The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.

GRAS transcription factors regulate cell division planes in moss overriding the default rule.

Plant cells are surrounded by a cell wall and do not migrate, which makes the regulation of cell division orientation crucial for development. Regulatory mechanisms controlling cell division orientation may have contributed to the evolution of body organization in land plants. The GRAS family of transcription factors was transferred horizontally from soil bacteria to an algal common ancestor of land plants. SHORTROOT (SHR) and SCARECROW (SCR) genes in this family regulate formative periclinal cell divisions in the roots of flowering plants, but their roles in nonflowering plants and their evolution have not been studied in relation to body organization. Here, we show that SHR cell autonomously inhibits formative periclinal cell divisions indispensable for leaf vein formation in the moss Physcomitrium patens, and SHR expression is positively and negatively regulated by SCR and the GRAS member LATERAL SUPPRESSOR, respectively. While precursor cells of a leaf vein lacking SHR usually follow the geometry rule of dividing along the division plane with the minimum surface area, SHR overrides this rule and forces cells to divide nonpericlinally. Together, these results imply that these bacterially derived GRAS transcription factors were involved in the establishment of the genetic regulatory networks modulating cell division orientation in the common ancestor of land plants and were later adapted to function in flowering plant and moss lineages for their specific body organizations.

Odd-Paired is Involved in Morphological Divergence of Snail-Feeding Beetles.

Body shape and size diversity and their evolutionary rates correlate with species richness at the macroevolutionary scale. However, the molecular genetic mechanisms underlying the morphological diversification across related species are poorly understood. In beetles, which account for one-fourth of the known species, adaptation to different trophic niches through morphological diversification appears to have contributed to species radiation. Here, we explored the key genes for the morphological divergence of the slender to stout body shape related to divergent feeding methods on large to small snails within the genus Carabus. We show that the zinc-finger transcription factor encoded by odd-paired (opa) controls morphological variation in the snail-feeding ground beetle Carabus blaptoides. Specifically, opa was identified as the gene underlying the slender to stout morphological difference between subspecies through genetic mapping and functional analysis via gene knockdown. Further analyses revealed that changes in opa cis-regulatory sequences likely contributed to the differences in body shape and size between C. blaptoides subspecies. Among opa cis-regulatory sequences, single nucleotide polymorphisms on the transcription factor binding sites may be associated with the morphological differences between C. blaptoides subspecies. opa was highly conserved in a wide range of taxa, especially in beetles. Therefore, opa may play an important role in adaptive morphological divergence in beetles.

Discovery of ultrafast myosin, its amino acid sequence, and structural features.

Cytoplasmic streaming with extremely high velocity (∼70 µm s-1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by myosin XI, it has been suggested that a myosin XI with a velocity of 70 µm s-1, the fastest myosin measured so far, exists in Chara cells. However, the velocity of the previously cloned Chara corallina myosin XI (CcXI) was about 20 µm s-1, one-third of the cytoplasmic streaming velocity in Chara Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. We cloned these four myosin XI (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 motor domains (MDs) were similar to that of CcXI MD, the velocities of CbXI-1 and CbXI-2 MDs were 3.2 times and 2.8 times faster than that of CcXI MD, respectively. The velocity of chimeric CbXI-1, a functional, full-length CbXI-1 construct, was 60 µm s-1 These results suggest that CbXI-1 and CbXI-2 would be the main contributors to cytoplasmic streaming in Chara cells and show that these myosins are ultrafast myosins with a velocity 10 times faster than fast skeletal muscle myosins in animals. We also report an atomic structure (2.8-Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-electron microscopy structure of acto-myosin XI at low resolution (4.3-Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loops support this hypothesis.

Diversity of genome size and chromosome number in homothallic and heterothallic strains of the Closterium peracerosum-strigosum-littorale complex (Desmidiales, Zygnematophyceae, Streptophyta).

