, and there are no reports on gene expression that can be used to assess whether the network predates the diversification of core eudicots. METHODS: Homologs of the RAD and DIV lineages were isolated from available genomes and transcriptomes, including those of three selected non-core eudicot species, the magnoliid Aristolochia fimbriata and the monocots Cattleya trianae and Hypoxis decumbens. Phylogenetic analyses for each gene lineage were performed. RT-PCR was used to evaluate the expression and putative contribution to floral symmetry in dissected floral organs of the selected species. KEY RESULTS: RAD-like genes have undergone at least two duplication events before eudicot diversification, three before monocots and at least four in Orchidaceae. DIV-like genes also duplicated twice before eudicot diversification and underwent independent duplications specific to Orchidaceae. RAD-like and DIV-like genes have differential dorsiventral expression only in C. trianae, which contrasts with the homogeneous expression in the perianth of A. fimbriata. CONCLUSIONS: Our results point to a common genetic regulatory network for floral symmetry in monocots and core eudicots, while alternative genetic mechanisms are likely driving the bilateral perianth symmetry in the early-diverging angiosperm Aristolochia.

Plastome reduction and gene content in New World Pilostyles (Apodanthaceae) unveils high similarities to African and Australian congeners.

Holoparasitism has led to extreme plastome reduction. Plastomes in the legume holoparasite Pilostyles (Apodanthaceae) are the most reduced in both size and gene content known so far in Embryophytes. Here, we found that the Pilostyles boyacensis plastome, the only American species sequenced so far, is reduced to seven functional genes, accD, rpl2, rrn16 (=16S), rrn23 (=23S), rps3, rps12 and a putative oxidoreductase (PbOx). An additional gene, not annotated in the genome, is actively transcribed between accD and rps12, and by synteny we predict corresponds to rps4. We present data on plastome assembly, transcriptomic data that confirm the transcriptional activity of all genes and describe for the first time six transcript variants of a putative ORF likely having oxidoreductase activity. Our data show that such extreme reduction in P. boyacensis is similar but not identical to that reported in one Australian and one African species of the genus. Such intercontinental similarity suggests that the legume-Pilostyles holoparasitism was already in place during the main African-Australian-South American break-up. We compare plastome content and synteny between the three sequenced species, perform phylogenetic analyses across angiosperms of the six annotated plastome genes, and discuss the odd phylogenetic affinities of 16S and 23S, likely caused by HGT prior the diversification of both legumes and Pilostyles.

Floral MADS-box protein interactions in the early diverging angiosperm Aristolochia fimbriata Cham. (Aristolochiaceae: Piperales).

Floral identity MADS-box A, B, C, D, E, and AGL6 class genes are predominantly single copy in Magnoliids, and predate the whole genome duplication (WGD) events in monocots and eudicots. By comparison with the model species Arabidopsis thaliana, the expression patterns of B-, C-, and D-class genes in stamen, carpel, and ovules are conserved in Aristolochia fimbriata, whereas A-, E-class, and AGL6 genes have different expression patterns. Nevertheless, the interactions of these proteins that act through multimeric complexes remain poorly known in early divergent angiosperms. This study evaluates protein interactions among all floral MADS-box A. fimbriata proteins using the Yeast Two Hybrid System (Y2H). We found no homodimers and less heterodimers formed by AfimFUL when compared to AfimAGL6, which allowed us to suggest AGL6 homodimers in combination with AfimSEP2 as the most likely tetramer in sepal identity. We found AfimAP3-AfimPI obligate heterodimers and AfimAG-AfimSEP2 protein interactions intact suggesting conserved stamen and carpel tetrameric complexes in A. fimbriata. We observed a broader interaction partner set for AfimSEP2 than for its paralog AfimSEP1. We show conserved and exclusive MADS-box protein interactions in A. fimbriata in comparison with other eudicot and monocot model species in order to establish plesiomorphic MADS-box protein floral networks in angiosperms.

Comparative inflorescence development in selected Andean Santalales.

