Mitsuyasu Hasebe

We report the draft genome sequence of the model moss Physcomitrella patens and compare its features with those of flowering plants, from which it is separated by more than 400 million years, and unicellular aquatic algae. This comparison reveals genomic changes concomitant with the evolutionary movement to land, including a general increase in gene family complexity; loss of genes associated with aquatic environments (e.g., flagellar arms); acquisition of genes for tolerating terrestrial stresses (e.g., variation in temperature and water availability); and the development of the auxin and abscisic acid signaling pathways for coordinating multicellular growth and dehydration response. The Physcomitrella genome provides a resource for phylogenetic inferences about gene function and for experimental analysis of plant processes through this plant's unique facility for reverse genetics.

The genome sequence of a lycophyte hints at ancient evolutionary transitions.

The ASYMMETRIC LEAVES2 (AS2) gene of Arabidopsis thaliana is involved in the establishment of the leaf venation system, which includes the prominent midvein, as well as in the development of a symmetric lamina. The gene product also represses the expression of class 1 knox homeobox genes in leaves. We have characterized the AS2 gene, which appears to encode a novel protein with cysteine repeats (designated the C-motif) and a leucine-zipper-like sequence in the amino-terminal half of the primary sequence. The Arabidopsis genome contains 42 putative genes that potentially encode proteins with conserved amino acid sequences that include the C-motif and the leucine-zipper-like sequence in the amino-terminal half. Thus, the AS2 protein belongs to a novel family of proteins that we have designated the AS2 family. Members of this family except AS2 also have been designated ASLs (AS2-like proteins). Transcripts of AS2 were detected mainly in adaxial domains of cotyledonary primordia. Green fluorescent protein-fused AS2 was concentrated in plant cell nuclei. Overexpression of AS2 cDNA in transgenic Arabidopsis plants resulted in upwardly curled leaves, which differed markedly from the downwardly curled leaves generated by loss-of-function mutation of AS2. Our results suggest that AS2 functions in the transcription of a certain gene(s) in plant nuclei and thereby controls the formation of a symmetric flat leaf lamina and the establishment of a prominent midvein and other patterns of venation.

The mosses and flowering plants diverged >400 million years ago. The mosses have haploid-dominant life cycles, whereas the flowering plants are diploid-dominant. The common ancestors of land plants have been inferred to be haploid-dominant, suggesting that genes used in the diploid body of flowering plants were recruited from the genes used in the haploid body of the ancestors during the evolution of land plants. To assess this evolutionary hypothesis, we constructed an EST library of the moss Physcomitrella patens, and compared the moss transcriptome to the genome of Arabidopsis thaliana. We constructed full-length enriched cDNA libraries from auxin-treated, cytokinin-treated, and untreated gametophytes of P. patens, and sequenced both ends of >40,000 clones. These data, together with the mRNA sequences in the public databases, were assembled into 15,883 putative transcripts. Sequence comparisons of A. thaliana and P. patens showed that at least 66% of the A. thaliana genes had homologues in P. patens. Comparison of the P. patens putative transcripts with all known proteins, revealed 9,907 putative transcripts with high levels of similarity to vascular plant genes, and 850 putative transcripts with high levels of similarity to other organisms. The haploid transcriptome of P. patens appears to be quite similar to the A. thaliana genome, supporting the evolutionary hypothesis. Our study also revealed that a number of genes are moss specific and were lost in the flowering plant lineage.

The development of cells specialized for water conduction or support is a striking innovation of plants that has enabled them to colonize land. The NAC transcription factors regulate the differentiation of these cells in vascular plants. However, the path by which plants with these cells have evolved from their nonvascular ancestors is unclear. We investigated genes of the moss Physcomitrella patens that encode NAC proteins. Loss-of-function mutants formed abnormal water-conducting and supporting cells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of water-conducting-like cells. Our results show conservation of transcriptional regulation and cellular function between moss and Arabidopsis thaliana water-conducting cells. The conserved genetic basis suggests roles for NAC proteins in the adaptation of plants to land.

Pteriodophytes have a longer evolutionary history than any other vascular land plant and, therefore, have endured greater loss of phylogenetically informative information. This factor has resulted in substantial disagreements in evaluating characters and, thus, controversy in establishing a stable classification. To compare competing classifications, we obtained DNA sequences of a chloroplast gene. The sequence of 1206 nt of the large subunit of the ribulose-bisphosphate carboxylase gene (rbcL) was determined from 58 species, representing almost all families of leptosporangiate ferns. Phlogenetic trees were inferred by the neighbor-joining and the parsimony methods. The two methods produced almost identical phylogenetic trees that provided insights concerning major general evolutionary trends in the leptosporangiate ferns. Interesting findings were as follows: (i) two morphologically distinct heterosporous water ferns, Marsilea and Salvinia, are sister genera; (ii) the tree ferns (Cyatheaceae, Dicksoniaceae, and Metaxyaceae) are monophyletic; and (iii) polypodioids are distantly related to the gleichenioids in spite of the similarity of their exindusiate soral morphology and are close to the higher indusiate ferns. In addition, the affinities of several "problematic genera" were assessed.

Characterization of seven MADS-box genes, termed PPM1-PPM4 and PpMADS1-PpMADS3, from the moss model species Physcomitrella patens is reported.Phylogeny reconstructions and comparison of exon-intron structures revealed that the genes described here represent two different classes of homologous, yet distinct, MIKC-type MADS-box genes, termed MIKC c -type genes-'' c '' stands for ''classic''-(PPM1, PPM2, PpMADS1) and MIKC*type genes (PPM3, PPM4, PpMADS2, PpMADS3).The two gene classes deviate from each other in a characteristic way, especially in a sequence stretch termed intervening region.MIKC c -type genes are abundantly present in all land plants which have been investigated in this respect, and give rise to well-known gene types such as floral meristem and organ identity genes.In contrast, LAMB1 from the clubmoss Lycopodium annotinum was identified as the only other MIKC*-type gene published so far.Our findings strongly suggest that the most recent common ancestor of mosses and vascular plants contained at least one MIKC c -type and one MIKC*-type gene.Our studies thus reveal an ancient duplication of an MIKC-type gene that occurred before the separation of the lineages that led to extant mosses and vascular plants more than about 450 MYA.The identification of bona fide K-domains in both MIKC*-type and MIKC c -type proteins suggests that the K-domain is more ancient than is suggested by a recent alternative hypothesis.MIKC*-type genes may have escaped identification in ferns and seed plants so far.It seems more likely, however, that they represent a class of genes which has been lost in the lineage which led to extant ferns and seed plants.The high number of P. patens MADS-box genes and the presence of a K-box in the coding region and of some potential binding sites for MADS-domain proteins and other transcription factors in the putative promoter regions of these genes suggest that MADS-box genes in mosses are involved in complex gene regulatory networks similar to those in flowering plants.

The plant-specific transcription factor LEAFY controls general aspects of the life cycle in a basal plant, the moss Physcomitrella patens. In contrast, LEAFY has more specialized functions in angiosperms, where it specifically induces floral fate during the reproductive phase. This raises the question of a concomitant change in the biochemical function of LEAFY during the evolution of land plants. We report that the DNA binding domain of LEAFY, although largely conserved, has diverged in activity. On the contrary, other, more rapidly evolving portions of the protein have few effects on LEAFY activity.

In rice (Oryza sativa) and Arabidopsis thaliana, gibberellin (GA) signaling is mediated by GIBBERELLIN-INSENSITIVE DWARF1 (GID1) and DELLA proteins in collaboration with a GA-specific F-box protein. To explore when plants evolved the ability to perceive GA by the GID1/DELLA pathway, we examined these GA signaling components in the lycophyte Selaginella moellendorffii and the bryophyte Physcomitrella patens. An in silico search identified several homologs of GID1, DELLA, and GID2, a GA-specific F-box protein in rice, in both species. Sm GID1a and Sm GID1b, GID1 proteins from S. moellendorffii, showed GA binding activity in vitro and interacted with DELLA proteins from S. moellendorffii in a GA-dependent manner in yeast. Introduction of constitutively expressed Sm GID1a, Sm G1D1b, and Sm GID2a transgenes rescued the dwarf phenotype of rice gid1 and gid2 mutants. Furthermore, treatment with GA(4), a major GA in S. moellendorffii, caused downregulation of Sm GID1b, Sm GA20 oxidase, and Sm GA3 oxidase and degradation of the Sm DELLA1 protein. These results demonstrate that the homologs of GID1, DELLA, and GID2 work in a similar manner in S. moellendorffii and in flowering plants. Biochemical studies revealed that Sm GID1s have different GA binding properties from GID1s in flowering plants. No evidence was found for the functional conservation of these genes in P. patens, indicating that GID1/DELLA-mediated GA signaling, if present, differs from that in vascular plants. Our results suggest that GID1/DELLA-mediated GA signaling appeared after the divergence of vascular plants from the moss lineage.

The AP2 (APETALA2)/EREBP (Ethylene Responsive Element Binding Protein) multigene family includes developmentally and physiologically important transcription factors. AP2/EREBP genes are divided into two subfamilies: AP2 genes with two AP2 domains and EREBP genes with a single AP2/ERF (Ethylene Responsive Element Binding Factor) domain. Based on previous phylogenetic analyses, AP2 genes can be divided into two clades, AP2 and ANT groups. To clarify the molecular evolution of the AP2 subfamily, we isolated and sequenced genes with two AP2 domains from three gymnosperms, Cycas revoluta, Ginkgo biloba, and Gnetum parvifolium,as well as from the moss Physcomitrella patens. Expressions of AP2-like genes, including AP2, in Arabidopsis thaliana are regulated by the microRNA miR172. We found that the target site of miR172 is significantly conserved in gymnosperm AP2 homologs, suggesting that regulatory mechanisms of gene expression using microRNA have been conserved over the three hundred million years since the divergence of gymnosperm and flowering plant lineages. We inferred a phylogenetic relationship of these genes with the green alga Chlamydomonas reinhardtii and seed–plant genes available in public DNA databases. The phylogenetic tree showed that the AP2 subfamily diverged into the AP2 and ANT groups before the last common ancestor of land plants and after C. reinhardtii diverged from the land-plant lineage. The tree also indicated that each AP2 and ANT group further diverged into several clades through gene duplications prior to the divergence of gymnosperms and angiosperms.

