Evolutionary studies of the bHLH transcription factors belonging to MBW complex: their role in seed development.

BACKGROUND AND AIMS: The MBW complex consist of proteins belonging to three major families (MYB, bHLH and WDR) involved in various processes throughout plant development: epidermal cell development, mucilage secretory cells and flavonoid biosynthesis. Recently, it has been reported that TT8, encoding a bHLH transcription factor, is involved in the biosynthesis of flavonoids in the seed coat and it also plays a role in bypassing the postzygotic barrier resulting from an unbalance in genetic loads of the parental lines. Here, we focus on the functional evolution, in seed development, of the bHLH proteins that are part of the MBW complex, complemented with a literature review. METHODS: Phylogenetic analyses performed across seed plants and expression analyses in the reproductive tissues of four selected angiosperms (Arabidopsis thaliana, Brassica napus, Capsella rubella and Solanum lycopersicum) allow us to hypothesize on the evolution of its functions. KEY RESULTS: TT8 expression in the innermost layer of the seed coat is conserved in the selected angiosperms. However, except for Arabidopsis, TT8 is also expressed in ovules, carpels and fruits. The homologues belonging to the sister clade of TT8, EGL3/GL3, involved in trichome development, are expressed in the outermost layer of the seed coat, suggesting potential roles in mucilage. CONCLUSIONS: The ancestral function of these genes appears to be flavonoid biosynthesis, and the conservation of TT8 expression patterns in the innermost layer of the seed coat in angiosperms suggests that their function in postzygotic barriers might also be conserved. Moreover, the literature review and the results of the present study suggest a sophisticated association, linking the mechanisms of action of these genes to the cross-communication activity between the different tissues of the seed. Thus, it provides avenues to study the mechanisms of action of TT8 in the postzygotic triploid block, which is crucial because it impacts seed development in unbalanced crosses.

The Evolution of euAPETALA2 Genes in Vascular Plants: From Plesiomorphic Roles in Sporangia to Acquired Functions in Ovules and Fruits.

The field of evolutionary developmental biology can help address how morphological novelties evolve, a key question in evolutionary biology. In Arabidopsis thaliana, APETALA2 (AP2) plays a role in the development of key plant innovations including seeds, flowers, and fruits. AP2 belongs to the AP2/ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR family which has members in all viridiplantae, making it one of the oldest and most diverse gene lineages. One key subclade, present across vascular plants is the euAPETALA2 (euAP2) clade, whose founding member is AP2. We reconstructed the evolution of the euAP2 gene lineage in vascular plants to better understand its impact on the morphological evolution of plants, identifying seven major duplication events. We also performed spatiotemporal expression analyses of euAP2/TOE3 genes focusing on less explored vascular plant lineages, including ferns, gymnosperms, early diverging angiosperms and early diverging eudicots. Altogether, our data suggest that euAP2 genes originally contributed to spore and sporangium development, and were subsequently recruited to ovule, fruit and floral organ development. Finally, euAP2 protein sequences are highly conserved; therefore, changes in the role of euAP2 homologs during development are most likely due to changes in regulatory regions.

Regulation of carotenoid and chlorophyll pools in hesperidia, anatomically unique fruits found only in Citrus.

Domesticated citrus varieties are woody perennials and interspecific hybrid crops of global economic and nutritional importance. The citrus fruit "hesperidium" is a unique morphological innovation not found in any other plant lineage. Efforts to improve the nutritional quality of the fruit are predicated on understanding the underlying regulatory mechanisms responsible for fruit development, including temporal control of chlorophyll degradation and carotenoid biosynthesis. Here, we investigated the molecular basis of the navel orange (Citrus sinensis) brown flavedo mutation, which conditions flavedo that is brown instead of orange. To overcome the limitations of using traditional genetic approaches in citrus and other woody perennials, we developed a strategy to elucidate the underlying genetic lesion. We used a multi-omics approach to collect data from several genetic sources and plant chimeras to successfully decipher this mutation. The multi-omics strategy applied here will be valuable in driving future gene discovery efforts in citrus as well as in other woody perennial plants. The comparison of transcriptomic and genomic data from multiple genotypes and plant sectors revealed an underlying lesion in the gene encoding STAY-GREEN (SGR) protein, which simultaneously regulates carotenoid biosynthesis and chlorophyll degradation. However, unlike SGR of other plant species, we found that the carotenoid and chlorophyll regulatory activities could be uncoupled in the case of certain SGR alleles in citrus and thus we propose a model for the molecular mechanism underlying the brown flavedo phenotype. The economic and nutritional value of citrus makes these findings of wide interest. The strategy implemented, and the results obtained, constitute an advance for agro-industry by driving opportunities for citrus crop improvement.

