Evolution of major flowering pathway integrators in Orchidaceae.

The Orchidaceae is a mega-diverse plant family with ca. 29,000 species with a large variety of life forms that can colonize transitory habitats. Despite this diversity, little is known about their flowering integrators in response to specific environmental factors. During the reproductive transition in flowering plants a vegetative apical meristem (SAM) transforms into an inflorescence meristem (IM) that forms bracts and flowers. In model grasses, like rice, a flowering genetic regulatory network (FGRN) controlling reproductive transitions has been identified, but little is known in the Orchidaceae. In order to analyze the players of the FRGN in orchids, we performed comprehensive phylogenetic analyses of CONSTANS-like/CONSTANS-like 4 (COL/COL4), FLOWERING LOCUS D (FD), FLOWERING LOCUS C/FRUITFULL (FLC/FUL) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) gene lineages. In addition to PEBP and AGL24/SVP genes previously analyzed, here we identify an increase of orchid homologs belonging to COL4, and FUL gene lineages in comparison with other monocots, including grasses, due to orchid-specific gene lineage duplications. Contrariwise, local duplications in Orchidaceae are less frequent in the COL, FD and SOC1 gene lineages, which points to a retention of key functions under strong purifying selection in essential signaling factors. We also identified changes in the protein sequences after such duplications, variation in the evolutionary rates of resulting paralogous clades and targeted expression of isolated homologs in different orchids. Interestingly, vernalization-response genes like VERNALIZATION1 (VRN1) and FLOWERING LOCUS C (FLC) are completely lacking in orchids, or alternatively are reduced in number, as is the case of VERNALIZATION2/GHD7 (VRN2). Our findings point to non-canonical factors sensing temperature changes in orchids during reproductive transition. Expression data of key factors gathered from Elleanthus auratiacus, a terrestrial orchid in high Andean mountains allow us to characterize which copies are actually active during flowering. Altogether, our data lays down a comprehensive framework to assess gene function of a restricted number of homologs identified more likely playing key roles during the flowering transition, and the changes of the FGRN in neotropical orchids in comparison with temperate grasses.

Global scenario of the RmtE pan-aminoglycoside-resistance mechanism: emergence of the rmtE4 gene in South America associated with a hospital-related IncL plasmid.

Antimicrobial resistance (AMR) mechanisms, especially those conferring resistance to critically important antibiotics, are a great concern for public health. 16S rRNA methyltransferases (16S-RMTases) abolish the effectiveness of most clinically used aminoglycosides, but some of them are considered sporadic, such as RmtE. The main goals of this work were the genomic analysis of bacteria producing 16S-RMTases from a 'One Health' perspective in Venezuela, and the study of the epidemiological and evolutionary scenario of RmtE variants and their related mobile genetic elements (MGEs) worldwide. A total of 21 samples were collected in 2014 from different animal and environmental sources in the Cumaná region (Venezuela). Highly aminoglycoside-resistant Enterobacteriaceae isolates were selected, identified and screened for 16S-RMTase genes. Illumina and Nanopore whole-genome sequencing data were combined to obtain hybrid assemblies and analyse their sequence type, resistome, plasmidome and pan-genome. Genomic collections of rmtE variants and their associated MGEs were generated to perform epidemiological and phylogenetic analyses. A single 16S-RMTase, the novel RmtE4, was identified in five Klebsiella isolates from wastewater samples of Cumaná. This variant possessed three amino acid modifications with respect to RmtE1-3 (Asn152Asp, Val216Ile and Lys267Ile), representing the most genetic distant among all known and novel variants described in this work, and the second most prevalent. rmtE variants were globally spread, and their geographical distribution was determined by the associated MGEs and the carrying bacterial species. Thus, rmtE4 was found to be confined to Klebsiella isolates from South America, where it was closely related to ISVsa3 and an uncommon IncL plasmid related with hospital environments. This work uncovered the global scenario of RmtE and the existence of RmtE4, which could potentially emerge from South America. Surveillance and control measures should be developed based on these findings in order to prevent the dissemination of this AMR mechanism and preserve public health worldwide.

Phylogenomics and plastome evolution of a Brazilian mycoheterotrophic orchid, Pogoniopsis schenckii.