Although the number and form of metazoan organs are determined in the embryo, plants continuously form organs via pluripotent stem cells contained within the meristem. Flowering plants have an indeterminate meristem in their diploid generation, whereas the common ancestor of land plants is inferred to have formed an indeterminate meristem in its haploid generation, as observed in the extant basal land plants, bryophytes, including mosses. It is hypothesized that the underlying gene networks for the diploid meristem were initially present in the haploid generation of the basal land plants and were eventually co-opted for expression in the diploid generation. In flowering plants, the class 1 KNOTTED1-LIKE HOMEOBOX (KNOX) transcription factors are essential for the function of the indeterminate apical meristem. Here, we show that the class 1 KNOX orthologs function in the diploid organ, with determinate growth in the moss Physcomitrella patens, but do not function in the haploid indeterminate meristem. We propose that the genetic networks governing the indeterminate meristem in land plants are variable, and the networks governing the diploid indeterminate meristem with the class 1 KNOX genes likely evolved de novo in the flowering plant lineage.

The moss Physcomitrella patens as a model species provides an important reference for early-diverging lineages of plants and the release of the genome in 2008 opened the doors to genome-wide studies. The usability of a reference genome greatly depends on the quality of the annotation and the availability of centralized community resources. Therefore, in the light of accumulating evidence for missing genes, fragmentary gene structures, false annotations and a low rate of functional annotations on the original release, we decided to improve the moss genome annotation.Here, we report the complete moss genome re-annotation (designated V1.6) incorporating the increased transcript availability from a multitude of developmental stages and tissue types. We demonstrate the utility of the improved P. patens genome annotation for comparative genomics and new extensions to the cosmoss.org resource as a central repository for this plant "flagship" genome. The structural annotation of 32,275 protein-coding genes results in 8387 additional loci including 1456 loci with known protein domains or homologs in Plantae. This is the first release to include information on transcript isoforms, suggesting alternative splicing events for at least 10.8% of the loci. Furthermore, this release now also provides information on non-protein-coding loci. Functional annotations were improved regarding quality and coverage, resulting in 58% annotated loci (previously: 41%) that comprise also 7200 additional loci with GO annotations. Access and manual curation of the functional and structural genome annotation is provided via the http://www.cosmoss.org model organism database.Comparative analysis of gene structure evolution along the green plant lineage provides novel insights, such as a comparatively high number of loci with 5'-UTR introns in the moss. Comparative analysis of functional annotations reveals expansions of moss house-keeping and metabolic genes and further possibly adaptive, lineage-specific expansions and gains including at least 13% orphan genes.

Land plants have distinct developmental programs in haploid (gametophyte) and diploid (sporophyte) generations. Although usually the two programs strictly alternate at fertilization and meiosis, one program can be induced during the other program. In a process called apogamy, cells of the gametophyte other than the egg cell initiate sporophyte development. Here, we report for the moss Physcomitrella patens that apogamy resulted from deletion of the gene orthologous to the Arabidopsis thaliana CURLY LEAF ( PpCLF ), which encodes a component of polycomb repressive complex 2 (PRC2). In the deletion lines, a gametophytic vegetative cell frequently gave rise to a sporophyte-like body. This body grew indeterminately from an apical cell with the character of a sporophytic pluripotent stem cell but did not form a sporangium. Furthermore, with continued culture, the sporophyte-like body branched. Sporophyte branching is almost unknown among extant bryophytes. When PpCLF was expressed in the deletion lines once the sporophyte-like bodies had formed, pluripotent stem cell activity was arrested and a sporangium-like organ formed. Supported by the observed pattern of PpCLF expression, these results demonstrate that, in the gametophyte, PpCLF represses initiation of a sporophytic pluripotent stem cell and, in the sporophyte, represses that stem cell activity and induces reproductive organ development. In land plants, branching, along with indeterminate apical growth and delayed initiation of spore-bearing reproductive organs, were conspicuous innovations for the evolution of a dominant sporophyte plant body. Our study provides insights into the role of PRC2 gene regulation for sustaining evolutionary innovation in land plants.

A Mossy Veil Land plants have two distinct generations: a haploid gametophyte responsible for producing the gametes, and a diploid sporophyte, that in most land plants is the dominant form observed. However, in mosses, a basal land plant, the primary biomass is composed of the haploid gametophyte. Sakakibara et al. (p. 1067 ; see the Perspective by Friedman ) analyzed loss-of-function mutants of KNOX2 (class 2 KNOTTED1-LIKE HOMEOBOX) genes in the moss Physcomitrella patens. Mutant plants exhibit apospory, where fertilization apparently occurs, but the normal sporophyte phase is bypassed and instead a diploid structure resembling a haploid gametophyte is produced. The results suggest that KNOX2 regulates the alternation of generations by suppressing the haploid body plan in the diploid phase.

Anhydrobiosis represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. Here we report the first comparative analysis examining the genomic background of extreme desiccation tolerance, which is exclusively found in larvae of the only anhydrobiotic insect, Polypedilum vanderplanki. We compare the genomes of P. vanderplanki and a congeneric desiccation-sensitive midge P. nubifer. We determine that the genome of the anhydrobiotic species specifically contains clusters of multi-copy genes with products that act as molecular shields. In addition, the genome possesses several groups of genes with high similarity to known protective proteins. However, these genes are located in distinct paralogous clusters in the genome apart from the classical orthologues of the corresponding genes shared by both chironomids and other insects. The transcripts of these clustered paralogues contribute to a large majority of the mRNA pool in the desiccating larvae and most likely define successful anhydrobiosis. Comparison of expression patterns of orthologues between two chironomid species provides evidence for the existence of desiccation-specific gene expression systems in P. vanderplanki. The African chironomid midge, Polypedilum vanderplanki, is able to withstand extreme desiccation. Here the authors sequence the genomes of a desiccation-tolerant and desiccation-sensitive species of chironomid midge and pinpoint genes that may have a role in conferring resistance to desiccation.

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.

Stem cells are formed at particular times and positions during the development of multicellular organisms. Whereas flowering plants form stem cells only in the sporophyte generation, non-seed plants form stem cells in both the sporophyte and gametophyte generations. Although the molecular mechanisms underlying stem cell formation in the sporophyte generation have been extensively studied, only a few transcription factors involved in the regulation of gametophyte stem cell formation have been reported. The moss Physcomitrella patens forms a hypha-like body (protonema) and a shoot-like body (gametophore) from a protonema apical cell and a gametophore apical cell, respectively. These apical cells have stem cell characteristics and are formed as side branches of differentiated protonema cells. Here, we show that four AP2-type transcription factors orthologous to Arabidopsis thaliana AINTEGUMENTA, PLETHORA and BABY BOOM (APB) are indispensable for the formation of gametophore apical cells from protonema cells. Quadruple disruption of all APB genes blocked gametophore formation, even in the presence of cytokinin, which enhances gametophore apical cell formation in the wild type. All APB genes were expressed in emerging gametophore apical cells, but not in protonema apical cells. Heat-shock induction of an APB4 transgene driven by a heat-shock promoter increased the number of gametophores. Expression of all APB genes was induced by auxin but not by cytokinin. Thus, the APB genes function synergistically with cytokinin signaling to determine the identity of the two types of stem cells.

Carnivorous plants exploit animals as a nutritional source and have inspired long-standing questions about the origin and evolution of carnivory-related traits. To investigate the molecular bases of carnivory, we sequenced the genome of the heterophyllous pitcher plant Cephalotus follicularis, in which we succeeded in regulating the developmental switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types and gene repertoire analysis identified genetic changes associated with prey attraction, capture, digestion and nutrient absorption. Analysis of digestive fluid proteins from C. follicularis and three other carnivorous plants with independent carnivorous origins revealed repeated co-options of stress-responsive protein lineages coupled with convergent amino acid substitutions to acquire digestive physiology. These results imply constraints on the available routes to evolve plant carnivory.

Stem cells self-renew and produce cells that differentiate to become the source of the plant body. The moss Physcomitrella patens forms eight types of stem cells during its life cycle and serves as a useful model in which to explore the evolution of such cells. The common ancestor of land plants is inferred to have been haplontic and to have formed stem cells only in the gametophyte generation. A single stem cell would have been maintained in the ancestral gametophyte meristem, as occurs in extant basal land plants. During land plant evolution, stem cells diverged in the gametophyte generation to form different types of body parts, including the protonema and rhizoid filaments, leafy-shoot and thalloid gametophores, and gametangia formed in moss. A simplex meristem with a single stem cell was acquired in the sporophyte generation early in land plant evolution. Subsequently, sporophyte stem cells became multiple in the meristem and were elaborated further in seed plant lineages, although the evolutionary origin of niche cells, which maintain stem cells is unknown. Comparisons of gene regulatory networks are expected to give insights into the general mechanisms of stem cell formation and maintenance in land plants and provide information about their evolution. P. patens develops at least seven types of simplex meristem in the gametophyte and at least one type in the sporophyte generation and is a good material for regulatory network comparisons. In this review, we summarize recently revealed molecular mechanisms of stem cell initiation and maintenance in the moss.

In land plants, the cell plate partitions the daughter cells at cytokinesis. The cell plate initially forms between daughter nuclei and expands centrifugally until reaching the plasma membrane. The centrifugal development of the cell plate is driven by the centrifugal expansion of the phragmoplast microtubule array, but the molecular mechanism underlying this expansion is unknown. Here, we show that the phragmoplast array comprises stable microtubule bundles and dynamic microtubules. We find that the dynamic microtubules are nucleated by γ-tubulin on stable bundles. The dynamic microtubules elongate at the plus ends and form new bundles preferentially at the leading edge of the phragmoplast. At the same time, they are moved away from the cell plate, maintaining a restricted distribution of minus ends. We propose that cycles of attachment of γ-tubulin complexes onto the microtubule bundles, microtubule nucleation and bundling, accompanied by minus-end-directed motility, drive the centrifugal development of the phragmoplast. Plant cell division is driven by the expansion of the phragmoplast, a characteristic structure that forms in the middle of the plant cell during cytokinesis. Murata et al. use genetic and cell imaging approaches to clarify the microtubule behaviour that leads to phragmoplast expansion.

Cell-to-cell communication is a fundamental mechanism for coordinating developmental and physiological events in multicellular organisms. Heterotrimeric G proteins are key molecules that transmit extracellular signals; similarly, CLAVATA signaling is a crucial regulator in plant development. Here, we show that Arabidopsis thaliana Gβ mutants exhibit an enlarged stem cell region, which is similar to that of clavata mutants. Our genetic and cell biological analyses suggest that the G protein beta-subunit1 AGB1 and RPK2, one of the major CLV3 peptide hormone receptors, work synergistically in stem cell homeostasis through their physical interactions. We propose that AGB1 and RPK2 compose a signaling module to facilitate meristem development.

