Structural and biochemical characterization of SmoPG1, an exo-polygalacturonase from Selaginella moellendorffii.

Polygalacturonases (PGs) can modulate chemistry and mechanical properties of the plant cell wall through the degradation of pectins, one of its major constituents. PGs are largely used in food, beverage, textile, and paper industries to increase processes' performances. To improve the use of PGs, knowledge of their biochemical, structural and functional features is of prime importance. Our study aims at characterizing SmoPG1, a polygalacturonase from Selaginella moellendorffii, that belongs to the lycophytes. Transcription data showed that SmoPG1 was mainly expressed in S. moellendorffii shoots while phylogenetic analyses suggested that SmoPG1 is an exo-PG, which was confirmed by the biochemical characterization following its expression in heterologous system. Indeed, LC-MS/MS oligoprofiling using various pectic substrates identified galacturonic acid (GalA) as the main hydrolysis product. We found that SmoPG1 was most active on polygalacturonic acid (PGA) at pH 5, and that its activity could be modulated by different cations (Ca2+, Cu2+, Fe2+, Mg2+, Mn2+, Na2+, Zn2+). In addition, SmoPG1 was inhibited by green tea catechins, including (-)-epigallocatechin-3-gallate (EGCG). Docking analyses and MD simulations showed in detail amino acids responsible for the SmoPG1-EGCG interaction. Considering its expression yield and activity, SmoPG1 appears as a prime candidate for the industrial production of GalA.

Structure-function analysis of plant G-protein regulatory mechanisms identifies key Gα-RGS protein interactions.

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.

Genome and transcriptome of Selaginella kraussiana reveal evolution of root apical meristems in vascular plants.

The evolution of roots allowed vascular plants to adapt to land environments. Fossil evidence indicates that roots evolved independently in euphyllophytes (ferns and seed plants) and lycophytes, the two lineages of extant vascular plants. Based on a high-quality genome assembly, mRNA sequencing (mRNA-seq) data, and single-cell RNA-seq data for the lycophyte Selaginella kraussiana, we show that the two root origin events in lycophytes and euphyllophytes adopted partially similar molecular modules in the regulation of root apical meristem (RAM) development. In S. kraussiana, the RAM initiates from the rhizophore primordium guided by auxin and duplicates itself by dichotomous branching. The auxin signaling pathway directly upregulates euAINTEGUMENTAb (SkeuANTb), and then SkeuANTb directly promotes the expression of SkeuANTa and the WUSCHEL-RELATED HOMEOBOX13b (SkWOX13b) for RAM maintenance, partially similar to the molecular pathway involving the euANT-branch PLETHORA (AtPLT) genes and AtWOX5 in root initiation in the seed plant Arabidopsis thaliana. Other molecular modules, e.g., SHORT-ROOT and SCARECROW, also have partially similar expression patterns in the RAMs of S. kraussiana and A. thaliana. Overall, our study not only provides genome and transcriptome tools of S. kraussiana but also indicates the employment of some common molecular modules in RAMs during root origins in lycophytes and euphyllophytes.

Mitogenome-based phylogenomics provides insights into the positions of the enigmatic sinensis group and the sanguinolenta group in Selaginellaceae (Lycophyte).

Spikemoss (Selaginellaceae) is one of the basal lineages of vascular plants. This family has a single genus Selaginella which consists of about 750 extant species. The phylogeny of Selaginellaceae has been extensively studied mainly based on plastid DNA and a few nuclear sequences. However, the placement of the enigmatic sinensis group is a long-term controversy because of the long branch in the plastid DNA phylogeny. The sanguinolenta group is also a phylogenetically problematic clade owing to two alternative positions resulted from different datasets. Here, we newly sequenced 34 mitochondrial genomes (mitogenomes) of individuals representing all seven subgenera and major clades in Selaginellaceae. We assembled the draft mitogenomes and annotated the genes and performed phylogenetic analyses based on the shared 17 mitochondrial genes. Our major results include: (1) all the assembled mitogenomes have complicated structures, unparalleled high GC content and a small gene content set, and the positive correlations among GC content, substitution rates and the number of RNA editing sites hold; (2) the sinensis group was well supported as a member of subg. Stachygynandrum; (3) the sanguinolenta group was strongly resolved as sister to all other Selaginella species except for subg. Selaginella. This study demonstrates the potential of mitogenome data in providing novel insights into phylogenetically recalcitrant problems.

Impacts of methyl jasmonate on Selaginella martensii: volatiles, transcriptomics, phytohormones, and gas exchange.