All type II classic MADS-box genes in the lycophyte Selaginella moellendorffii are broadly yet discretely expressed in vegetative and reproductive tissues.

The MADS-box genes constitute a large transcription factor family that appear to have evolved by duplication and diversification of function. Two types of MADS-box genes are distinguished throughout eukaryotes, types I and II. Type II classic MADS-box genes, also known as MIKC-type, are key developmental regulators in flowering plants and are particularly well-studied for their role in floral organ specification. However, very little is known about the role that these genes might play outside of the flowering plants. We investigated the evolution of type II classic MADS-box genes across land plants by performing a maximum likelihood analysis with a particular focus on lycophytes. Here, we present the expression patterns of all three type II classic MADS-box homologs throughout plant development in the lycophyte Selaginella moellendorffii: SmMADS1, SmMADS3, and SmMADS6. We used scanning electron microscopy and histological analyses to define stages of sporangia development in S. moellendorffii. We performed phylogenetic analyses of this gene lineage across land plants and found that lycophyte sequences appeared before the multiple duplication events that gave rise to the major MADS-box gene lineages in seed plants. Our expression analyses by in situ hybridization show that all type II classic MADS-box genes in S. moellendorffii have broad but distinct patterns of expression in vegetative and reproductive tissues, where SmMADS1 and SmMADS6 only differ during late sporangia development. The broad expression during S. moellendorffii development suggests that MADS-box genes have undergone neofunctionalization and subfunctionalization after duplication events in seed plants.

Deciphering the evolution of the ovule genetic network through expression analyses in Gnetum gnemon.

BACKGROUND AND AIMS: The ovule is a synapomorphy of all seed plants (gymnosperms and angiosperms); however, there are some striking differences in ovules among the major seed plant lineages, such as the number of integuments or the orientation of the ovule. The genetics involved in ovule development have been well studied in the model species Arabidopsis thaliana, which has two integuments and anatropous orientation. This study is approached from what is known in arabidopsis, focusing on the expression patterns of homologues of four genes known to be key for the proper development of the integuments in arabidopsis: AINTEGUMENTA (ANT), BELL1, (BEL1), KANADIs (KANs) and UNICORN (UCN). METHODS: We used histology to describe the morphoanatomical development from ovules to seeds in Gnetum gnemon. We carried out spatiotemporal expression analyses in G. gnemon, a gymnosperm, which has a unique ovule morphology with an integument covering the nucellus, two additional envelopes where the outermost becomes fleshy as the seed matures, and an orthotropous orientation. KEY RESULTS: Our anatomical and developmental descriptions provide a framework for expression analyses in the ovule of G. gnemon. Our expression results show that although ANT, KAN and UCN homologues are expressed in the inner integument, their spatiotemporal patterns differ from those found in angiosperms. Furthermore, all homologues studied here are expressed in the nucellus, revealing major differences in seed plants. Finally, no expression of the studied homologues was detected in the outer envelopes. CONCLUSIONS: Altogether, these analyses provide significant comparative data that allows us to better understand the functional evolution of these gene lineages, providing a compelling framework for evolutionary and developmental studies of seeds. Our findings suggest that these genes were most likely recruited from the sporangium development network and became restricted to the integuments of angiosperm ovules.

Phylogenetic analyses of key developmental genes provide insight into the complex evolution of seeds.