Sex chromosomes turn over rapidly in some taxonomic groups, where closely related species have different sex chromosomes. Although there are many examples of sex chromosome turnover, we know little about the functional roles of sex chromosome turnover in phenotypic diversification and genomic evolution. The sympatric pair of Japanese threespine stickleback (Gasterosteus aculeatus) provides an excellent system to address these questions: the Japan Sea species has a neo-sex chromosome system resulting from a fusion between an ancestral Y chromosome and an autosome, while the sympatric Pacific Ocean species has a simple XY sex chromosome system. Furthermore, previous quantitative trait locus (QTL) mapping demonstrated that the Japan Sea neo-X chromosome contributes to phenotypic divergence and reproductive isolation between these sympatric species. To investigate the genomic basis for the accumulation of genes important for speciation on the neo-X chromosome, we conducted whole genome sequencing of males and females of both the Japan Sea and the Pacific Ocean species. No substantial degeneration has yet occurred on the neo-Y chromosome, but the nucleotide sequence of the neo-X and the neo-Y has started to diverge, particularly at regions near the fusion. The neo-sex chromosomes also harbor an excess of genes with sex-biased expression. Furthermore, genes on the neo-X chromosome showed higher non-synonymous substitution rates than autosomal genes in the Japan Sea lineage. Genomic regions of higher sequence divergence between species, genes with divergent expression between species, and QTL for inter-species phenotypic differences were found not only at the regions near the fusion site, but also at other regions along the neo-X chromosome. Neo-sex chromosomes can therefore accumulate substitutions causing species differences even in the absence of substantial neo-Y degeneration.

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.

Most plants grow and develop by taking up nutrients from the soil while continuously under threat from foraging animals. Carnivorous plants have turned the tables by capturing and consuming nutrient-rich animal prey, enabling them to thrive in nutrient-poor soil. To better understand the evolution of botanical carnivory, we compared the draft genome of the Venus flytrap (Dionaea muscipula) with that of its aquatic sister, the waterwheel plant Aldrovanda vesiculosa, and the sundew Drosera spatulata. We identified an early whole-genome duplication in the family as source for carnivory-associated genes. Recruitment of genes to the trap from the root especially was a major mechanism in the evolution of carnivory, supported by family-specific duplications. Still, these genomes belong to the gene poorest land plants sequenced thus far, suggesting reduction of selective pressure on different processes, including non-carnivorous nutrient acquisition. Our results show how non-carnivorous plants evolved into the most skillful green hunters on the planet.

A stable multimodal system is developed by combining two common-path digital holographic microscopes (DHMs): coherent and incoherent, for simultaneous recording and retrieval of three-dimensional (3-D) phase and 3-D fluorescence imaging (FI), respectively, of a biological specimen. The 3-D FI is realized by a single-shot common-path off-axis fluorescent DHM developed recently by our group. In addition, we accomplish, the phase imaging by another single-shot, highly stable common-path off-axis DHM based on a beam splitter. In this DHM configuration, a beam splitter is used to divide the incoming object beam into two beams. One beam serves as the object beam carrying the useful information of the object under study, whereas another beam is spatially filtered at its Fourier plane by using a pinhole and it serves as a reference beam. This DHM setup, owing to a common-path geometry, is less vibration-sensitive and compact, having a similar field of view but with high temporal phase stability in comparison to a two-beam Mach–Zehnder-type DHM. The performance of the proposed common-path DHM and the multimodal system is verified by conducting various experiments on fluorescent microspheres and fluorescent protein-labeled living cells of the moss <italic>Physcomitrella patens</italic>. Moreover, the potential capability of the proposed multimodal system for 3-D live fluorescence and phase imaging of the fluorescent beads is also demonstrated. The obtained experimental results corroborate the feasibility of the proposed multimodal system and indicate its potential applications for the analysis of functional and structural behaviors of a biological specimen and enhancement of the understanding of physiological mechanisms and various biological diseases.

Animals possess specialized systems, e.g., neuromuscular systems, to sense the environment and then move their bodies quickly in response. Mimosa pudica, the sensitive plant, moves its leaves within seconds in response to external stimuli; e.g., touch or wounding. However, neither the plant-wide signaling network that triggers these rapid movements nor the physiological roles of the movements themselves have been determined. Here by simultaneous recording of cytosolic Ca2+ and electrical signals, we show that rapid changes in Ca2+ coupled with action and variation potentials trigger rapid movements in wounded M. pudica. Furthermore, pharmacological manipulation of cytosolic Ca2+ dynamics and CRISPR-Cas9 genome editing technology revealed that an immotile M. pudica is more vulnerable to attacks by herbivorous insects. Our findings provide evidence that rapid movements based on propagating Ca2+ and electrical signals protect this plant from insect attacks.

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.

The blue light receptors termed cryptochromes mediate photomorphological responses in seed plants. However, the mechanisms by which cryptochrome signals regulate plant development remain obscure. In this study, cryptochrome functions were analyzed using the moss Physcomitrella patens. This moss has recently become known as the only plant species in which gene replacement occurs at a high frequency by homologous recombination. Two cryptochrome genes were identified in Physcomitrella, and single and double disruptants of these genes were generated. Using these disruptants, it was revealed that cryptochrome signals regulate many steps in moss development, including induction of side branching on protonema and gametophore induction and development. In addition, the disruption of cryptochromes altered auxin responses, including the expression of auxin-inducible genes. Cryptochrome disruptants were more sensitive to external auxin than wild type in a blue light-specific manner, suggesting that cryptochrome light signals repress auxin signals to control plant development.

After fertilization, the zygote undergoes dynamic changes in chromosomal and cytoplasmic organization, and begins the cell cycles that eventually lead to formation of the multicellular embryo. Specific transcription factors that initiate this cascade of events in land plants have not been identified. We have identified two FLO/LFY genes, PpLFY1 and PpLFY2, that regulate the first cell division after formation of the zygote in the moss Physcomitrella patens. The deduced amino acid sequences of the two PpLFY genes are 94.8% identical to each other and show similar expression patterns. While fertilization occurred in the PpLFY disruptants, the development of double disruptant zygotes was arrested at the single-cell stage. When the double disruptants, as the female parent, were crossed with the wild type, as the male parent, normal sporophytes were formed, supporting the notion that the PpLFY genes function after fertilization to regulate the first mitotic cell division in zygotes. The rare sporophytes that formed on the PpLFY double disruptants showed mostly normal organogenesis, but had abnormalities in the pattern of cell division, supporting a role of PpLFY genes in regulating cell division. The FLO/LFY genes in angiosperms are conserved master regulators of floral identity without any obvious effects on cell division. By contrast, our study suggests that FLO/LFY genes have functions throughout sporophyte development in the basal land plant lineages.

The flagellar basal body of Salmonella typhimurium consists of four rings surrounding a rod. The rod, which is believed to transmit motor rotation to the filament, is not well characterized in terms of its structure and composition. FlgG is known to lie within the distal portion of the rod, in the region where it is surrounded by the L and P rings, just before the rod-hook junction. The FlgC and FlgF proteins are also known to be flagellar basal-body components; by comparison of deduced and experimental N-terminal amino acid sequences we show here that FlgB is a basal-body protein. The flgB, flgC, flgF and flgG gene sequences and the deduced protein sequences are presented. The four proteins are clearly related to each other in primary sequence, especially toward the N and C termini, supporting the hypothesis (based on examination of basal-body subfractions) that FlgB, FlgC and FlgF are, like FlgG, rod proteins. From this and other information we suggest that the rod is the cell-proximal part of a segmented axial structure of the flagellum, with FlgB, FlgC and FlgF located (in unknown order) in successive segments of the proximal rod, followed by FlgG located in the distal rod; the axial structure then continues with the hook, HAPs and filament. Although the rod is external to the cell membrane, none of the four rod proteins contains a consensus signal sequence for the primary export pathway; comparison with the experimentally determined N-terminal amino acid sequence indicates that FlgB has had its N-terminal methionine removed, while the other three are not processed at all. This demonstrates that these proteins are not exported by the primary cellular pathway, and suggests that they are exported by the same flagellum-specific pathway as the flagellar filament protein flagellin. The observed sequence similarities among the rod proteins, especially a six-residue consensus motif about 30 residues in from the N terminus, may constitute a recognition signal for this pathway or they may reflect higher-order structural similarities within the rod.

The MADS-box genes of land plants are extensively diverged to form a superfamily and are important in various aspects of development including the specification of floral organs as homeotic selector genes. The closest relatives of land plants are the freshwater green algae charophyceans. To study the origin and evolution of land plant MADS-box genes, we characterized these genes in three charophycean green algae: the stonewort Chara globularis, the coleochaete Coleochaete scutata, and the desmid Closterium peracerosum-strigosum-littorale complex. Phylogenetic analyses suggested that MADS-box genes diverged extensively in the land plant lineage after the separation of charophyceans from land plants. The stonewort C. globularis mRNA was specifically detected in the oogonium and antheridium together with the egg and spermatozoid during their differentiation. The expression of the C. peracerosum-strigosum-littorale-complex gene increased when vegetative cells began to differentiate into gametangial cells and decreased after fertilization. These expression patterns suggest that the precursors of land plant MADS-box genes originally functioned in haploid reproductive cell differentiation and that the haploid MADS-box genes were recruited into a diploid generation during the evolution of land plants.

Differentiation of epidermal cells is important for plants because they are in direct contact with the environment. Rhizoids are multicellular filaments that develop from the epidermis in a wide range of plants, including pteridophytes, bryophytes, and green algae; they have similar functions to root hairs in vascular plants in that they support the plant body and are involved in water and nutrient absorption. In this study, we examined mechanisms underlying rhizoid development in the moss, Physcomitrella patens, which is the only land plant in which high-frequency gene targeting is possible. We found that rhizoid development can be split into two processes: determination and differentiation. Two types of rhizoids with distinct developmental patterns (basal and mid-stem rhizoids) were recognized. The development of basal rhizoids from epidermal cells was induced by exogenous auxin, while that of mid-stem rhizoids required an unknown factor in addition to exogenous auxin. Once an epidermal cell had acquired a rhizoid initial cell fate, expression of the homeodomain-leucine zipper I gene Pphb7 was induced. Analysis of Pphb7 disruptant lines showed that Pphb7 affects the induction of pigmentation and the increase in the number and size of chloroplasts, but not the position or number of rhizoids. This is the first report on the involvement of a homeodomain-leucine zipper I gene in epidermal cell differentiation.