The evolutionary transitions of mating systems between outcrossing and self-fertilization are often suggested to associate with the cytological and genomic changes, but the empirical reports are limited in multicellular organisms. Here we used the unicellular zygnematophycean algae, the Closterium peracerosum-strigosum-littorale (C. psl.) complex, to address whether genomic properties such as genome sizes and chromosome numbers are associated with mating system transitions between homothallism (self-fertility) and heterothallism (self-sterility). Phylogenetic analysis revealed the polyphyly of homothallic strains, suggesting multiple transitions between homothallism and heterothallism in the C. psl. complex. Flow cytometry analysis identified a more than 2-fold genome size variation, ranging from 0.53 to 1.42 Gbp, which was positively correlated with chromosome number variation between strains. Although we did not find consistent trends in genome size change and mating system transitions, the mean chromosome sizes tend to be smaller in homothallic strains than in their relative heterothallic strains. This result suggests that homothallic strains possibly have more fragmented chromosomes, which is consistent with the argument that self-fertilizing populations may tolerate more chromosomal rearrangements.

A divergent RWP-RK transcription factor determines mating type in heterothallic Closterium.

The Closterium peracerosum-strigosum-littorale complex (Closterium, Zygnematophyceae) has an isogamous mating system. Members of the Zygnematophyceae are the closest relatives to extant land plants and are distantly related to chlorophytic models, for which a genetic basis of mating type (MT) determination has been reported. We thus investigated MT determination in Closterium. We sequenced genomes representing the two MTs, mt+ and mt-, in Closterium and identified CpMinus1, a gene linked to the mt- phenotype. We analyzed its function using reverse genetics methods. CpMinus1 encodes a divergent RWP-RK domain-containing-like transcription factor and is specifically expressed during gamete differentiation. Introduction of CpMinus1 into an mt+ strain was sufficient to convert it to a phenotypically mt- strain, while CpMinus1-knockout mt- strains were phenotypically mt+. We propose that CpMinus1 is the major MT determinant that acts by evoking the mt- phenotype and suppressing the mt+ phenotype in heterothallic Closterium. CpMinus1 likely evolved independently in the Zygnematophyceae lineage, which lost an egg-sperm anisogamous mating system. mt- specific regions possibly constitute an MT locus flanked by common sequences that undergo some recombination.

Upregulation of the Long Noncoding RNA HULC by Hepatitis C Virus and Its Regulation of Viral Replication.

BACKGROUND: Many long noncoding RNAs (lncRNAs) have important roles in biological processes. The lncRNA HULC was found to be upregulated in human hepatoma tissues. HULC is thought to be involved in multiple steps of hepatoma development and progression; however, the relationship between HULC and hepatitis C virus (HCV) infection, which is a leading cause of hepatoma, remains unclear. METHODS: We examined the effect of HCV replication on HULC expression and the underlying mechanism using cell culture systems. Subsequently, we tested the effect of HULC suppression and overexpression on HCV replication. Finally, we examined the impact of HCV eradication on HULC expression using human liver tissue and blood samples. RESULTS: HCV replication increased HULC expression in cell cultures. A promoter assay showed that an HCV nonstructural protein, NS5A, increased HULC transcription. HULC suppression inhibited HCV replication; conversely, its overexpression enhanced HCV replication. These effects on HCV replication seemed to occur by the modification of HCV translation. Measurements from human liver and blood samples showed that HCV eradication significantly reduced HULC levels in the liver and blood. CONCLUSIONS: HCV infection increases HULC expression in vitro and in vivo. HULC modulates HCV replication through an HCV internal ribosome entry site-directed translation step.

Phylogenetic distribution and expression pattern analyses identified a divergent basal body assembly protein involved in land plant spermatogenesis.

Land plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is a continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning spermatogenesis remain to be elucidated. We successfully identified candidate genes involved in spermatogenesis, deeply divergent BLD10s, by computational analyses combining multiple methods and omics data. We then examined the functions of BLD10s in the liverwort Marchantia polymorpha and the moss Physcomitrium patens. MpBLD10 and PpBLD10 are required for normal basal body (BB) and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, and thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. We identified orthologs of MpBLD10 and PpBLD10 in diverse Streptophyta and found that MpBLD10 and PpBLD10 are orthologous to BLD10/CEP135 family proteins, which function in BB assembly. However, BLD10s evolved especially quickly in land plants and MpBLD10 might have acquired additional functions in spermatozoid formation through rapid molecular evolution.