PREMISE OF THE STUDY: Loranthaceae, Santalaceae, and Viscaceae are the most diversified hemiparasitic families of Santalales in the Andes. Their partial inflorescences (PIs) vary from solitary flowers, or dichasia in most Santalales, to congested floral groups along articles in most Viscaceae. The atypical articled inflorescences in Phoradendreae (Viscaceae), a phylogenetic novelty restricted to this tribe, have been variously described as racemes, spikes, fascicles, or as intercalary inflorescences, but no developmental studies have been performed to compare them with the construction of PIs across Santalales. METHODS: We used standard light microscopy and scanning electron microscopy to record the inflorescence development in members of Phoradendreae (Viscaceae) in comparison to those in species of Aetanthus, Gaiadendron, Oryctanthus, Passovia, and Peristethium (Loranthaceae) and Antidaphne (Santalaceae). KEY RESULTS: Morphological and developmental comparisons as well as optimization onto a phylogenetic framework indicate that individual inflorescences in Santalales are indeterminate and are formed by axillary cymose PIs. The latter correspond to dichasia, either simple, compound, or variously reduced by abortion of lateral flowers, abortion of the terminal flower, or loss of bracteoles. CONCLUSIONS: Dichasia are plesiomorphic in Santalales. These results favor the interpretation that inflorescences in Phoradendreae are formed by the fusion of serial dichasia (=floral rows) with the main inflorescence axis via syndesmy. We compared this interpretation with the competing one based on the co-occurrence of collateral and serial floral buds.

Deep into the Aristolochia Flower: Expression of C, D, and E-Class Genes in Aristolochia fimbriata (Aristolochiaceae).

Aristolochia fimbriata (Aristolochiaceae) is a member of an early diverging lineage of flowering plants and a promising candidate for evo-devo studies. Aristolochia flowers exhibit a unique floral synorganization that consists of a monosymmetric and petaloid calyx formed by three congenitally fused sepals, and a gynostemium formed by the congenital fusion between stamens and the stigmatic region of the carpels. This floral ground plan atypical in the magnoliids can be used to evaluate the role of floral organ identity MADS-box genes during early flower evolution. In this study, we present in situ hybridization experiments for the homologs of the canonical C-, D-, and E-class genes. Spatiotemporal expression of the C-class gene AfimAG is restricted to stamens, ovary, and ovules, suggesting a conserved stamen and carpel identity function, consistent with that reported in core-eudicots and monocots. The D-class gene AfimSTK is detected in the anthers, the stigmas, the ovary, the ovules, the fruit, and the seeds, suggesting conserved roles in ovule and seed identity and unique roles in stamens, ovary, and fruit development. In addition, AfimSTK expression patterns in areas of organ abscission and dehiscence zones suggest putative roles linked to senescence processes. We found that both E-class genes are expressed in the anthers and the ovary; however, AfimSEP2 exhibits higher expression compared to AfimSEP1. These findings provide a comprehensive picture of the ancestral expression patterns of the canonical MADS-box floral organ identity genes and the foundations for further comparative analyses in other magnoliids.

Evolutionary Developmental Biology (Evo-Devo) Research in Latin America.

Famous for its blind cavefish and Darwin's finches, Latin America is home to some of the richest biodiversity hotspots of our planet. The Latin American fauna and flora inspired and captivated naturalists from the nineteenth and twentieth centuries, including such notable pioneers such as Fritz Müller, Florentino Ameghino, and Léon Croizat who made a significant contribution to the study of embryology and evolutionary thinking. But, what are the historical and present contributions of the Latin American scientific community to Evo-Devo? Here, we provide the first comprehensive overview of the Evo-Devo laboratories based in Latin America and describe current lines of research based on endemic species, focusing on body plans and patterning, systematics, physiology, computational modeling approaches, ecology, and domestication. Literature searches reveal that Evo-Devo in Latin America is still in its early days; while showing encouraging indicators of productivity, it has not stabilized yet, because it relies on few and sparsely distributed laboratories. Coping with the rapid changes in national scientific policies and contributing to solve social and health issues specific to each region are among the main challenges faced by Latin American researchers. The 2015 inaugural meeting of the Pan-American Society for Evolutionary Developmental Biology played a pivotal role in bringing together Latin American researchers eager to initiate and consolidate regional and worldwide collaborative networks. Such networks will undoubtedly advance research on the extremely high genetic and phenotypic biodiversity of Latin America, bound to be an almost infinite source of amazement and fascinating findings for the Evo-Devo community.

Limited mitogenomic degradation in response to a parasitic lifestyle in Orobanchaceae.