We sequenced transcripts from all putative genes for proteins, rRNAs, and a selection of gene-encoding tRNAs in the chloroplast genome of the fern Adiantum capillus-veneris. We detected 350 RNA editing sites when the cDNA sequence was compared to that of the genomic DNA. Of these sites, 10% were U-to-C edits and 90% were C-to-U edits. RNA editing created 19 new start codons, three new stop codons, and "repaired" 26 internal stop codons. Of the 332 editing sites that altered a codon, 26% were in the first codon position, 68% in the second, and 6% in the third. We also detected 21 silent edits, as well as 19 edits that were in untranslated regions, including introns and the anticodon of tRNA(Leu). The latter edit provided a tRNA that is not otherwise encoded in this genome and accounts for a heavily used leucine codon. The level of RNA editing in this fern is more than ten times that of any other vascular plant examined across an entire chloroplast genome. A previous study found even higher levels of editing in a hornwort (942 sites). This suggests that the relatively low levels of editing in seed plants (less than 0.05%) may not be typical for land plants, and that RNA editing may play a major role in chloroplast genome processing. Additionally, we found that 53 editing sites in A. capillus-veneris are homologous to editing sites in the hornwort, and some other land plants. This implies that a major component of RNA editing sites have been conserved for hundreds of millions of years.

Opinions on the basal relationship of land plants vary considerably and no phylogenetic tree with significant statistical support has been obtained. Here, we report phylogenetic analyses using 51 genes from the entire chloroplast genome sequences of 20 representative green plant species. The analyses, using translated amino acid sequences, indicated that extant bryophytes (mosses, liverworts, and hornworts) form a monophyletic group with high statistical confidence and that extant bryophytes are likely sisters to extant vascular plants, although the support for monophyletic vascular plants was not strong. Analyses at the nucleotide level could not resolve the basal relationship with statistical confidence. Bryophyte monophyly inferred using amino acid sequences has a good statistical foundation and is not rejected statistically by other data sets. We propose bryophyte monophyly as the currently best hypothesis.

The shoot is a repeated structure made up of stems and leaves and is the basic body plan in land plants. Vascular plants form a shoot in the diploid generation, whereas nonvascular plants such as mosses form a shoot in the haploid generation. It is not clear whether all land plants use similar molecular mechanisms in shoot development or how the genetic networks for shoot development evolved. The control of auxin distribution, especially by polar auxin transport, is essential for shoot development in flowering plants. We did not detect polar auxin transport in the gametophytic shoots of several mosses, but did detect it in the sporophytes of mosses without shoot structure. Treatment with auxin transport inhibitors resulted in abnormal embryo development, as in flowering plants, but did not cause any morphological changes in the haploid shoots. We fused the soybean auxin-inducible promoter GH3 with a GUS reporter gene and used it to indirectly detect auxin distribution in the moss Physcomitrella patens. An auxin transport inhibitor NPA did not cause any changes in the putative distribution of auxin in the haploid shoot. These results indicate that polar auxin transport is not involved in haploid shoot development in mosses and that shoots in vascular plants and mosses are most likely regulated differently during development.

The sundew genus Drosera consists of carnivorous plants with active flypaper traps and includes nearly 150 species distributed mainly in Australia, Africa, and South America, with some Northern Hemisphere species. In addition to confused intrageneric classification of Drosera, the intergeneric relationships among the Drosera and two other genera in the Droseraceae with snap traps, Dionaea and Aldrovanda, are problematic. We conducted phylogenetic analyses of DNA sequences of the chloroplast rbcL gene for 59 species of Drosera, covering all sections except one. These analyses revealed that five of 11 sections, including three monotypic sections, are polyphyletic. Combined rbcL and 18S rDNA sequence data were used to infer phylogenetic relationships among Drosera, Dionaea, and Aldrovanda. This analysis revealed that all Drosera species form a clade sister to a clade including Dionaea and Aldrovanda, suggesting that the snap traps of Aldrovanda and Dionaea are homologous despite their morphological differences. MacClade reconstructions indicated that multiple episodes of aneuploidy occurred in a clade that includes mainly Australian species, while the chromosome numbers in the other clades are not as variable. Drosera regia, which is native to South Africa, and most species native to Australia, were clustered basally, suggesting that Drosera originated in Africa or Australia. The rbcL tree indicates that Australian species expanded their distribution to South America and then to Africa. Expansion of distribution to the Northern Hemisphere from the Southern Hemispere occurred in a few different lineages.

Summary Members of the class 1 knotted‐like homeobox (KNOX) gene family are important regulators of shoot apical meristem development in angiosperms. To determine whether they function similarly in seedless plants, three KNOX genes (two class 1 genes and one class 2 gene) from the fern Ceratopteris richardii were characterized. Expression of both class 1 genes was detected in the shoot apical cell, leaf primordia, marginal part of the leaves, and vascular bundles by in situ hybridization, a pattern that closely resembles that of class 1 KNOX genes in angiosperms with compound leaves. The fern class 2 gene was expressed in all sporophyte tissues examined, which is characteristic of class 2 gene expression in angiosperms. All three CRKNOX genes were not detected in gametophyte tissues by RNA gel blot analysis. Arabidopsis plants overexpressing the fern class 1 genes resembled plants that overexpress seed plant class 1 KNOX genes in leaf morphology. Ectopic expression of the class 2 gene in Arabidopsis did not result in any unusual phenotypes. Taken together with phylogenetic analysis, our results suggest that (a) the class 1 and 2 KNOX genes diverged prior to the divergence of fern and seed plant lineages, (b) the class 1 KNOX genes function similarly in seed plant and fern sporophyte meristem development despite their differences in structure, (c) KNOX gene expression is not required for the development of the fern gametophyte, and (d) the sporophyte and gametophyte meristems of ferns are not regulated by the same developmental mechanisms at the molecular level.

Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurene via ent-kaurenoic acid. GAs are ubiquitously present in seed plants. The GA signal is perceived and transduced by the GID1 GA receptor/DELLA repressor pathway. The lycopod Selaginella moellendorffii biosynthesizes GA and has functional GID1-DELLA signaling components. In contrast, no GAs or functionally orthologous GID1-DELLA components have been found in the moss Physcomitrella patens. However, P. patens produces ent-kaurene, a common precursor for GAs, and possesses a functional ent-kaurene synthase, PpCPS/KS. To assess the biological role of ent-kaurene in P. patens, we generated a PpCPS/KS disruption mutant that does not accumulate ent-kaurene. Phenotypic analysis demonstrates that the mutant has a defect in the protonemal differentiation of the chloronemata to caulonemata. Gas chromatography-mass spectrometry analysis shows that P. patens produces ent-kaurenoic acid, an ent-kaurene metabolite in the GA biosynthesis pathway. The phenotypic defect of the disruptant was recovered by the application of ent-kaurene or ent-kaurenoic acid, suggesting that ent-kaurenoic acid, or a downstream metabolite, is involved in protonemal differentiation. Treatment with uniconazole, an inhibitor of ent-kaurene oxidase in GA biosynthesis, mimics the protonemal phenotypes of the PpCPS/KS mutant, which were also restored by ent-kaurenoic acid treatment. Interestingly, the GA(9) methyl ester, a fern antheridiogen, rescued the protonemal defect of the disruption mutant, while GA(3) and GA(4), both of which are active GAs in angiosperms, did not. Our results suggest that the moss P. patens utilizes a diterpene metabolite from ent-kaurene as an endogenous developmental regulator and provide insights into the evolution of GA functions in land plants.

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.

Microtubules form arrays with parallel and antiparallel bundles and function in various cellular processes, including subcellular transport and cell division. The antiparallel bundles in phragmoplasts, plant-unique microtubule arrays, are mostly unexplored and potentially offer new cellular insights. Here, we report that the Physcomitrella patens kinesins KINID1a and KINID1b (for kinesin for interdigitated microtubules 1a and 1b), which are specific to land plants and orthologous to Arabidopsis thaliana PAKRP2, are novel factors indispensable for the generation of interdigitated antiparallel microtubules in the phragmoplasts of the moss P. patens. KINID1a and KINID1b are predominantly localized to the putative interdigitated parts of antiparallel microtubules. This interdigitation disappeared in double-deletion mutants of both genes, indicating that both KINID1a and 1b are indispensable for interdigitation of the antiparallel microtubule array. Furthermore, cell plates formed by these phragmoplasts did not reach the plasma membrane in approximately 20% of the mutant cells examined. We observed that in the double-deletion mutant lines, chloroplasts remained between the plasma membrane and the expanding margins of the cell plate, while chloroplasts were absent from the margins of the cell plates in the wild type. This suggests that the kinesins, the antiparallel microtubule bundles with interdigitation, or both are necessary for proper progression of cell wall expansion.

Gibberellin (GA) controls pollen development in flowering plants via the GAMYB transcription factor. Here we show that GAMYB is conserved in Selaginella moellendorffii (lycophyte) and Physcomitrella patens (moss), although the former contains the GA signalling pathway, the latter does not. In the lycophyte, GA treatment promotes the outer wall development on microspores, whereas treatment with GA biosynthesis inhibitors disturbs its development. Contrary, in the moss, GAMYB homologue knockouts also produce abnormal spores that resemble Selaginella microspores treated with GA biosynthesis inhibitors and pollen grains of rice gamyb mutant. Moreover, the knockouts fail to develop male organs, instead ectopically forming female organs. Thus, before the establishment of the GA signalling pathway, basal land plants, including mosses, contained a GAMYB-based system for spore and sexual organ development. Subsequently, during the evolution from mosses to basal vascular plants including lycophytes, GA signalling might have merged to regulate this pre-existing GAMYB-based system.

Ferns are the only major lineage of vascular plants not represented by a sequenced nuclear genome. This lack of genome sequence information significantly impedes our ability to understand and reconstruct genome evolution not only in ferns, but across all land plants. Azolla and Ceratopteris are ideal and complementary candidates to be the first ferns to have their nuclear genomes sequenced. They differ dramatically in genome size, life history, and habit, and thus represent the immense diversity of extant ferns. Together, this pair of genomes will facilitate myriad large-scale comparative analyses across ferns and all land plants. Here we review the unique biological characteristics of ferns and describe a number of outstanding questions in plant biology that will benefit from the addition of ferns to the set of taxa with sequenced nuclear genomes. We explain why the fern clade is pivotal for understanding genome evolution across land plants, and we provide a rationale for how knowledge of fern genomes will enable progress in research beyond the ferns themselves.