The complete plastid genome sequence of the enigmatic moss, Takakia lepidozioides (Takakiopsida, Bryophyta): evolutionary perspectives on the largest collection of genes in mosses and the intensive RNA editing.

KEY MESSAGE: Complete chloroplast genome sequence of a moss, Takakia lepidozioides (Takakiopsida) is reported. The largest collection of genes in mosses and the intensive RNA editing were discussed from evolutionary perspectives. We assembled the entire plastid genome sequence of Takakia lepidozioides (Takakiopsida), emerging from the first phylogenetic split among extant mosses. The genome sequences were assembled into a circular molecule 149,016 bp in length, with a quadripartite structure comprising a large and a small single-copy region separated by inverted repeats. It contained 88 genes coding for proteins, 32 for tRNA, four for rRNA, two open reading frames, and at least one pseudogene (tufA). This is the largest number of genes of all sequenced plastid genomes in mosses and Takakia is the only moss that retains the seven coding genes ccsA, cysA, cysT, petN rpoA, rps16 and trnPGGG. Parsimonious interpretation of gene loss suggests that the last common ancestor of bryophytes had all seven genes and that mosses lost at least three of them during their diversification. Analyses of the plastid transcriptome identified the extraordinary frequency of RNA editing with more than 1100 sites. We indicated a close correlation between the monoplastidy of vegetative tissue and the intensive RNA editing sites in the plastid genome in land plant lineages. Here, we proposed a hypothesis that the small population size of plastids in each vegetative cell of some early diverging land plants, including Takakia, might cause the frequent fixation of mutations in plastid genome through the intracellular genetic drift and that deleterious mutations might be continuously compensated by RNA editing during or following transcription.

An Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis.

Despite their key phylogenetic position and their unique biology, hornworts have been widely overlooked. Until recently there was no hornwort model species amenable to systematic experimental investigation. Anthoceros agrestis has been proposed as the model species to study hornwort biology. We have developed an Agrobacterium-mediated method for the stable transformation of A. agrestis, a hornwort model species for which a genetic manipulation technique was not yet available. High transformation efficiency was achieved by using thallus tissue grown under low light conditions. We generated a total of 274 transgenic A. agrestis lines expressing the ß-glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing and land plant evolution in general.

Single-cell transcriptome analysis of Physcomitrella leaf cells during reprogramming using microcapillary manipulation.

Next-generation sequencing technologies have made it possible to carry out transcriptome analysis at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) data provide insights into cellular dynamics, including intercellular heterogeneity as well as inter- and intra-cellular fluctuations in gene expression that cannot be studied using populations of cells. The utilization of scRNA-seq is, however, restricted to cell types that can be isolated from their original tissues, and it can be difficult to obtain precise positional information for these cells in situ. Here, we established single cell-digital gene expression (1cell-DGE), a method of scRNA-seq that uses micromanipulation to extract the contents of individual living cells in intact tissue while recording their positional information. With 1cell-DGE, we could detect differentially expressed genes (DEGs) during the reprogramming of leaf cells of the moss Physcomitrella patens, identifying 6382 DEGs between cells at 0 and 24 h after excision. Furthermore, we identified a subpopulation of reprogramming cells based on their pseudotimes, which were calculated using transcriptome profiles at 24 h. 1cell-DGE with microcapillary manipulation can be used to analyze the gene expression of individual cells without detaching them from their tightly associated tissues, enabling us to retain positional information and investigate cell-cell interactions.

Algal ancestor of land plants was preadapted for symbiosis.

Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

The Polycomb group protein CLF emerges as a specific tri-methylase of H3K27 regulating gene expression and development in Physcomitrella patens.