Gene duplication plays a decisive role in organismal diversification and in the appearance of novel structures. In plants the megagametophyte covered by the integuments, which after fertilization becomes the seed constitutes a novel structure: the ovule. In Arabidopsis thaliana, genetic mechanisms regulating ovule development, including the genetics underlying ovule initiation, ovule patterning and integument development, have been identified. Among seed plants, integuments are not only a novelty in evolution, but integuments also present an enormous morphological variation. This study is focused on the evolution of gene families that play a role in the proper morphological development of the integuments, BELL1 (BEL1), KANADIs (KAN1, KAN2, and KAN4/ATS), UNICORN (UCN) and SHORT INTEGUMENTS1 (SIN1). In Arabidopsis, BEL1 establishes the initiation of integument development. KAN1 and 2 act in the proper development of the outer integument. While ABERRANT TESTA SHAPE (ATS), is involved in the correct separation of both integuments. UCN acts in planar growth of the outer integument repressing ATS. SIN1 is involved in cell elongation in the integuments. The results of our analyses show that each of these genes has a different evolutionary history and that while gymnosperms appear to have a simpler ovule morphology, they have more homologues of these candidate genes than angiosperms. In addition, we present the conserved and novel motifs for each of these genes among seed plants and their selection constraints, which may be related to functional changes and to the diversity of ovule morphologies.

Evolution of the APETALA2 Gene Lineage in Seed Plants.

Gene duplication is a fundamental source of functional evolutionary change and has been associated with organismal diversification and the acquisition of novel features. The APETALA2/ETHYLENE RESPONSIVE ELEMENT-BINDING FACTOR (AP2/ERF) genes are exclusive to vascular plants and have been classified into the AP2-like and ERF-like clades. The AP2-like clade includes the AINTEGUMENTA (ANT) and the euAPETALA2 (euAP2) genes, both regulated by miR172 Arabidopsis has two paralogs in the euAP2 clade, namely APETALA2 (AP2) and TARGET OF EAT3 (TOE3) that control flowering time, meristem determinacy, sepal and petal identity and fruit development. euAP2 genes are likely functionally divergent outside Brassicaceae, as they control fruit development in tomato, and regulate inflorescence meristematic activity in maize. We studied the evolution and expression patterns of euAP2/TOE3 genes to assess large scale and local duplications and evaluate protein motifs likely related with functional changes across seed plants. We sampled euAP2/TOE3 genes from vascular plants and have found three major duplications and a few taxon-specific duplications. Here, we report conserved and new motifs across euAP2/TOE3 proteins and conclude that proteins predating the Brassicaceae duplication are more similar to AP2 than TOE3. Expression data show a shift from restricted expression in leaves, carpels, and fruits in non-core eudicots and asterids to a broader expression of euAP2 genes in leaves, all floral organs and fruits in rosids. Altogether, our data show a functional trend where the canonical A-function (sepal and petal identity) is exclusive to Brassicaceae and it is likely not maintained outside of rosids.

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.

Maternal control of triploid seed development by the TRANSPARENT TESTA 8 (TT8) transcription factor in Arabidopsis thaliana.

The balance between parental genome dosage is critical to offspring development in both animals and plants. In some angiosperm species, despite the imbalance between maternally and paternally inherited chromosome sets, crosses between parental lines of different ploidy may result in viable offspring. However, many plant species, like Arabidopsis thaliana, present a post-zygotic reproductive barrier, known as triploid block which results in the inability of crosses between individuals of different ploidy to generate viable seeds but also, in defective development of the seed. Several paternal regulators have been proposed as active players in establishing the triploid block. Maternal regulators known to be involved in this process are some flavonoid biosynthetic (FB) genes, expressed in the innermost layer of the seed coat. Here we explore the role of selected flavonoid pathway genes in triploid block, including TRANSPARENT TESTA 4 (TT4), TRANSPARENT TESTA 7 (TT7), SEEDSTICK (STK), TRANSPARENT TESTA 16 (TT16), TT8 and TRANSPARENT TESTA 13 (TT13). This approach allowed us to detect that TT8, a bHLH transcription factor, member of this FB pathway is required for the paternal genome dosage, as loss of function tt8, leads to complete rescue of the triploid block to seed development.

The genome of the Wollemi pine, a critically endangered "living fossil" unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

Fleshy or dry: transcriptome analyses reveal the genetic mechanisms underlying bract development in Ephedra.