Inducible transgene expression provides a useful tool to analyze gene function. The moss Physcomitrella patens is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with β-estradiol. The P. patens system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing β-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This β-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in P. patens.

Summary Ligand receptor‐based signaling is a means of cell‐to‐cell communication for coordinating developmental and physiological processes in multicellular organisms. In plants, cell‐producing meristems utilize this signaling to regulate their activities and ensure for proper development. Shoot and root systems share common requirements for carrying out this process; however, its molecular basis is largely unclear. It has been suggested that synthetic CLV 3/ EMBRYO SURROUNDING REGION ( CLE ) peptide shrinks the root meristem through the actions of CLAVATA 2 ( CLV 2 ) and the RECEPTOR ‐ LIKE PROTEIN KINASE 2 ( RPK 2 ) pathway in Arabidopsis thaliana . Our genetic screening for mutations that resist CLE peptide signaling in roots determined that BAM 1, which is a member of the leucine‐rich repeat receptor‐like kinase ( LRR ‐ RLK ) family, is also involved in this pathway. BAM 1 is preferentially expressed in the root tip, including the quiescent center and its surrounding stem cells. Our genetic analysis revealed that BAM 1 functions together with RPK 2. Using coimmunoprecipitation assay, we showed that BAM 1 is capable of forming heteromeric complexes with RPK 2. These findings suggest that the BAM 1 and RPK 2 receptors constitute a signaling pathway that modulates cell proliferation in the root meristem and that related molecules are employed in root and shoot meristems.

The root meristem (RM) is a fundamental structure that is responsible for postembryonic root growth. The RM contains the quiescent center (QC), stem cells and frequently dividing meristematic cells, in which the timing and the frequency of cell division are tightly regulated. In Arabidopsis thaliana, several gain-of-function analyses have demonstrated that peptide ligands of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-RELATED (CLE) family are important for maintaining RM size. Here, we demonstrate that a plant U-box E3 ubiquitin ligase, PUB4, is a novel downstream component of CLV3/CLE signaling in the RM. Mutations in PUB4 reduced the inhibitory effect of exogenous CLV3/CLE peptide on root cell proliferation and columella stem cell maintenance. Moreover, pub4 mutants grown without exogenous CLV3/CLE peptide exhibited characteristic phenotypes in the RM, such as enhanced root growth, increased number of cortex/endodermis stem cells and decreased number of columella layers. Our phenotypic and gene expression analyses indicated that PUB4 promotes expression of a cell cycle regulatory gene, CYCD6;1, and regulates formative periclinal asymmetric cell divisions in endodermis and cortex/endodermis initial daughters. These data suggest that PUB4 functions as a global regulator of cell proliferation and the timing of asymmetric cell division that are important for final root architecture.

Complex morphology is an evolutionary outcome of phenotypic diversification. In some carnivorous plants, the ancestral planar leaf has been modified to form a pitcher shape. However, how leaf development was altered during evolution remains unknown. Here we show that the pitcher leaves of Sarracenia purpurea develop through cell division patterns of adaxial tissues that are distinct from those in bifacial and peltate leaves, subsequent to standard expression of adaxial and abaxial marker genes. Differences in the orientation of cell divisions in the adaxial domain cause bifacial growth in the distal region and adaxial ridge protrusion in the middle region. These different growth patterns establish pitcher morphology. A computer simulation suggests that the cell division plane is critical for the pitcher morphogenesis. Our results imply that tissue-specific changes in the orientation of cell division underlie the development of a morphologically complex leaf.

Novel developmental programs often evolve via cooption of existing genetic networks. To understand this process, we explored cooption of the TAS3 tasiRNA pathway in the moss Physcomitrella patens. We find an ancestral function for this repeatedly redeployed pathway in the spatial regulation of a conserved set of Auxin Response Factors. In moss, this results in stochastic patterning of the filamentous protonemal tissue. Through modeling and experimentation, we demonstrate that tasiRNA regulation confers sensitivity and robustness onto the auxin response. Increased auxin sensitivity parallels increased developmental sensitivity to nitrogen, a key environmental signal. We propose that the properties lent to the auxin response network, along with the ability to stochastically modulate development in response to environmental cues, have contributed to repeated cooption of the tasiRNA-ARF module during evolution. The signaling properties of a genetic network, and not just its developmental output, are thus critical to understanding evolution of multicellular forms.

Abstract Elysia chlorotica , a sacoglossan sea slug found off the East Coast of the United States, is well-known for its ability to sequester chloroplasts from its algal prey and survive by photosynthesis for up to 12 months in the absence of food supply. Here we present a draft genome assembly of E. chlorotica that was generated using a hybrid assembly strategy with Illumina short reads and PacBio long reads. The genome assembly comprised 9,989 scaffolds, with a total length of 557 Mb and a scaffold N50 of 442 kb. BUSCO assessment indicated that 93.3% of the expected metazoan genes were completely present in the genome assembly. Annotation of the E. chlorotica genome assembly identified 176 Mb (32.6%) of repetitive sequences and a total of 24,980 protein-coding genes. We anticipate that the annotated draft genome assembly of the E. chlorotica sea slug will promote the investigation of sacoglossan genetics, evolution, and particularly, the genetic signatures accounting for the long-term functioning of algal chloroplasts in an animal.

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus , and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.

The most extreme environments are the most vulnerable to transformation under a rapidly changing climate. These ecosystems harbor some of the most specialized species, which will likely suffer the highest extinction rates. We document the steepest temperature increase (2010-2021) on record at altitudes of above 4,000 m, triggering a decline of the relictual and highly adapted moss Takakia lepidozioides. Its de-novo-sequenced genome with 27,467 protein-coding genes includes distinct adaptations to abiotic stresses and comprises the largest number of fast-evolving genes under positive selection. The uplift of the study site in the last 65 million years has resulted in life-threatening UV-B radiation and drastically reduced temperatures, and we detected several of the molecular adaptations of Takakia to these environmental changes. Surprisingly, specific morphological features likely occurred earlier than 165 mya in much warmer environments. Following nearly 400 million years of evolution and resilience, this species is now facing extinction.

The MADS genes encode a family of transcription factors, some of which control the identities of floral organs in flowering plants. To understand the role of MADS genes in the evolution of floral organs, five MADS genes (CMADS1, 2, 3, 4, and 6) were cloned from the fern Ceratopteris richardii , a nonflowering plant. A gene tree of partial amino acid sequences of seed plant and fern MADS genes showed that the fern genes form three subfamilies. All members of one of the fern MADS subfamilies have additional amino-terminal amino acids, which is a synapomorphic character of the AGAMOUS subfamily of the flowering plant MADS genes. Their structural similarity indicates a sister relationship between the two subfamilies. The temporal and spatial patterns of expression of the five fern MADS genes were assessed by Northern blot analyses and in situ hybridizations. CMADS1, 2, 3, and 4 are expressed similarly in the meristematic regions and primordia of sporophyte shoots and roots, as well as in reproductive structures, including sporophylls and sporangial initials, although the amount of expression in each tissue is different in each gene. CMADS6 is expressed in gametophytic tissues but not in sporophytic tissues. The lack of organ-specific expression of MADS genes in the reproductive structures of the fern sporophyte may indicate that the restriction of MADS gene expression to specific reproductive organs and the specialization of MADS gene functions as homeotic selector genes in the flowering plant lineage were important in floral organ evolution.

The general consensus is that a cyanobacterium phagocytosed by a host cell evolved into the plastids of red and green algae, land plants, and glaucophytes. In contrast to the plastids of glaucophytes, which retain a cyanobacterial-type peptidoglycan layer, no wall-like structures have been detected in plastids from other sources. Although the genome of Arabidopsis thaliana contains five genes that are essential for peptidoglycan synthesis, MurE, MurG, two genes for D-Ala-D-Ala ligase (Ddl), and the gene for translocase I (MraY), their functions have not been determined. We report that the moss Physcomitrella patens has nine homologous genes related to peptidoglycan biosynthesis: MurA, B, C, D, E, and F, Ddl, genes for the penicillin-binding protein Pbp, and dd-carboxypeptidase (Dac). Corroborating a computer prediction, analysis of the GFP fusion proteins with the N terminus of PpMurE or of PpPbp suggests that these proteins are located in the chloroplasts. Gene disruption of the PpMurE gene in P. patens resulted in the appearance of macrochloroplasts both in protonema and in leaf cells. Moreover, gene knockout of the P. patens Pbp gene showed inhibition of chloroplast division in this moss; however, no Pbp gene was found in A. thaliana.

Summary Leaves of flowering plants are diverse in shape. Part of this morphological diversity can be attributed to differences in spatiotemporal regulation of polarity in the upper (adaxial) and lower (abaxial) sides of developing leaves. In a leaf primordium, antagonistic interactions between polarity determinants specify the adaxial and abaxial domains in a mutually exclusive manner. The patterning of those domains is critical for leaf morphogenesis. In this review, we first summarize the gene networks regulating adaxial–abaxial polarity in conventional bifacial leaves and then discuss how patterning is modified in different leaf type categories. genesis 52:1–18, 2014. © 2013 The Authors. Genesis Published byWiley Periodicals, Inc.

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.

The complex body plan of modern vascular plants evolved by modification of simple systems of branching axes which originated from the determinate vegetative axis of a bryophyte-grade ancestor. Understanding body plan evolution and homologies has implications for land plant phylogeny and requires resolution of the specific developmental changes and their evolutionary sequence. The branched sporophyte may have evolved from a sterilized bryophyte sporangium, but prolongation of embryonic vegetative growth is a more parsimonious explanation. Research in the bryophyte model system Physcomitrella points to mechanisms regulating sporophyte meristem maintenance, indeterminacy, branching and the transition to reproductive development. These results can form the basis for hypotheses to identify and refine the nature and sequence of changes in development that occurred during the evolution of the indeterminate branched sporophyte from an unbranched bryophyte-grade sporophyte.

Significance Cell division axis orientation is critical for differentiation and morphogenesis. In animal cells, centrosome-driven mitotic spindle orientation is key to orient cell divisions. However, in naturally acentrosomal plants, the mechanism underlying spindle orientation is poorly understood. Using two model systems, asymmetrically dividing cells in the moss Physcomitrella patens and tobacco tissue culture cells, we identified de novo assembled microtubule organizing centers during mitotic prophase as a common critical mechanism for spindle orientation. Disruption of these microtubule organizing centers caused misoriented spindles and cell plates. The “phragmoplast guidance” mechanism, which directs cell plate orientation, could not fully restore initial spindle orientation defects. Thus, this study identifies spindle orientation as a conserved factor in land plants to assist division plane orientation.