PREMISE: Pogoniopsis likely represents an independent photosynthesis loss in orchids. We use phylogenomic data to better identify the phylogenetic placement of this fully mycoheterotrophic taxon, and investigate its molecular evolution. METHODS: We performed likelihood analysis of plastid and mitochondrial phylogenomic data to localize the position of Pogoniopsis schenckii in orchid phylogeny, and investigated the evolution of its plastid genome. RESULTS: All analyses place Pogoniopsis in subfamily Epidendroideae, with strongest support from mitochondrial data, which also place it near tribe Sobralieae with moderately strong support. Extreme rate elevation in Pogoniopsis plastid genes broadly depresses branch support; in contrast, mitochondrial genes are only mildly rate elevated and display very modest and localized reductions in bootstrap support. Despite considerable genome reduction, including loss of photosynthesis genes and multiple translation apparatus genes, gene order in Pogoniopsis plastomes is identical to related autotrophs, apart from moderately shifted inverted repeat (IR) boundaries. All cis-spliced introns have been lost in retained genes. Two plastid genes (accD, rpl2) show significant strengthening of purifying selection. A retained plastid tRNA gene (trnE-UUC) of Pogoniopsis lacks an anticodon; we predict that it no longer functions in translation but retains a secondary role in heme biosynthesis. CONCLUSIONS: Slowly evolving mitochondrial genes clarify the placement of Pogoniopsis in orchid phylogeny, a strong contrast with analysis of rate-elevated plastome data. We documented the effects of the novel loss of photosynthesis: for example, despite massive gene loss, its plastome is fully colinear with other orchids, and it displays only moderate shifts in selective pressure in retained genes.

Transcriptional analysis of Ceratopteris richardii young sporophyte reveals conservation of stem cell factors in the root apical meristem.

Gene expression in roots has been assessed in different plant species in studies ranging from complete organs to specific cell layers, and more recently at the single cell level. While certain genes or functional categories are expressed in the root of all or most plant species, lineage-specific genes have also been discovered. An increasing amount of transcriptomic data is available for angiosperms, while a limited amount of data is available for ferns, and few studies have focused on fern roots. Here, we present a de novo transcriptome assembly from three different parts of the Ceratopteris richardii young sporophyte. Differential gene expression analysis of the root tip transcriptional program showed an enrichment of functional categories related to histogenesis and cell division, indicating an active apical meristem. Analysis of a diverse set of orthologous genes revealed conserved expression in the root meristem, suggesting a preserved role for different developmental roles in this tissue, including stem cell maintenance. The reconstruction of evolutionary trajectories for ground tissue specification genes suggests a high degree of conservation in vascular plants, but not for genes involved in root cap development, showing that certain genes are absent in Ceratopteris or have intricate evolutionary paths difficult to track. Overall, our results suggest different processes of conservation and divergence of genes involved in root development.

Glutamine synthetase evolutionary history revisited: Tracing back beyond the Last Universal Common Ancestor.

Glutamine synthetase (GS; EC 6.3.1.2, L-glutamate: ammonia ligase) is an essential enzyme in nitrogen assimilation. It catalyzes glutamine synthesis using glutamate and ammonium with ATP hydrolysis. Four forms of GSs have been described in literature. These enzyme types are discriminated based on their primary and quaternary structures. GS-encoding genes are believed to be of the oldest functioning genes studied, and its evolutionary history was explored in classic studies in the 90s. Here, we evaluated GS-homologous sequences from the three life domains to revisit their origins and evolutionary history. There are clear examples of ancient duplications and interdomain horizontal gene transfers. We present GS-encoding genes as one multigenic family that comprises three distinct groups. Our findings are presented in light of two main hypotheses for GS origins and evolutions, and we argue in favor of gene duplications giving rise to the three genes in the Last Universal Common Ancestral. Type I family is the most diverse one, presenting a subgroup of polyamine metabolizing enzymes, besides many examples of noncatalytic GS homologs. Many instances of gene loss, duplication, and transfer have occurred after life diversification, contributing to GS complex evolutionary history.

Phylogenomic and Microsynteny Analysis Provides Evidence of Genome Arrangements of High-Affinity Nitrate Transporter Gene Families of Plants.

Nitrate transporter 2 (NRT2) and NRT3 or nitrate-assimilation-related 2 (NAR2) proteins families form a two-component, high-affinity nitrate transport system, which is essential for the acquisition of nitrate from soils with low N availability. An extensive phylogenomic analysis across land plants for these families has not been performed. In this study, we performed a microsynteny and orthology analysis on the NRT2 and NRT3 genes families across 132 plants (Sensu lato) to decipher their evolutionary history. We identified significant differences in the number of sequences per taxonomic group and different genomic contexts within the NRT2 family that might have contributed to N acquisition by the plants. We hypothesized that the greater losses of NRT2 sequences correlate with specialized ecological adaptations, such as aquatic, epiphytic, and carnivory lifestyles. We also detected expansion on the NRT2 family in specific lineages that could be a source of key innovations for colonizing contrasting niches in N availability. Microsyntenic analysis on NRT3 family showed a deep conservation on land plants, suggesting a high evolutionary constraint to preserve their function. Our study provides novel information that could be used as guide for functional characterization of these gene families across plant lineages.