BACKGROUND: Gnetales have a key phylogenetic position in the evolution of seed plants. Among the Gnetales, there is an extraordinary morphological diversity of seeds, the genus Ephedra, in particular, exhibits fleshy, coriaceous or winged (dry) seeds. Despite this striking diversity, its underlying genetic mechanisms remain poorly understood due to the limited studies in gymnosperms. Expanding the genomic and developmental data from gymnosperms contributes to a better understanding of seed evolution and development. RESULTS: We performed transcriptome analyses on different plant tissues of two Ephedra species with different seed morphologies. Anatomical observations in early developing ovules, show that differences in the seed morphologies are established early in their development. The transcriptomic analyses in dry-seeded Ephedra californica and fleshy-seeded Ephedra antisyphilitica, allowed us to identify the major differences between the differentially expressed genes in these species. We detected several genes known to be involved in fruit ripening as upregulated in the fleshy seed of Ephedra antisyphilitica. CONCLUSIONS: This study allowed us to determine the differentially expressed genes involved in seed development of two Ephedra species. Furthermore, the results of this study of seeds with the enigmatic morphology in Ephedra californica and Ephedra antisyphilitica, allowed us to corroborate the hypothesis which suggest that the extra envelopes covering the seeds of Gnetales are not genetically similar to integument. Our results highlight the importance of carrying out studies on less explored species such as gymnosperms, to gain a better understanding of the evolutionary history of plants.

Dynamic genome evolution in a model fern.

The large size and complexity of most fern genomes have hampered efforts to elucidate fundamental aspects of fern biology and land plant evolution through genome-enabled research. Here we present a chromosomal genome assembly and associated methylome, transcriptome and metabolome analyses for the model fern species Ceratopteris richardii. The assembly reveals a history of remarkably dynamic genome evolution including rapid changes in genome content and structure following the most recent whole-genome duplication approximately 60 million years ago. These changes include massive gene loss, rampant tandem duplications and multiple horizontal gene transfers from bacteria, contributing to the diversification of defence-related gene families. The insertion of transposable elements into introns has led to the large size of the Ceratopteris genome and to exceptionally long genes relative to other plants. Gene family analyses indicate that genes directing seed development were co-opted from those controlling the development of fern sporangia, providing insights into seed plant evolution. Our findings and annotated genome assembly extend the utility of Ceratopteris as a model for investigating and teaching plant biology.

Expression analyses in Ginkgo biloba provide new insights into the evolution and development of the seed.

Although the seed is a key morphological innovation, its origin remains unknown and molecular data outside angiosperms is still limited. Ginkgo biloba, with a unique place in plant evolution, being one of the first extant gymnosperms where seeds evolved, can testify to the evolution and development of the seed. Initially, to better understand the development of the ovules in Ginkgo biloba ovules, we performed spatio-temporal expression analyses in seeds at early developing stages, of six candidate gene homologues known in angiosperms: WUSCHEL, AINTEGUMENTA, BELL1, KANADI, UNICORN, and C3HDZip. Surprisingly, the expression patterns of most these ovule homologues indicate that they are not wholly conserved between angiosperms and Ginkgo biloba. Consistent with previous studies on early diverging seedless plant lineages, ferns, lycophytes, and bryophytes, many of these candidate genes are mainly expressed in mega- and micro-sporangia. Through in-depth comparative transcriptome analyses of Ginkgo biloba developing ovules, pollen cones, and megagametophytes we have been able to identify novel genes, likely involved in ovule development. Finally, our expression analyses support the synangial or neo-synangial hypotheses for the origin of the seed, where the sporangium developmental network was likely co-opted and restricted during integument evolution.

The Evolution of the KANADI Gene Family and Leaf Development in Lycophytes and Ferns.

Leaves constitute the main photosynthetic plant organ and even though their importance is not debated, the origin and development of leaves still is. The leaf developmental network has been elucidated for angiosperms, from genes controlling leaf initiation, to leaf polarity and shape. There are four KANADI (KAN) paralogs in Arabidopsis thaliana needed for organ polarity with KAN1 and KAN2 specifying abaxial leaf identity. Yet, studies of this gene lineage outside angiosperms are required to better understand the evolutionary patterns of leaf development and the role of KAN homologs. We studied the evolution of KAN genes across vascular plants and their expression by in situ hybridization in the fern, Equisetum hyemale and the lycophyte Selaginella moellendorffii. Our results show that the expression of KAN genes in leaves is similar between ferns and angiosperms. However, the expression patterns observed in the lycophyte S. moellendorffii are significantly different compared to all other vascular plants, suggesting that the KAN function in leaf polarity is likely only conserved across ferns, gymnosperms, and angiosperms. This study indicates that mechanisms for leaf development are different in lycophytes compared to other vascular plants.