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.

Parasitic plants form a specialized organ, a haustorium, to invade host tissues and acquire water and nutrients. To understand the molecular mechanism of haustorium development, we performed a forward genetics screening to isolate mutants exhibiting haustorial defects in the model parasitic plant Phtheirospermum japonicum. We isolated two mutants that show prolonged and sometimes aberrant meristematic activity in the haustorium apex, resulting in severe defects on host invasion. Whole-genome sequencing revealed that the two mutants respectively have point mutations in homologs of ETHYLENE RESPONSE 1 (ETR1) and ETHYLENE INSENSITIVE 2 (EIN2), signaling components in response to the gaseous phytohormone ethylene. Application of the ethylene signaling inhibitors also caused similar haustorial defects, indicating that ethylene signaling regulates cell proliferation and differentiation of parasite cells. Genetic disruption of host ethylene production also perturbs parasite invasion. We propose that parasitic plants use ethylene as a signal to invade host roots.

Homeobox genes encode transcription factors involved in many aspects of developmental processes.The homeodomain-leucine zipper (HD-Zip) genes, which are characterized by the presence of both a homeodomain and a leucine zipper motif, form a clade within the homeobox superfamily and were previously reported only from vascular plants.Here we report the isolation of 10 HD-Zip genes (named Pphb1-Pphb10) from the moss Physcomitrella patens.Based on a phylogenetic analysis of the 10 Pphb genes and previously reported vascular plant HD-Zip genes, all of the Pphb genes except Pphb3 belong to three of the four HD-Zip subfamilies (HD-Zip I, II, and III), indicating that these subfamilies originated before the divergence of the vascular plant and moss lineages.Pphb3 is sister to the HD-Zip II subfamily and has some distinctive characteristics, including the difference of the a 1 and d 1 sites of its leucine zipper motif, which are well conserved in each HD-Zip subfamily.Comparison of the genetic divergence of representative HD-Zip I and II genes showed that the evolutionary rate of HD-Zip I genes was faster than that of HD-Zip II genes.

Coriaria, which has the most conspicuously disjunct distribution of the flowering plants, is distributed in four separate areas of the world. The phylogenetic relationships of 12 Coriaria species collected from the representative disjunct areas were inferred by comparing 2416 bp of the combined data set of rbcL (a large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase) and matK (maturase K) genes. The phylogenetic tree shows that the Chile-Papua New Guinea-New Zealand-Pacific islands species and the Central America-northern South America species form a sister group, and the Eurasian clade is more basal to them. The divergence time between the Eurasian group and the other species was estimated as 63 or 59 million years ago using rbcL and matK molecular clocks, respectively. These results do not support previously proposed hypotheses which explain the disjunct distribution on the basis of continental drift but suggest that the distribution pattern was formed by several geographical migrations and separations in the Cenozoic.

Restriction fragment length polymorphism (RFLP) analysis of chloroplast DNA (cpDNA) has been applied successfully to solve systematic problems in various groups of angiosperms (reviewed in Palmer, 1987, 1988). It is now widely accepted that this method can provide more definite evidence of systematic relationships than can morphological, cytological, or chemical (secondary plant products) analyses. Two major approaches are used to detect cpDNA RFLPs. One is that of preparing purified cpDNA (Palmer & Zamir, 1982; Ogihara & Tsunewaki, 1982; Palmer et al., 1983), and the other is that of preparing total cellular DNA, in which cpDNA RFLPs are detected by means of heterologous cpDNA probes (Sytsma & Schaal, 1985; Sytsma & Gottlieb, 1986a,b; Jansen & Palmer, 1988). Since, in pteridophyte molecular systematics, we usually use field-collected living materials and often have to extract DNA from small quantities of leaf tissue, the second method is the more suitable. In contrast to the wide use of cpDNA analysis for angiosperm systematics, for pteridophyte cpDNA systematic studies only two groups, Osmunda (Stein et al., 1986) and some polystichoid ferns (Yatskievych et al., 1988; Stein et al., 1989; Soltis et al., 1990), have been analyzed. These pioneering studies have clearly demonstrated the usefulness of cpDNA analysis in pteridophyte systematics. One problem encountered when applying RFLP analysis of cpDNA to pteridophyte systematics is that currently available angiosperm heterologous cpDNA probes hybridize to fern cpDNA only weakly, even in low-stringency conditions, and give only slight signals. Improvement of probe DNA is thus desirable to ensure stronger signals. This will be possible by preparing better-matched probes from fern cpDNAs, since the maximum rate of hybridization at the optimal temperature is expected to increase as the homology between probe and sample DNAs increases (Bonner et al., 1973; Anderson & Young, 1985). In this paper, we report application of our newly prepared Adiantum capillus-veneris cpDNA clone bank (Hasebe & Iwatsuki, 1990) to pteridophyte systematics. This is the first nearly complete clone bank from a pteridophyte that has been characterized by both physical and gene maps.

Summary NimA‐related kinase 6 (NEK6) has been implicated in microtubule regulation to suppress the ectopic outgrowth of epidermal cells; however, its molecular functions remain to be elucidated. Here, we analyze the function of NEK6 and other members of the NEK family with regard to epidermal cell expansion and cortical microtubule organization. The functional NEK6–green fluorescent protein fusion localizes to cortical microtubules, predominantly in particles that exhibit dynamic movement along microtubules. The kinase‐dead mutant of NEK6 ( ibo1‐1 ) exhibits a disturbance of the cortical microtubule array at the site of ectopic protrusions in epidermal cells. Pharmacological studies with microtubule inhibitors and quantitative analysis of microtubule dynamics indicate excessive stabilization of cortical microtubules in ibo1 / nek6 mutants. In addition, NEK6 directly binds to microtubules in vitro and phosphorylates β‐tubulin. NEK6 interacts and co‐localizes with NEK4 and NEK5 in a transient expression assay. The ibo1‐3 mutation markedly reduces the interaction between NEK6 and NEK4 and increases the interaction between NEK6 and NEK5. NEK4 and NEK5 are required for the ibo1 / nek6 ectopic outgrowth phenotype in epidermal cells. These results demonstrate that NEK6 homodimerizes and forms heterodimers with NEK4 and NEK5 to regulate cortical microtubule organization possibly through the phosphorylation of β‐tubulins.

Chloroplasts require association with the plasma membrane for movement in response to light and for appropriate positioning within the cell to capture photosynthetic light efficiently. In Arabidopsis, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts. Here, we show that KAC and CHUP1 orthologs (AcKAC1, AcCHUP1A and AcCHUP1B, and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern Adiantum capillus-veneris and the moss Physcomitrella patens. The knockdown of AcKAC1 and two AcCHUP1 genes induced the aggregation of chloroplasts around the nucleus. Analyses of A. capillus-veneris mutants containing perinuclear-aggregated chloroplasts confirmed that AcKAC1 is required for chloroplast-plasma membrane association. In addition, P. patens lines in which two KAC genes had been knocked out showed an aggregated chloroplast phenotype similar to that of the fern kac1 mutants. These results indicate that chloroplast positioning and movement are mediated through the activities of KAC and CHUP1 proteins, which are conserved in land plants.

Abstract Both land plants and metazoa have the capacity to reprogram differentiated cells to stem cells. Here we show that the moss Physcomitrella patens Cold-Shock Domain Protein 1 (PpCSP1) regulates reprogramming of differentiated leaf cells to chloronema apical stem cells and shares conserved domains with the induced pluripotent stem cell factor Lin28 in mammals. PpCSP1 accumulates in the reprogramming cells and is maintained throughout the reprogramming process and in the resultant stem cells. Expression of PpCSP1 is negatively regulated by its 3′-untranslated region (3′-UTR). Removal of the 3′-UTR stabilizes PpCSP1 transcripts, results in accumulation of PpCSP1 protein and enhances reprogramming. A quadruple deletion mutant of PpCSP1 and three closely related PpCSP genes exhibits attenuated reprogramming indicating that the PpCSP genes function redundantly in cellular reprogramming. Taken together, these data demonstrate a positive role of PpCSP1 in reprogramming, which is similar to the function of mammalian Lin28.

Plant movements are generally slow, but some plant species have evolved the ability to move very rapidly at speeds comparable to those of animals. Whereas movement in animals relies on the contraction machinery of muscles, many plant movements use turgor pressure as the primary driving force together with secondarily generated elastic forces. The movement of stomata is the best-characterized model system for studying turgor-driven movement, and many gene products responsible for this movement, especially those related to ion transport, have been identified. Similar gene products were recently shown to function in the daily sleep movements of pulvini, the motor organs for macroscopic leaf movements. However, it is difficult to explain the mechanisms behind rapid multicellular movements as a simple extension of the mechanisms used for unicellular or slow movements. For example, water transport through plant tissues imposes a limit on the speed of plant movements, which becomes more severe as the size of the moving part increases. Rapidly moving traps in carnivorous plants overcome this limitation with the aid of the mechanical behaviors of their three-dimensional structures. In addition to a mechanism for rapid deformation, rapid multicellular movements also require a molecular system for rapid cell-cell communication, along with a mechanosensing system that initiates the response. Electrical activities similar to animal action potentials are found in many plant species, representing promising candidates for the rapid cell-cell signaling behind rapid movements, but the molecular entities of these electrical signals remain obscure. Here we review the current understanding of rapid plant movements with the aim of encouraging further biological studies into this fascinating, challenging topic.

Introduction Half a century since the creation of the International Association of Bryologists, we carried out a review to identify outstanding challenges and future perspectives in bryology. Specifically, we have identified 50 fundamental questions that are critical in advancing the discipline.Methods We have adapted a deep-rooted methodology of horizon scanning to identify key research foci. An initial pool of 258 questions was prepared by a multidisciplinary and international working group of 32 bryologists. A series of online surveys completed by a broader community of researchers in bryology, followed by quality-control steps implemented by the working group, were used to create a list of top-priority questions. This final list was restricted to 50 questions with a broad conceptual scope and answerable through realistic research approaches.Key results The top list of 50 fundamental questions was organised into four general topics: Bryophyte Biodiversity and Biogeography; Bryophyte Ecology, Physiology and Reproductive Biology; Bryophyte Conservation and Management; and Bryophyte Evolution and Systematics. These topics included 9, 19, 14 and 8 questions, respectively.Conclusions Although many of the research challenges identified are not newly conceived, our horizon-scanning exercise has established a significant foundation for future bryological research. We suggest analytical and conceptual strategies and novel developments for potential use in advancing the research agenda for bryology.