Methyl jasmonate (MeJA) induces various defence responses in seed plants, but for early plant lineages, information on the potential of jasmonates to elicit stress signalling and trigger physiological modifications is limited. The spikemoss Selaginella martensii was exposed to a range of MeJA concentrations (0, 10, 25, and 50 mM), and biogenic volatile organic compound (BVOC) emissions, photosynthetic rate (A), and stomatal conductance (gs) were continuously measured. In addition, changes in phytohormone concentrations and gene expression were studied. Enhancement of methanol, lipoxygenase pathway volatiles and linalool emissions, and reductions in A and gs, were MeJA dose-dependent. Before MeJA treatment, the concentration of 12-oxo-phytodienoic acid (OPDA) was 7-fold higher than jasmonic acid (JA). MeJA treatment rapidly increased OPDA and JA concentrations (within 30 min), with the latter more responsive. Some genes involved in BVOC biosynthesis and OPDA-specific response were up-regulated at 30 min after MeJA spraying, whereas those in the JA signalling pathway were not affected. Although JA was synthesized in S. martensii, OPDA was prioritized as a signalling molecule upon MeJA application. MeJA inhibited primary and enhanced secondary metabolism; we propose that fast-emitted linalool could serve as a marker of elicitation of stress-induced metabolism in lycophytes.

Cellular and gene expression patterns associated with root bifurcation in Selaginella.

The roots of lycophytes branch through dichotomy or bifurcation, during which the root apex splits into two daughter roots. This is morphologically distinct from lateral root (LR) branching in the extant euphyllophytes, with LRs developing along the root axis at different distances from the apex. Although the process of root bifurcation is poorly understood, such knowledge can be important, because it may represent an evolutionarily ancient strategy that roots recruited to form new stem cells or meristems. In this study, we examined root bifurcation in the lycophyte Selaginella moellendorffii. We characterized an in vitro developmental time frame based on repetitive apex bifurcations, allowing us to sample different stages of dichotomous root branching and analyze the root meristem and root branching in S. moellendorffii at the microscopic and transcriptomic level. Our results showed that, in contrast to previous assumptions, initial cells (ICs) in the root meristem are mostly not tetrahedral but rather show an irregular shape. Tracking down the early stages of root branching argues for the occurrence of a symmetric division of the single IC, resulting in two apical stem cells that initiate root meristem bifurcation. Moreover, we generated a S. moellendorffii root branching transcriptome that resulted in the delineation of a subset of core meristem genes. The occurrence of multiple putative orthologs of meristem genes in this dataset suggests the presence of conserved pathways in the control of meristem and root stem cell establishment or maintenance.

The evolution of extremely diverged plastomes in Selaginellaceae (lycophyte) is driven by repeat patterns and the underlying DNA maintenance machinery.

Two factors are proposed to account for the unusual features of organellar genomes: the disruptions of organelle-targeted DNA replication, repair, and recombination (DNA-RRR) systems in the nuclear genome and repetitive elements in organellar genomes. Little is known about how these factors affect organellar genome evolution. The deep-branching vascular plant family Selaginellaceae is known to have a deficient DNA-RRR system and convergently evolved organellar genomes. However, we found that the plastid genome (plastome) of Selaginella sinensis has extremely accelerated substitution rates, a low GC content, pervasive repeat elements, a dynamic network structure, and it lacks direct or inverted repeats. Unexpectedly, its organelle DNA-RRR system is short of a plastid-targeted Recombinase A1 (RecA1) and a mitochondrion-targeted RecA3, in line with other explored Selaginella species. The plastome contains a large collection of short- and medium-sized repeats. Given the absence of RecA1 surveillance, we propose that these repeats trigger illegitimate recombination, accelerated mutation rates, and structural instability. The correlations between repeat quantity and architectural complexity in the Selaginella plastomes support these conclusions. We, therefore, hypothesize that the interplay of the deficient DNA-RRR system and the high repeat content has led to the extraordinary divergence of the S. sinensis plastome. Our study not only sheds new light on the mechanism of plastome divergence by emphasizing the power of cytonuclear integration, but it also reconciles the longstanding contradiction on the effects of DNA-RRR system disruption on genome structure evolution.

Plastid phylogenomic analyses of the Selaginella sanguinolenta group (Selaginellaceae) reveal conflict signatures resulting from sequence types, outlier genes, and pervasive RNA editing.