Duplication and Diversification of REPLUMLESS - A Case Study in the Papaveraceae.

There is a vast amount of fruit morphological diversity in terms of their texture, the number of carpels, if those carpels are fused or not and how fruits open to disperse the seeds. Arabidopsis thaliana, a model eudicot, has a dry bicarpellate silique, when the fruit matures, the two valves fall apart through the dehiscence zone leaving the seeds attached to the remaining medial tissue, called the replum. Proper replum development in A. thaliana is mediated by REPLUMLESS (RPL), a TALE Homeodomain protein. RPL represses the valve margin genetic program and the downstream dehiscence zone formation in the medial tissue of the siliques and RPL orthologs have conserved roles across the Brassicaceae eudicots. A RPL homolog, qSH1, has been studied in rice, a monocot, and plays a role in fruit shedding making it difficult to predict functional evolution of this gene lineage across angiosperms. Although RPL orthologs have been identified across all angiosperms, expression and functional analyses are scarce. In order to fill the phylogenetic gap between the Brassicaceae and monocots we have characterized the expression patterns of RPL homologs in two poppies with different fruit types, Bocconia frutescens with operculate valvate dehiscence and a persistent medial tissue, similar to a replum, and Papaver somniferum, a poppy with persistent medial tissue in between the multicarpellate gynoecia. We found that RPL homologs in Papaveraceae have broad expression patterns during plant development; in the shoot apical meristem, during flowering transition and in many floral organs, especially the carpels. These patterns are similar to those of RPL in A. thaliana. However, our results suggest that RPL does not have conserved roles in the maintenance of medial persistent tissues of fruits but may be involved with establishing the putative dehiscence zone in dry poppy fruits.

Evolution of the SPATULA/ALCATRAZ gene lineage and expression analyses in the basal eudicot, Bocconia frutescens L. (Papaveraceae).

BACKGROUND: SPATULA (SPT) and ALCATRAZ (ALC) are recent paralogs that belong to the large bHLH transcription factor family. Orthologs of these genes have been found in all core eudicots, whereas pre-duplication genes, named paleoSPATULA/ALCATRAZ, have been found in basal eudicots, monocots, basal angiosperms and gymnosperms. Nevertheless, functional studies have only been performed in Arabidopsis thaliana, where SPT and ALC are partially redundant in carpel and valve margin development and ALC has a unique role in the dehiscence zone. Further analyses of pre-duplication genes are necessary to assess the functional evolution of this gene lineage. RESULTS: We isolated additional paleoSPT/ALC genes from Aristolochia fimbriata, Bocconia frutescens, Cattleya trianae and Hypoxis decumbens from our transcriptome libraries and performed phylogenetic analyses. We identified the previously described bHLH domain in all analyzed sequences and also new conserved motifs using the MEME suite. Finally, we analyzed the expression of three paleoSPT/ALC genes (BofrSPT1/2/3) from Bocconia frutescens, a basal eudicot in the Papaveraceae. To determine the developmental stages at which these genes were expressed, pre- and post-anthesis carpels and fruits of B. frutescens were collected, sectioned, stained, and examined using light microscopy. Using in situ hybridization we detected that BofrSPT1/2/3 genes are expressed in floral buds, early sepal initiation, stamens and carpel primordia and later during fruit development in the dehiscence zone of the opercular fruit. CONCLUSIONS: Our expression results, in comparison with those available for core eudicots, suggest conserved roles of members of the SPT/ALC gene lineage across eudicots in the specification of carpel margins and the dehiscence zone of the mature fruits. Although there is some redundancy between ALC and SPT, these gene clades seem to have undergone some degree of sub-functionalization in the core eudicots, likely by changes in cis regulatory regions and to some extent in coding sequences, at least in Brassicaceae. Our results also indicate that in Bocconia frutescens, paleoSPT/ALC genes may play a role in early floral organ specification that was subsequently lost in core eudicot lineages.