Ancestry analysis indicates two different sets of essential genes in eukaryotic model species.

Essential genes are so-called because they are crucial for organism perpetuation. Those genes are usually related to essential functions to cellular metabolism or multicellular homeostasis. Deleterious alterations on essential genes produce a spectrum of phenotypes in multicellular organisms. The effects range from the impairment of the fertilization process, disruption of fetal development, to loss of reproductive capacity. Essential genes are described as more evolutionarily conserved than non-essential genes. However, there is no consensus about the relationship between gene essentiality and gene age. Here, we identified essential genes in five model eukaryotic species (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus) and estimate their evolutionary ancestry and their network properties. We observed that essential genes, on average, are older than other genes in all species investigated. The relationship of network properties and gene essentiality convey with previous findings, showing essential genes as important nodes in biological networks. As expected, we also observed that essential orthologs shared by the five species evaluated here are old. However, all the species evaluated here have a specific set of young essential genes not shared among them. Additionally, these two groups of essential genes are involved with distinct biological functions, suggesting two sets of essential genes: (i) a set of old essential genes common to all the evaluated species, regulating basic cellular functions, and (ii) a set of young essential genes exclusive to each species, which perform specific essential functions in each species.

Adaptive evolution of Moniliophthora PR-1 proteins towards its pathogenic lifestyle.

BACKGROUND: Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches' broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. RESULTS: Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host-pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. CONCLUSIONS: Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.

Evolution and Expression of Reproductive Transition Regulatory Genes FT/TFL1 With Emphasis in Selected Neotropical Orchids.

Flowering is a rigorously timed and morphologically complex shift in plant development. This change depends on endogenous as well as environmental factors. FLOWERING LOCUS T (FT) integrates several cues from different pathways acting as a flowering promoter. Contrary to the role of FT, its paralog TERMINAL FLOWER 1 (TFL1) delays floral transition. Although FT/TFL1 homologs have been studied in model eudicots and monocots, scarce studies are available in non-model monocots like the Orchidaceae. Orchids are very diverse and their floral complexity is translated into a unique aesthetic display, which appeals the ornamental plant market. Nonetheless, orchid trade faces huge limitations due to their long vegetative phase and intractable indoor flowering seasons. Little is known about the genetic basis that control reproductive transition in orchids and, consequently, manipulating their flowering time remains a challenge. In order to contribute to the understanding of the genetic bases that control flowering in orchids we present here the first broad-scale analysis of FT/TFL1-like genes in monocots with an expanded sampling in Orchidaceae. We also compare expression patterns in three selected species and propose hypotheses on the putative role of these genes in their reproductive transition. Our findings show that FT-like genes are by far more diversified than TFL1-like genes in monocots with six subclades in the former and only one in the latter. Within MonFT1, the comparative protein sequences of MonFT1A and MonFT1B suggest that they could have recruited functional roles in delaying flowering, a role typically assigned to TFL1-like proteins. On the other hand, MonFT2 proteins have retained their canonical motifs and roles in promoting flowering transition. This is also shown by their increased expression levels from the shoot apical meristem (SAM) and leaves to inflorescence meristems (IM) and floral buds (FBs). Finally, TFL1-like genes are retained as single copy and often times are lost. Their loss could be linked to the parallel recruitment of MonFT1A and MonFT1B homologs in delaying flowering and maintaining indeterminacy of the inflorescence meristem. These hypotheses lay the foundation for future functional validation in emerging model orchid species and comparative analyses in orchids with high horticultural potential in the market.

Characterization of Histone Genes from the Bivalve Lucina Pectinata.

Lucina pectinata is a clam that lives in sulfide-rich environments and houses intracellular sulfide-oxidizing endosymbionts. To identify new Lucina pectinata proteins, we produced libraries for genome and transcriptome sequencing and assembled them de novo. We searched for histone-like sequences using the Lucina pectinata histone H3 partial nucleotide sequence against our previously described genome assembly to obtain the complete coding region and identify H3 coding sequences from mollusk sequences in Genbank. Solen marginatus histone nucleotide sequences were used as query sequences using the genome and transcriptome assemblies to identify the Lucina pectinata H1, H2A, H2B and H4 genes and mRNAs and obtained the complete coding regions of the five histone genes by RT-PCR combined with automated Sanger DNA sequencing. The amino acid sequence conservation between the Lucina pectinata and Solen marginatus histones was: 77%, 93%, 83%, 96% and 97% for H1, H2A, H2B, H3 and H4, respectively. As expected, the H3 and H4 proteins were the most conserved and the H1 proteins were most similar to H1's from aquatic organisms like Crassostrea gigas, Aplysia californica, Mytilus trossulus and Biomphalaria glabrata. The Lucina pectinata draft genome and transcriptome assemblies, obtained by semiconductor sequencing, were adequate for identification of conserved proteins as evidenced by our results for the histone genes.