Different from the generally conserved plastomes (plastid genomes) of most land plants, the Selaginellaceae plastomes exhibit dynamic structure, high GC content and high substitution rates. Previous plastome analyses identified strong conflict on several clades in Selaginella, however the factors causing the conflictions and the impact on the phylogenetic inference have not been sufficiently investigated. Here, we dissect the distribution of phylogenetic signals and conflicts in Selaginella sanguinolenta group, the plastome of which is DR (direct repeats) structure and with genome-wide RNA editing. We analyzed the data sets including 22 plastomes representing all species of the S. sanguinolenta group, covering the entire geographical distribution from the Himalayas to Siberia and the Russian Far East regions. We recovered four different topologies by applying multispecies coalescent (ASTRAL) and concatenation methods (IQ-TREE and RAxML) on four data sets of PC (protein-coding genes), NC (non-coding sequences), PCN (the concatenated PC and NC), and RC (predicted RNA editing sites "C" were corrected by "T"), respectively. Six monophyletic clades, S. nummularifolia clade, S. rossii clade, S. sajanensis clade, S. sanguinolenta I clade, S. sanguinolenta II clade, and S. sanguinolenta III clade, were consistently resolved and supported by the characteristics of GC content, RNA editing frequency, and gene content. However, the relationships among these clades varied across the four topologies. To explore the underlying causes of the uncertainty, we compared the phylogenetic signals of the four topologies. We identified that the sequence types (coding versus non-coding), outlier genes (genes with extremely high |ΔGLS| values), and C-to-U RNA editing frequency in the protein-coding genes were responsible for the unstable phylogenomic relationship. We further revealed a significant positive correlation between the |ΔGLS| values and the variation coefficient of the RNA editing number. Our results demonstrated that the coalescent method performed better than the concatenation method in overcoming the problems caused by outlier genes and extreme RNA editing events. Our study particularly focused on the importance of exploring the plastid phylogenomic conflicts and suggested conducting concatenated analyses cautiously when adopting organelle genome data.

Backbone and sidechain 1H, 15N and 13C resonance assignments of the free and RNA-bound tandem zinc finger domain of the tristetraprolin family member from Selaginella moellendorffii.

Members of the tristetraprolin (TTP) family of RNA binding proteins (RBPs) regulate the metabolism of a variety of mRNA targets. In mammals, these proteins modulate many physiological processes, including immune cell activation, hematopoiesis, and embryonic development. Regulation of mRNA stability by these proteins requires that the tandem zinc finger (TZF) domain binds initially and directly to target mRNAs, ultimately leading to their deadenylation and decay. Proteins of this type throughout eukarya possess a highly conserved TZF domain, suggesting that they are all capable of high-affinity RNA binding. However, the mechanism of TTP-mediated mRNA decay is largely undefined. Given the vital role that these TTP family proteins play in maintaining RNA homeostasis throughout eukaryotes, we focused here on the first, key step in this process: recognition and binding of the TZF domain to target RNA. For these studies, we chose a primitive plant, the spikemoss Selaginella moellendorffii, which last shared a common ancestor with humans more than a billion years ago. Here we report the near complete backbone and side chain resonance assignments of the spikemoss TZF domain, including: (1) the assignment of the RNA-TZF domain complex, representing one of only two data sets currently available for the entire TTP family of proteins; and (2) the first NMR resonance assignments of the entire TZF domain, in the RNA-free form. This work will serve as the basis for further NMR structural investigations aimed at gaining insights into the process of RNA recognition and the mechanisms of TTP-mediated mRNA decay.

Evolution and conserved functionality of organ size and shape regulator PEAPOD.

Transcriptional regulator PEAPOD (PPD) and its binding partners comprise a complex that is conserved throughout many core eudicot plants with regard to protein domain sequence and the function of controlling organ size and shape. Orthologues of PPD also exist in the basal angiosperm Amborella trichopoda, various gymnosperm species, the lycophyte Selaginella moellendorffii and several monocot genera, although until now it was not known if these are functional sequences. Here we report constitutive expression of orthologues from species representing diverse taxa of plant phylogeny in the Arabidopsis Δppd mutant. PPD orthologues from S. moellendorffii, gymnosperm Picea abies, A. trichopoda, monocot Musa acuminata, and dicot Trifolium repens were able to complement the mutant and return it to the wild-type phenotype, demonstrating the conserved functionality of PPD throughout vascular plants. In addition, analysis of bryophyte genomes revealed potential PPD orthologues in model liverwort and moss species, suggesting a more primitive lineage for this conserved regulator. The Poaceae (grasses) lack the genes for the PPD module and the reason for loss of the complex from this economically significant family is unclear, given that grasses were the last of the flowering plants to evolve. Bioinformatic analyses identified putative PPD orthologues in close relatives of the Poaceae, indicating that the explanation for absence of PPD in the grasses may be more complex than previously considered. Understanding the mechanisms which led to loss of PPD from the grasses will provide insight into evolution of the Poaceae.