In parasitic plants, the reduction in plastid genome (plastome) size and content is driven predominantly by the loss of photosynthetic genes. The first completed mitochondrial genomes (mitogenomes) from parasitic mistletoes also exhibit significant degradation, but the generality of this observation for other parasitic plants is unclear. We sequenced the complete mitogenome and plastome of the hemiparasite Castilleja paramensis (Orobanchaceae) and compared them with additional holoparasitic, hemiparasitic and nonparasitic species from Orobanchaceae. Comparative mitogenomic analysis revealed minimal gene loss among the seven Orobanchaceae species, indicating the retention of typical mitochondrial function among Orobanchaceae species. Phylogenetic analysis demonstrated that the mobile cox1 intron was acquired vertically from a nonparasitic ancestor, arguing against a role for Orobanchaceae parasites in the horizontal acquisition or distribution of this intron. The C. paramensis plastome has retained nearly all genes except for the recent pseudogenization of four subunits of the NAD(P)H dehydrogenase complex, indicating a very early stage of plastome degradation. These results lend support to the notion that loss of ndh gene function is the first step of plastome degradation in the transition to a parasitic lifestyle.

The developmental and genetic bases of apetaly in Bocconia frutescens (Chelidonieae: Papaveraceae).

BACKGROUND: Bocconia and Macleaya are the only genera of the poppy family (Papaveraceae) lacking petals; however, the developmental and genetic processes underlying such evolutionary shift have not yet been studied. RESULTS: We studied floral development in two species of petal-less poppies Bocconia frutescens and Macleaya cordata as well as in the closely related petal-bearing Stylophorum diphyllum. We generated a floral transcriptome of B. frutescens to identify MADS-box ABCE floral organ identity genes expressed during early floral development. We performed phylogenetic analyses of these genes across Ranunculales as well as RT-PCR and qRT-PCR to assess loci-specific expression patterns. We found that petal-to-stamen homeosis in petal-less poppies occurs through distinct developmental pathways. Transcriptomic analyses of B. frutescens floral buds showed that homologs of all MADS-box genes are expressed except for the APETALA3-3 ortholog. Species-specific duplications of other ABCE genes in B. frutescens have resulted in functional copies with expanded expression patterns than those predicted by the model. CONCLUSIONS: Petal loss in B. frutescens is likely associated with the lack of expression of AP3-3 and an expanded expression of AGAMOUS. The genetic basis of petal identity is conserved in Ranunculaceae and Papaveraceae although they have different number of AP3 paralogs and exhibit dissimilar floral groundplans.

Flower Development and Perianth Identity Candidate Genes in the Basal Angiosperm Aristolochia fimbriata (Piperales: Aristolochiaceae).

Aristolochia fimbriata (Aristolochiaceae: Piperales) exhibits highly synorganized flowers with a single convoluted structure forming a petaloid perianth that surrounds the gynostemium, putatively formed by the congenital fusion between stamens and the upper portion of the carpels. Here we present the flower development and morphology of A. fimbriata, together with the expression of the key regulatory genes that participate in flower development, particularly those likely controlling perianth identity. A. fimbriata is a member of the magnoliids, and thus gene expression detected for all ABCE MADS-box genes in this taxon, can also help to elucidate patterns of gene expression prior the independent duplications of these genes in eudicots and monocots. Using both floral development and anatomy in combination with the isolation of MADS-box gene homologs, gene phylogenetic analyses and expression studies (both by reverse transcription PCR and in situ hybridization), we present hypotheses on floral organ identity genes involved in the formation of this bizarre flower. We found that most MADS-box genes were expressed in vegetative and reproductive tissues with the exception of AfimSEP2, AfimAGL6, and AfimSTK transcripts that are only found in flowers and capsules but are not detected in leaves. Two genes show ubiquitous expression; AfimFUL that is found in all floral organs at all developmental stages as well as in leaves and capsules, and AfimAG that has low expression in leaves and is found in all floral organs at all stages with a considerable reduction of expression in the limb of anthetic flowers. Our results indicate that expression of AfimFUL is indicative of pleiotropic roles and not of a perianth identity specific function. On the other hand, expression of B-class genes, AfimAP3 and AfimPI, suggests their conserved role in stamen identity and corroborates that the perianth is sepal and not petal-derived. Our data also postulates an AGL6 ortholog as a candidate gene for sepal identity in the Aristolochiaceae and provides testable hypothesis for a modified ABCE model in synorganized magnoliid flowers.