Packaging of eukaryotic DNA largely depends on histone modifications that affect the accessibility of DNA to transcriptional regulators, thus controlling gene expression. The Polycomb group (PcG) chromatin remodeling complex deposits a methyl group on lysine 27 of histone 3 leading to repressed gene expression. Plants encode homologs of the Enhancer of zeste (E(z)), a component of the PcG complex from Drosophila, one of which is a SET domain protein designated CURLY LEAF (CLF). Although this SET domain protein exhibits a strong correlation with the presence of the H3K27me3 mark in plants, the methyl-transferase activity and specificity of its SET domain have not been directly tested in-vivo. Using the evolutionary early-diverged land plant model species Physcomitrella patens we show that abolishment of a single copy gene PpCLF, as well as an additional member of the PcG complex, FERTILIZATION-INDEPENDENT ENDOSPERM (PpFIE), results in a specific loss of tri-methylation of H3K27. Using site-directed mutagenesis of key residues, we revealed that H3K27 tri-methylation is mediated by the SET domain of the CLF protein. Moreover, the abolishment of H3K27me3 led to enhanced expression of transcription factor genes. This in turn led to the development of fertilization-independent sporophyte-like structures, as observed in PpCLF and PpFIE null mutants. Overall, our results demonstrate the role of PpCLF as a SET protein in tri-methylation of H3K27 in-vivo and the importance of this modification in regulating the expression of transcription factor genes involved in developmental programs of P. patens.

WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens.

Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

Methylome diversification through changes in DNA methyltransferase sequence specificity.

Epigenetic modifications such as DNA methylation have large effects on gene expression and genome maintenance. Helicobacter pylori, a human gastric pathogen, has a large number of DNA methyltransferase genes, with different strains having unique repertoires. Previous genome comparisons suggested that these methyltransferases often change DNA sequence specificity through domain movement--the movement between and within genes of coding sequences of target recognition domains. Using single-molecule real-time sequencing technology, which detects N6-methyladenines and N4-methylcytosines with single-base resolution, we studied methylated DNA sites throughout the H. pylori genome for several closely related strains. Overall, the methylome was highly variable among closely related strains. Hypermethylated regions were found, for example, in rpoB gene for RNA polymerase. We identified DNA sequence motifs for methylation and then assigned each of them to a specific homology group of the target recognition domains in the specificity-determining genes for Type I and other restriction-modification systems. These results supported proposed mechanisms for sequence-specificity changes in DNA methyltransferases. Knocking out one of the Type I specificity genes led to transcriptome changes, which suggested its role in gene expression. These results are consistent with the concept of evolution driven by DNA methylation, in which changes in the methylome lead to changes in the transcriptome and potentially to changes in phenotype, providing targets for natural or artificial selection.

SNARE Molecules in Marchantia polymorpha: Unique and Conserved Features of the Membrane Fusion Machinery.

The membrane trafficking pathway has been diversified in a specific way for each eukaryotic lineage, probably to fulfill specific functions in the organisms. In green plants, comparative genomics has supported the possibility that terrestrialization and/or multicellularization could be associated with the elaboration and diversification of membrane trafficking pathways, which have been accomplished by an expansion of the numbers of genes required for machinery components of membrane trafficking, including soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, information regarding membrane trafficking pathways in basal land plant lineages remains limited. In the present study, we conducted extensive analyses of SNARE molecules, which mediate membrane fusion between target membranes and transport vesicles or donor organelles, in the liverwort, Marchantia polymorpha. The M. polymorpha genome contained at least 34 genes for 36 SNARE proteins, comprising fundamental sets of SNARE proteins that are shared among land plant lineages with low degrees of redundancy. We examined the subcellular distribution of a major portion of these SNARE proteins by expressing Citrine-tagged SNARE proteins in M. polymorpha, and the results showed that some of the SNARE proteins were targeted to different compartments from their orthologous products in Arabidopsis thaliana. For example, MpSYP12B was localized to the surface of the oil body, which is a unique organelle in liverworts. Furthermore, we identified three VAMP72 members with distinctive structural characteristics, whose N-terminal extensions contain consensus sequences for N-myristoylation. These results suggest that M. polymorpha has acquired unique membrane trafficking pathways associated with newly acquired machinery components during evolution.

Genome sequencing of Metrosideros polymorpha (Myrtaceae), a dominant species in various habitats in the Hawaiian Islands with remarkable phenotypic variations.