Gnetales, one of the extant gymnosperm orders, has traditionally been recognized to be most closely related to flowering plants, because the reproductive organ of Gnetales has some morphological characteristics similar to flowering plants. Most recent molecular phylogenetic studies do not support the sister relationship of the Gnetales and flowering plants, but instead support a close relationship between Gnetales and other extant gymnosperms. The MADS genes are transcription factors, some of which are involved in reproductive organ development in flowering plants. To resolve the discrepancy in phylogenetic inferences, and to provide insights into the evolution of reproductive organs in seed plants, four MADS genes (GpMADS1-4) were cloned from Gnetum parvifolium. GpMADS2 is likely to be a pseudogene and the other three genes were characterized. A MADS gene tree based on partial amino acid sequences showed that GpMADS3 is included in the AGL6 group, but the other two genes do not cluster with any previously reported MADS gene. The three GpMADS genes were expressed during the early stage of ovule development in the differentiating nucellus and three envelopes. A comparison of MADS gene expression among conifers, Gnetum, and flowering plants suggests that the comparable reproductive organs in Gnetum and flowering plants evolved in parallel, and is likely to support the homology between the ovule-ovuliferous scale complex of conifers and the Gnetum ovules, including the three envelopes.

The homeodomain-leucine zipper (HD-Zip) genes encode transcription factors that are characterized by the presence of both a homeodomain and a leucine zipper motif. They belong to the homeobox gene superfamily and have been reported only from flowering plants. This article is the first report on the ferm HD-Zip genes (named Crhb1-Crhb11) isolated from the homosporous ferm Ceratopteris richardii. Phylogenetic analyses of the II Crhb genes with previously reported angiosperm HD-Zip genes show that the Crhb genes belong to three of the four different angiosperm HD-Zip subfamilies (HD-Zip I, II, and IV), indicating that these subfamilies of HD-Zip genes originated before the diversification of the ferm and seed plant lineages. The Crhb4-Crhb8 and Crhb11 genes belong to the HD-Zip I subfamily but differ from angiosperm HD-Zip I genes by the presence of a seven-amino-acid indel in the leucine zipper motif. By the northern analyses, Crhb1 and Crhb3 were expressed only in gametophyte tissue. Expression of Crhb2 and Crhb11 genes could not be detected in any tissue examined, while all other Crhb genes were expressed in most sporophytic and gametophytic tissues. Although the functions of the Crhb genes in Ceratopteris are unknown, their patterns of expression suggest that they regulate developmental or physiological processes common to both the gametophyte and the sporophyte generations of the fern. Differences in the expression of Crhb1 between male gametophytes and male-hermaphrodite mixed populations of gametophytes suggests that the Crhb1 gene is involved in gametophytic sex determination.

Nucleotide sequences of the chloroplast gene rbcL from 42 species of the fern tribe Physematieae (Dryopteridaceae) were analyzed to gain insights into the inter- and intrageneric relationships and the generic circumscriptions in the tribe. The phylogenetic relationships were inferred using the neighbor-joining and maximum-parsimony methods, and both methods produced largely congruent trees. These trees reveal that: (1) Athyrium, Cornopteris, Pseudocystopteris, and Anisocampium form a clade and Athyrium is polyphyletic; (2) Deparia sensu lato is monophyletic and Dictyodroma formosana is included in the Deparia clade; (3) Diplaziopsis forms a clade with Homalosorus, which is isolated from the other genera of the Physematieae; (4) Monomelangium is included in the monophyletic Diplazium clade; and (5) Rhachidosorus is not closely related to either Athyrium or Diplazium.

The chloroplast genome of most land plants is highly conserved. In contrast, physical and gene mapping studies have revealed a highly rearranged chloroplast genome in species representing four families of ferns. In all four, there has been a rare duplication of the psbA gene and the order of the psbA, 16S, and 23S rRNA genes has been inverted. Our analysis shows that the described rearrangement results from a minimum of two inversions within the inverted repeat. This chloroplast DNA structure provides unambiguous evidence that phylogenetically links families of ferns once thought to belong to different major evolutionary lineages.

Ceratopteris richardii is a useful experimental system for studying gametophyte development and sexual reproduction in plants. However, few tools for cloning mutant genes or disrupting gene function exist for this species. The feasibility of systemic gene silencing as a reverse genetics tool was examined in this study. Several DNA constructs targeting a Ceratopteris protoporphyrin IX magnesium chelatase (CrChlI) gene that is required for chlorophyll biosynthesis were each introduced into young gametophytes by biolistic delivery. Their transient expression in individual cells resulted in a colorless cell phenotype that affected most cells of the mature gametophyte, including the meristem and gametangia. The colorless phenotype was associated with a 7-fold decrease in the abundance of the endogenous transcript. While a construct designed to promote the transient expression of a CrChlI double stranded, potentially hairpin-forming RNA was found to be the most efficient in systemically silencing the endogenous gene, a plasmid containing the CrChlI cDNA insert alone was sufficient to induce silencing. Bombarded, colorless hermaphroditic gametophytes produced colorless embryos following self-fertilization, demonstrating that the silencing signal could be transmitted through gametogenesis and fertilization. Bombardment of young gametophytes with constructs targeting the Ceratopteris filamentous temperature sensitive (CrFtsZ) and uroporphyrin dehydrogenase (CrUrod) genes also produced the expected mutant phenotypes. A method that induces the systemic silencing of target genes in the Ceratopteris gametophyte is described. It provides a simple, inexpensive and rapid means to test the functions of genes involved in gametophyte development, especially those involved in cellular processes common to all plants.

Partial nucleotide sequences of the large subunit of ribulose-1,5-bisphosphate carboxylase (rubisco) gene (1333 base pairs: about 90% of the gene) from several seed plants were determined. Phylogenetic trees based on amino acid sequences were inferred by using the neighbor joining and maximum likelihood methods. The results indicate (1) monophyly of gnetum group (Ephedra, Gnetum, Welwitschia), (2) monophyly of extant gymnosperms containing gnetum group, which contradicts the results of morphological data.

This comprehensive overview of the xyloglucan endotransglucosylase/hydrolase (XTH) family of genes and proteins in bryophytes, based on research using genomic resources that are newly available for the moss Physcomitrella patens, provides new insights into plant evolution. In angiosperms, the XTH genes are found in large multi-gene families, probably reflecting the diverse roles of individual XTHs in various cell types. As there are fewer cell types in P. patens than in angiosperms such as Arabidopsis and rice, it is tempting to deduce that there are fewer XTH family genes in bryophytes. However, the present study unexpectedly identified as many as 32 genes that potentially encode XTH family proteins in the genome of P. patens, constituting a fairly large multi-gene family that is comparable in size with those of Arabidopsis and rice. In situ localization of xyloglucan endotransglucosylase activity in this moss indicates that some P. patens XTH proteins exhibit biochemical functions similar to those found in angiosperms, and that their expression profiles are tissue-dependent. However, comparison of structural features of families of XTH genes between P. patens and angiosperms demonstrated the existence of several bryophyte-specific XTH genes with distinct structural and functional features that are not found in angiosperms. These bryophyte-specific XTH genes might have evolved to meet morphological and functional needs specific to the bryophyte. These findings raise interesting questions about the biological implications of the XTH family of proteins in non-seed plants.

We isolated Burkholderia symbiont strain RPE64 from the bean bug Riptortus pedestris. Analysis of the complete 6.96-Mb genome, which consists of three chromosomes and two plasmids, will facilitate further understanding of insect-microbe symbiosis and the development of pest-control technologies.

Next-generation sequencing (NGS) technologies enable the rapid production of an enormous quantity of sequence data. These powerful new technologies allow the identification of mutations by whole-genome sequencing. However, most reported NGS-based mapping methods, which are based on bulked segregant analysis, are costly and laborious. To address these limitations, we designed a versatile NGS-based mapping method that consists of a combination of low- to medium-coverage multiplex SOLiD (Sequencing by Oligonucleotide Ligation and Detection) and classical genetic rough mapping. Using only low to medium coverage reduces the SOLiD sequencing costs and, since just 10 to 20 mutant F 2 plants are required for rough mapping, the operation is simple enough to handle in a laboratory with limited space and funding. As a proof of principle, we successfully applied this method to identify the CTR1, which is involved in boron-mediated root development, from among a population of high boron requiring Arabidopsis thaliana mutants. Our work demonstrates that this NGS-based mapping method is a moderately priced and versatile method that can readily be applied to other model organisms.

The temporal resolution of a two-photon excitation laser scanning microscopy (TPLSM) system is limited by the excitation laser beam's scanning speed. To improve the temporal resolution, the TPLSM system is equipped with a spinning-disk confocal scanning unit. However, the insufficient energy of a conventional Ti:sapphire laser source restricts the field of view (FOV) for TPLSM images to a narrow region. Therefore, we introduced a high-peak-power Yb-based laser in order to enlarge the FOV. This system provided three-dimensional imaging of a sufficiently deep and wide region of fixed mouse brain slices, clear four-dimensional imaging of actin dynamics in live mammalian cells and microtubule dynamics during mitosis and cytokinesis in live plant cells.

New cDNA sequences for chalcone synthase (CHS), a key enzyme in the flavonoid biosynthesis, were obtained from the Japanese and common morning glories; they are more closely related to other CHS sequences than the six previously described CHS genes from the same plants. The newly isolated CHS-D gene is abundantly expressed in the pigmented flower buds, while its expression is drastically reduced in the white flower buds. Thus CHS-D appears to produce major CHS transcripts for flower pigmentation.

The monocot coleoptile tip region has been generally supposed to be the source of IAA to supply IAA to basal parts by the polar IAA transport system, which results in gravi- and phototropic curvature of coleoptiles. Based on this IAA transport system and gravitropism of maize coleoptiles, we have developed two screening methods to identify small molecules from a large chemical library that inhibit IAA transport. The methods detect molecules that affect (i) gravitropic curvature of coleoptiles; and (ii) the amount of IAA transported from the tip. From 10,000 chemicals, eight compounds were identified and categorized into two groups. Four chemicals in group A decreased IAA transport from the tip, and increased endogenous IAA levels in the tip. The structures of two compounds resembled that of 1-N-naphthylphthalamic acid (NPA), but those of the other two differed from structures of known IAA transport inhibitors. Four chemicals in group B strongly inhibited IAA transport from the tip, but IAA levels at the tip were only slightly affected. At higher concentrations, group B compounds inhibited germination of Arabidopsis, similarly to brefeldin A (BFA). Analysis of the cellular distribution of PIN2-green fluorescent protein (GFP) and PIN1-GFP in Arabidopsis revealed that one of the four chemicals in group B induced internalization of PIN1 and PIN2 proteins into vesicles smaller than BFA bodies, suggesting that this compound affects cellular vesicle trafficking systems related to PIN trafficking. The eight chemicals identified here will be a useful tool for understanding the mechanisms of IAA transport in plants.