Overexpression of the Selaginella lepidophylla bHLH transcription factor enhances water-use efficiency, growth, and development in Arabidopsis.

Abiotic stresses have the greatest impact on the growth and productivity of crops, especially under current and future extreme weather events due to climate change. Thus, it is vital to explore novel strategies to improve crop plant abiotic stress tolerance to feed an ever-growing world population. Selaginella lepidophylla is a desiccation-tolerant spike moss with specialized adaptations that allow it to tolerate water loss down to 4% relative water content. A candidate basic helix-loop-helix (bHLH) transcription factor was highly expressed at 4% relative water content in S. lepidophylla (SlbHLH). This SlbHLH gene was codon-optimized (SlbHLHopt) and overexpressed in Arabidopsis for functional characterization. Overexpression of the SlbHLHopt gene not only significantly increased plant growth, development, and integrated water-use efficiency, but also significantly increased seed germination and green cotyledon emergence rates under water-deficit stress and salt stress conditions. Under a 150 mM NaCl salt stress condition, SlbHLHopt-overexpressing lines increased primary root length, the number of lateral roots, and fresh and dry biomass at the seedling stage compared to control lines. Interestingly, SlbHLHopt-overexpressing lines also have significantly higher flavonoid content. Altogether, these results suggest that SlbHLH functions as an important regulator of plant growth, development, abiotic stress tolerance, and water-use efficiency.

Plastome structure, evolution, and phylogeny of Selaginella.

As one of the earliest land plant lineages, Selaginella is important for studying land plant evolution. It is the largest genus of lycophytes containing 700-800 species. Some unique characters of Selaginella plastomes have been reported, but based only on 20 species. There have been no plastome phylogenies of Selaginella based on a relatively large sampling, and no efforts have been made to resolve the phylogeny of the enigmatic Sinensis group whose relationships have been unclear based on small datasets. Here we investigated the structures of 59 plastomes representing 51 species covering all six subgenera and 18 sections of Selaginella except two sections and including the intriguing Sinensis group for the first time. Our major results include: (1) the plastome size of Selaginella ranges tremendously from 78,492 bp to 187,632 bp; (2) there are numerous gene losses in Selaginella comparing with other lycophytes, Isoëtaceae and Lycopodiaceae; (3) the gene contents and plastome structures in Selaginella vary lineage-specifically and all infrageneric taxa are well supported in the plastome phylogeny; (4) the ndh gene family tends to lose or pseudogenize in those species with DR structure and without other short or medium repeats; (5) the short and medium repeat regions in SC mediate many conformations causing diverse and complex plastome structures, and six new conformations are discovered; (6) forty-eight species sampled have high GC content (>50%) but three species in the Sinensis group have âˆ¼ 30% GC content in plastomes, similar to most vascular plants; (7) the Sinensis group is monophyletic, includes at least two subgroups, and has the smallest plastomes in land plants except some parasitic plants, and their plastomes do not contain any tRNAs; (8) the younger lineages in Selaginella tend to have higher GC content, whereas the older lineages tend to have lower GC content; and (9) because of incomplete genomic data and abnormal structures or some unknown reasons, even the concatenated plastomes could not well resolve the phylogenetic relationships in Selaginella with confidence, highlighting the difficulty in resolving the phylogeny and evolution of this particularly important land plant lineage.

Identification of resurrection genes from the transcriptome of dehydrated and rehydrated Selaginella tamariscina.