Papain-like cysteine protease encoding genes in rubber (Hevea brasiliensis): comparative genomics, phylogenetic, and transcriptional profiling analysis.

MAIN CONCLUSION: 43 HbPLCPs representing nine subfamilies or 20 orthologous groups were found in rubber, where paralogs were resulted from the recent WGD and local duplication. Several senescence-associated genes were also identified. Papain-like cysteine proteases (PLCPs) comprise a large family of proteolytic enzymes involved in plant growth and development, seed germination, organ senescence, immunity, and stress response. Despite their importance and the extensive research in the model plant Arabidopsis thaliana, little information is available on rubber tree (Hevea brasiliensis), a rubber-producing plant of the Euphorbiaceae family. This study performed a genome-wide identification of PLCP family genes in rubber, resulting in a relatively high number of 43 members. The phylogenetic analysis assigned these genes into nine subfamilies, i.e., RD21 (6), CEP (4), XCP (4), XBCP3 (2), THI (1), SAG12 (18), RD19 (4), ALP (2), and CTB (2). Most of them were shown to have orthologs in Arabidopsis; however, several members in SAG12, CEP and XBCP3 subfamilies form new groups as observed in other core eudicots such as Manihot esculenta, Ricinus communis, Populus trichocarpa, and Vitis vinifera. Based on an expert sequence comparison, 20 orthologous groups (OGs) were proposed for core eudicots, and rubber paralogs were shown to be resulted from the recent whole-genome duplication (WGD) as well as local duplication. Transcriptional profiling showed distinct expression pattern of different members across various tissues, e.g., root, leaf, bark, laticifer, flower, and seed. By using the senescence-specific HbSAG12H1 as the indicator, the transcriptome of senescent rubber leaves was deeply sequenced and several senescence-associated PLCP genes were identified. Results obtained from this study provide valuable information for future functional analysis and utilization of PLCP genes in Hevea and other species.

Distribution patterns and impact of transposable elements in genes of green algae.

Transposable elements (TEs) are DNA sequences able to transpose in the host genome, a remarkable feature that enables them to influence evolutive trajectories of species. An investigation about the TE distribution and TE impact in different gene regions of the green algae species Chlamydomonas reinhardtii and Volvox carteri was performed. Our results indicate that TEs are very scarce near introns boundaries, suggesting that insertions in this region are negatively selected. This contrasts with previous results showing enrichment of tandem repeats in introns boundaries and suggests that different evolutionary forces are acting in these different classes of repeats. Despite the relatively low abundance of TEs in the genome of green algae when compared to mammals, the proportion of poly(A) sites derived from TEs found in C. reinhardtii was similar to that described in human and mice. This fact, associated with the enrichment of TEs in gene 5' and 3' flanks of C. reinhardtii, opens up the possibility that TEs may have considerably contributed for gene regulatory sequences evolution in this species. Moreover, it was possible identify several instances of TE exonization for C. reinhardtii, with a particularly interesting case from a gene coding for Condensin II, a protein involved in the maintenance of chromosomal structure, where the addition of a transposomal PHD finger may contribute to binding specificity of this protein. Taken together, our results suggest that the low abundance of TEs in green algae genomes is correlated with a strict negative selection process, combined with the retention of copies that contribute positively with gene structures.

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.

Accelerated evolution of schistosome genes coding for proteins located at the host-parasite interface.

Study of proteins located at the host-parasite interface in schistosomes might provide clues about the mechanisms utilized by the parasite to escape the host immune system attack. Micro-exon gene (MEG) protein products and venom allergen-like (VAL) proteins have been shown to be present in schistosome secretions or associated with glands, which led to the hypothesis that they are important components in the molecular interaction of the parasite with the host. Phylogenetic and structural analysis of genes and their transcripts in these two classes shows that recent species-specific expansion of gene number for these families occurred separately in three different species of schistosomes. Enrichment of transposable elements in MEG and VAL genes in Schistosoma mansoni provides a credible mechanism for preferential expansion of gene numbers for these families. Analysis of the ratio between synonymous and nonsynonymous substitution rates (dN/dS) in the comparison between schistosome orthologs for the two classes of genes reveals significantly higher values when compared with a set of a control genes coding for secreted proteins, and for proteins previously localized in the tegument. Additional analyses of paralog genes indicate that exposure of the protein to the definitive host immune system is a determining factor leading to the higher than usual dN/dS values in those genes. The observation that two genes encoding S. mansoni vaccine candidate proteins, known to be exposed at the parasite surface, also display similar evolutionary dynamics suggests a broad response of the parasite to evolutionary pressure imposed by the definitive host immune system.