Whole genome sequences, which can be provided even for non-model organisms owing to high-throughput sequencers, are valuable in enhancing the understanding of adaptive evolution. Metrosideros polymorpha, a tree species endemic to the Hawaiian Islands, occupies a wide range of ecological habitats and shows remarkable polymorphism in phenotypes among/within populations. The biological functions of genetic variations observed within this species could provide significant insights into the adaptive radiation found in a single species. Here de novo assembled genome sequences of M. polymorpha are presented to reveal basic genomic parameters about this species and to develop our knowledge of ecological divergences. The assembly yielded 304-Mbp genome sequences, half of which were covered by 19 scaffolds with >5 Mbp, and contained 30 K protein-coding genes. Demographic history inferred from the genome-wide heterozygosity indicated that this species experienced a dramatic rise and fall in the effective population size, possibly owing to past geographic or climatic changes in the Hawaiian Islands. This M. polymorpha genome assembly represents a high-quality genome resource useful for future functional analyses of both intra- and interspecies genetic variations or comparative genomics.

ppdb: plant promoter database version 3.0.

ppdb (http://ppdb.agr.gifu-u.ac.jp) is a plant promoter database that provides information on transcription start sites (TSSs), core promoter structure (TATA boxes, Initiators, Y Patches, GA and CA elements) and regulatory element groups (REGs) as putative and comprehensive transcriptional regulatory elements. Since the last report in this journal, the database has been updated in three areas to version 3.0. First, new genomes have been included in the database, and now ppdb provides information on Arabidopsis thaliana, rice, Physcomitrella patens and poplar. Second, new TSS tag data (34 million) from A. thaliana, determined by a high throughput sequencer, has been added to give a ∼200-fold increase in TSS data compared with version 1.0. This results in a much higher coverage of ∼27,000 A. thaliana genes and finer positioning of promoters even for genes with low expression levels. Third, microarray data-based predictions have been appended as REG annotations which inform their putative physiological roles.

Characterization of the heterotrimeric G-protein complex and its regulator from the green alga Chara braunii expands the evolutionary breadth of plant G-protein signaling.

The lack of heterotrimeric G-protein homologs in the sequenced genomes of green algae has led to the hypothesis that, in plants, this signaling mechanism coevolved with the embryophytic life cycle and the acquisition of terrestrial habitat. Given the large evolutionary gap that exists between the chlorophyte green algae and most basal land plants, the bryophytes, we evaluated the presence of this signaling complex in a charophyte green alga, Chara braunii, proposed to be the closest living relative of land plants. The C. braunii genome encodes for the entire G-protein complex, the Gα, Gß, and Gγ subunits, and the REGULATOR OF G-PROTEIN SIGNALING (RGS) protein. The biochemical properties of these proteins and their cross-species functionality show that they are functional homologs of canonical G-proteins. The subunit-specific interactions between CbGα and CbGß, CbGß and CbGγ, and CbGα and CbRGS are also conserved, establishing the existence of functional G-protein complex-based signaling mechanisms in green algae.

Physcomitrella cyclin-dependent kinase A links cell cycle reactivation to other cellular changes during reprogramming of leaf cells.

During regeneration, differentiated plant cells can be reprogrammed to produce stem cells, a process that requires coordination of cell cycle reactivation with acquisition of other cellular characteristics. However, the factors that coordinate the two functions during reprogramming have not been determined. Here, we report a link between cell cycle reactivation and the acquisition of new cell-type characteristics through the activity of cyclin-dependent kinase A (CDKA) during reprogramming in the moss Physcomitrella patens. Excised gametophore leaf cells of P. patens are readily reprogrammed, initiate tip growth, and form chloronema apical cells with stem cell characteristics at their first cell division. We found that leaf cells facing the cut undergo CDK activation along with induction of a D-type cyclin, tip growth, and transcriptional activation of protonema-specific genes. A DNA synthesis inhibitor, aphidicolin, inhibited cell cycle progression but prevented neither tip growth nor protonemal gene expression, indicating that cell cycle progression is not required for acquisition of protonema cell-type characteristics. By contrast, treatment with a CDK inhibitor or induction of dominant-negative CDKA;1 protein inhibited not only cell cycle progression but also tip growth and protonemal gene expression. These findings indicate that cell cycle progression is coordinated with other cellular changes by the concomitant regulation through CDKA;1.