Selaginella tamariscina is a lycophyta species that survives under extremely dry conditions via the mechanism of resurrection. This phenomenon involves the regulation of numerous genes that play vital roles in desiccation tolerance and subsequent rehydration. To identify resurrection-related genes, we analyzed the transcriptome between dehydration conditions and rehydration conditions of S. tamariscina. The de novo assembly generated 124,417 transcripts with an average size of 1,000 bp and 87,754 unigenes. Among these genes, 1,267 genes and 634 genes were up and down regulated by rehydration compared to dehydration. To understand gene function, we annotated Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The unigenes encoding early light-inducible protein (ELIP) were down-regulated, whereas pentatricopeptide repeat-containing protein (PPR), late embryogenesis abundant proteins (LEA), sucrose nonfermenting protein (SNF), trehalose phosphate phosphatase (TPP), trehalose phosphate synthase (TPS), and ABC transporter G family (ABCG) were significantly up-regulated in response to rehydration conditions by differentially expressed genes (DEGs) analysis. Several studies provide evidence that these genes play a role in stress environment. The ELIP and PPR genes are involved in chloroplast protection during dehydration and rehydration. LEA, SNF, and trehalose genes are known to be oxidant scavengers that protect the cell structure from the deleterious effect of drought. TPP and TPS genes were found in the starch and sucrose metabolism pathways, which are essential sugar-signaling metabolites regulating plant metabolism and other biological processes. ABC-G gene interacts with abscisic acid (ABA) phytohormone in the stomata opening during stress conditions. Our findings provide valuable information and candidate resurrection genes for future functional analysis aimed at improving the drought tolerance of crop plants.

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.

Plastid Genomes of the Early Vascular Plant Genus Selaginella Have Unusual Direct Repeat Structures and Drastically Reduced Gene Numbers.

The early vascular plants in the genus Selaginella, which is the sole genus of the Selaginellaceae family, have an important place in evolutionary history, along with ferns, as such plants are valuable resources for deciphering plant evolution. In this study, we sequenced and assembled the plastid genome (plastome) sequences of two Selaginella tamariscina individuals, as well as Selaginella stauntoniana and Selaginella involvens. Unlike the inverted repeat (IR) structures typically found in plant plastomes, Selaginella species had direct repeat (DR) structures, which were confirmed by Oxford Nanopore long-read sequence assembly. Comparative analyses of 19 lycophytes, including two Huperzia and one Isoetes species, revealed unique phylogenetic relationships between Selaginella species and related lycophytes, reflected by structural rearrangements involving two rounds of large inversions that resulted in dynamic changes between IR and DR blocks in the plastome sequence. Furthermore, we present other uncommon characteristics, including a small genome size, drastic reductions in gene and intron numbers, a high GC content, and extensive RNA editing. Although the 16 Selaginella species examined may not fully represent the genus, our findings suggest that Selaginella plastomes have undergone unique evolutionary events yielding genomic features unparalleled in other lycophytes, ferns, or seed plants.

Biochemical Characterization and Function of Eight Microbial Type Terpene Synthases from Lycophyte Selaginella moellendorffii.

Selaginella moellendorffii is a lycophyte, a member of an ancient vascular plant lineage. Two distinct types of terpene synthase (TPS) genes were identified from this species, including S. moellendorffii TPS genes (SmTPSs) and S. moellendorffii microbial TPS-like genes (SmMTPSLs). The goal of this study was to investigate the biochemical functions of SmMTPSLs. Here, eight full-length SmMTPSL genes (SmMTPSL5, -15, -19, -23, -33, -37, -46, and -47) were functionally characterized from S. moellendorffii. Escherichia coli-expressed recombinant SmMTPSLs were tested for monoterpenes synthase and sesquiterpenes synthase activities. These enzymatic products were typical monoterpenes and sesquiterpenes that have been previous shown to be generated by typical plant TPSs when provided with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as the substrates. Meanwhile, SmMTPSL23, -33, and -37 were up-regulated when induced by alamethicin (ALA) and methyl jasmonate (MeJA), suggesting a role for these genes in plants response to abiotic stresses. Furthermore, this study pointed out that the terpenoids products of SmMTPSL23, -33, and -37 have an antibacterial effect on Pseudomonas syringae pv. tomato DC3000 and Staphylococcus aureus. Taken together, these results provide more information about the catalytic and biochemical function of SmMTPSLs in S. moellendorffii plants.

A Synthetic Approach to Reconstruct the Evolutionary and Functional Innovations of the Plant Histone Variant H2A.W.

Diversification of histone variants is marked by the acquisition of distinct motifs and functional properties through convergent evolution.1-4 H2A variants are distinguished by specific C-terminal motifs and tend to be segregated within defined domains of the genome.5,6 Whether evolution of these motifs pre-dated the evolution of segregation mechanisms or vice versa has remained unclear. A suitable model to address this question is the variant H2A.W, which evolved in plants through acquisition of a KSPK motif7 and is tightly associated with heterochromatin.4 We used fission yeast, where chromatin is naturally devoid of H2A.W, to study the impact of engineered chimeras combining yeast H2A with the KSPK motif. Biochemical assays showed that the KSPK motif conferred nucleosomes with specific properties. Despite uniform incorporation of the engineered H2A chimeras in the yeast genome, the KSPK motif specifically affected heterochromatin composition and function. We conclude that the KSPK motif promotes chromatin properties in yeast that are comparable to the properties and function of H2A.W in plant heterochromatin. We propose that the selection of functional motifs confer histone variants with properties that impact primarily a specific chromatin state. The association between a new histone variant and a preferred chromatin state can thus provide a setting for the evolution of mechanisms that segregate the new variant to this state, thereby enhancing the impact of the selected properties of the variant on genome activity.

Paleo-polyploidization in Lycophytes.

Lycophytes and seed plants constitute the typical vascular plants. Lycophytes have been thought to have no paleo-polyploidization although the event is known to be critical for the fast expansion of seed plants. Here, genomic analyses including the homologous gene dot plot analysis detected multiple paleo-polyploidization events, with one occurring approximately 13-15 million years ago (MYA) and another about 125-142 MYA, during the evolution of the genome of Selaginella moellendorffii, a model lycophyte. In addition, comparative analysis of reconstructed ancestral genomes of lycophytes and angiosperms suggested that lycophytes were affected by more paleo-polyploidization events than seed plants. Results from the present genomic analyses indicate that paleo-polyploidization has contributed to the successful establishment of both lineages-lycophytes and seed plants-of vascular plants.

Distinctive evolutionary pattern of organelle genomes linked to the nuclear genome in Selaginellaceae.

Plastids and mitochondria are endosymbiotic organelles that store genetic information. The genomes of these organelles generally exhibit contrasting patterns regarding genome architecture and genetic content. However, they have similar genetic features in Selaginellaceae, and little is known about what causes parallel evolution. Here, we document the multipartite plastid genomes (plastomes) and the highly divergent mitochondrial genomes (mitogenomes) from spikemoss obtained by combining short- and long-reads. The 188-kb multipartite plastome has three ribosomal operon copies in the master genomic conformation, creating the alternative subgenomic conformation composed of 110- and 78-kb subgenomes. The long-read data indicated that the two different genomic conformations were present in almost equal proportions in the plastomes of Selaginella nipponica. The mitogenome of S. nipponica was assembled into 27 contigs with a total size of 110 kb. All contigs contained directly arranged repeats at both ends, which introduced multiple conformations. Our results showed that plastomes and mitogenomes share high tRNA losses, GC-biased nucleotides, elevated substitution rates and complicated organization. The exploration of nuclear-encoded organelle DNA replication, recombination and repair proteins indicated that, several single-targeted proteins, particularly plastid-targeted recombinase A1, have been lost in Selaginellaceae; conversely, the dual-targeted proteins remain intact. According to the reported function of recombinase A1, we propose that the plastomes of spikemoss often fail to pair homologous sequences during recombination, and the dual-targeted proteins play a key role in the convergent genetic features of plastomes and mitogenomes. Our results provide a distinctive evolutionary pattern of the organelle genomes in Selaginellaceae and evidence of their convergent evolution.

Comparative transcriptome analysis suggests convergent evolution of desiccation tolerance in Selaginella species.

BACKGROUND: Desiccation tolerant Selaginella species evolved to survive extreme environmental conditions. Studies to determine the mechanisms involved in the acquisition of desiccation tolerance (DT) have focused on only a few Selaginella species. Due to the large diversity in morphology and the wide range of responses to desiccation within the genus, the understanding of the molecular basis of DT in Selaginella species is still limited. RESULTS: Here we present a reference transcriptome for the desiccation tolerant species S. sellowii and the desiccation sensitive species S. denticulata. The analysis also included transcriptome data for the well-studied S. lepidophylla (desiccation tolerant), in order to identify DT mechanisms that are independent of morphological adaptations. We used a comparative approach to discriminate between DT responses and the common water loss response in Selaginella species. Predicted proteomes show strong homology, but most of the desiccation responsive genes differ between species. Despite such differences, functional analysis revealed that tolerant species with different morphologies employ similar mechanisms to survive desiccation. Significant functions involved in DT and shared by both tolerant species included induction of antioxidant systems, amino acid and secondary metabolism, whereas species-specific responses included cell wall modification and carbohydrate metabolism. CONCLUSIONS: Reference transcriptomes generated in this work represent a valuable resource to study Selaginella biology and plant evolution in relation to DT. Our results provide evidence of convergent evolution of S. sellowii and S. lepidophylla due to the different gene sets that underwent selection to acquire DT.