Stem cells self-renew and repeatedly produce differentiated cells during development and growth. The differentiated cells can be converted into stem cells in some metazoans and land plants with appropriate treatments. After leaves of the moss Physcomitrella patens are excised, leaf cells reenter the cell cycle and commence tip growth, which is characteristic of stem cells called chloronema apical cells. To understand the underlying molecular mechanisms, a digital gene expression profiling method using mRNA 5'-end tags (5'-DGE) was established. The 5'-DGE method produced reproducible data with a dynamic range of four orders that correlated well with qRT-PCR measurements. After the excision of leaves, the expression levels of 11% of the transcripts changed significantly within 6 h. Genes involved in stress responses and proteolysis were induced and those involved in metabolism, including photosynthesis, were reduced. The later processes of reprogramming involved photosynthesis recovery and higher macromolecule biosynthesis, including of RNA and proteins. Auxin and cytokinin signaling pathways, which are activated during stem cell formation via callus in flowering plants, are also activated during reprogramming in P. patens, although no exogenous phytohormone is applied in the moss system, suggesting that an intrinsic phytohormone regulatory system may be used in the moss.

Polyamines are small basic compounds present in all living organisms and act in a variety of biological processes. However, the mechanism of polyamine sensing, signaling and response in relation to other metabolic pathways remains to be fully addressed in plant cells. As one approach, we isolated Arabidopsis mutants that show increased resistance to spermine in terms of chlorosis. We show here that two of the mutants have a point mutation in a nitrate transporter gene of the NRT1/PTR family (NPF), NRT1.3 (AtNPF6.4). These mutants also exhibit increased resistance to putrescine and spermidine while loss-of-function mutants of the two closest homologs of NRT1.3, root-specific NRT1.1 (AtNPF6.3) and petiole-specific NRT1.4 (AtNPF6.2), were shown to have a normal sensitivity to polyamines. When the GUS reporter gene was expressed under the control of the NRT1.3 promoter, GUS staining was observed in leaf mesophyll cells and stem cortex cells but not in the epidermis, suggesting that NRT1.3 specifically functions in parenchymal tissues. We further found that the aerial part of the mutant seedling has normal levels of polyamines but shows reduced uptake of norspermidine compared with the wild type. These results suggest that polyamine transport or metabolism is associated with nitrate transport in the parenchymal tissue of the shoot.

Cytokinesis is fundamental for cell proliferation [1, 2]. In plants, a bipolar short-microtubule array forms the phragmoplast, which mediates vesicle transport to the midzone and guides the formation of cell walls that separate the mother cell into two daughter cells [2]. The phragmoplast centrifugally expands toward the cell cortex to guide cell-plate formation at the cortical division site [3, 4]. Several proteins in the phragmoplast midzone facilitate the anti-parallel bundling of microtubules and vesicle accumulation [5]. However, the mechanisms by which short microtubules are maintained during phragmoplast development, in particular, the behavior of microtubules at the distal zone of phragmoplasts, are poorly understood. Here, we show that a plant-specific protein, CORTICAL MICROTUBULE DISORDERING 4 (CORD4), tethers the conserved microtubule-severing protein katanin to facilitate formation of the short-microtubule array in phragmoplasts. CORD4 was specifically expressed during mitosis and localized to preprophase bands and phragmoplast microtubules. Custom-made two-photon spinning disk confocal microscopy revealed that CORD4 rapidly localized to microtubules in the distal phragmoplast zone during phragmoplast assembly at late anaphase and persisted throughout phragmoplast expansion. Loss of CORD4 caused abnormally long and oblique phragmoplast microtubules and slow expansion of phragmoplasts. The p60 katanin subunit, KTN1, localized to the distal phragmoplast zone in a CORD4-dependent manner. These results suggest that CORD4 tethers KTN1 at phragmoplasts to modulate microtubule length, thereby accelerating phragmoplast growth. This reveals the presence of a distinct machinery to accelerate cytokinesis by regulating the action of katanin.

Summary Because of its simple body plan and ease of gene knockout and allele replacement, the moss Physcomitrella patens is often used as a model system for studies in plant physiology and developmental biology. Gene‐trap and enhancer‐trap systems are useful techniques for cloning genes and enhancers that function in specific tissues or cells. Additionally, these systems are convenient for obtaining molecular markers specific for certain developmental processes. Elements for gene‐trap and enhancer‐trap systems were constructed using the uidA reporter gene with either a splice acceptor or a minimal promoter. Through a high rate of transformation conferred by a method utilizing homologous recombination, 235 gene‐trap and 1073 enhancer‐trap lines were obtained from 5637 and 3726 transgenic lines, respectively. The expression patterns of these trap lines in the moss gametophyte varied. The candidate gene trapped in a gene‐trap line YH209, which shows rhizoid‐specific expression, was obtained by 5′ and 3′ RACE. This gene was named PpGLU , and forms a clade with plant acidic α‐glucosidase genes. Thus, these gene‐trap and enhancer‐trap systems should prove useful to identify tissue‐ and cell‐specific genes in Physcomitrella .

Summary Cleistogamy is a mating system found in approximately 300 species of flowering plants. Cleistogamous plants produce closed (cleistogamous, CL) flowers that require obligate self‐pollination and open (chasmogamous, CH) flowers that allow for outcross‐pollination. CL and CH flowers are induced by various environmental factors; this appears to be an adaptive mating strategy in unpredictable environments. We examined the molecular basis of CL and CH flowering in Cardamine kokaiensis , which is closely related to the model flowering plant Arabidopsis thaliana . CL and CH flowering should be regulated by gene expression that is dependent on environmental conditions. By elucidating the molecular basis of CL and CH flowering, we can determine the changes in gene regulatory networks involved in the transition from CH to CL flowering. Furthermore, these results may help clarify the molecular evolutionary mechanisms leading to cleistogamy. We regulated CL and CH flowering of C. kokaiensis using chilling treatments in a growth chamber. In a control treatment without chilling, C. kokaiensis produced CH and intermediate (INT) flowers. Long chilling of seedlings led to INT flowers, while long chilling of seeds induced the formation of CL flowers. Chilling seeds, and to a lesser extent of seedlings, induced early flowering and small plant size at flowering. We also conducted a cross‐species microarray analysis to compare gene expression patterns between CL and CH flowers using genomic DNA‐based probe‐selection strategy in an A. thaliana microarray. In this result, 69 genes, including genes related to floral development, auxin, flowering time, cold‐stress, and drought‐stress, were differentially expressed between CL and CH flowers. Synthesis . This is the first report on the molecular basis of cleistogamy. We hypothesize that the interaction between the genetic network of the chilling response and that of floral development has been important in the evolution of cleistogamy in C. kokaiensis . Our results help to clarify the molecular basis for the evolution of plant mating systems that depend on environmental conditions.

Eukaryotic cells have developed several essential membrane components. In flowering plants, appropriate structures and distributions of the major membrane components are predominantly regulated by actin microfilaments. In this study, we have focused on the regulatory mechanism of vacuolar structures in the moss, Physcomitrella patens. The high ability of P. patens to undergo homologous recombination enabled us stably to express green fluorescent protein (GFP) or red fluorescent protein (RFP) fusion proteins, and the simple body structure of P. patens enabled us to perform detailed visualization of the intracellular vacuolar and cytoskeletal structures. Three-dimensional analysis and high-speed time-lapse observations revealed surprisingly complex structures and dynamics of the vacuole, with inner sheets and tubular protrusions, and frequent rearrangements by separation and fusion of the membranes. Depolymerization of microtubules dramatically affected these structures and movements. Dual observation of microtubules and vacuolar membranes revealed that microtubules induced tubular protrusions and cytoplasmic strands of the vacuoles, indicative of interactions between microtubules and vacuolar membranes. These results demonstrate a novel function of microtubules in maintaining the distribution of the vacuole and suggest a functional divergence of cytoskeletal functions in land plant evolution.

Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested for methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5 μM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC–MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.

In astronomy, adaptive optics (AO) can be used to cancel aberrations caused by atmospheric turbulence and to perform diffraction-limited observation of astronomical objects from the ground. AO can also be applied to microscopy, to cancel aberrations caused by cellular structures and to perform high-resolution live imaging. As a step toward the application of AO to microscopy, here we analyzed the optical properties of plant cells. We used leaves of the moss Physcomitrella patens, which have a single layer of cells and are thus suitable for optical analysis. Observation of the cells with bright field and phase contrast microscopy, and image degradation analysis using fluorescent beads demonstrated that chloroplasts provide the main source of optical degradations. Unexpectedly, the cell wall, which was thought to be a major obstacle, has only a minor effect. Such information provides the basis for the application of AO to microscopy for the observation of plant cells.

The plant type III polyketide synthases (PKSs), which produce diverse secondary metabolites with different biological activities, have successfully co-evolved with land plants. To gain insight into the roles that ancestral type III PKSs played during the early evolution of land plants, we cloned and characterized PpORS from the moss Physcomitrella. PpORS has been proposed to closely resemble the most recent common ancestor of the plant type III PKSs. PpORS condenses a very long chain fatty acyl-CoA with four molecules of malonyl-CoA and catalyzes decarboxylative aldol cyclization to yield the pentaketide 2'-oxoalkylresorcinol. Therefore, PpORS is a 2'-oxoalkylresorcinol synthase. Structure modeling and sequence alignments identified a unique set of amino acid residues (Gln(218), Val(277), and Ala(286)) at the putative PpORS active site. Substitution of the Ala(286) to Phe apparently constricted the active site cavity, and the A286F mutant instead produced triketide alkylpyrones from fatty acyl-CoA substrates with shorter chain lengths. Phylogenetic analysis and comparison of the active sites of PpORS and alkylresorcinol synthases from sorghum and rice suggested that the gramineous enzymes evolved independently from PpORS to have similar functions but with distinct active site architecture. Microarray analysis revealed that PpORS is exclusively expressed in nonprotonemal moss cells. The in planta function of PpORS, therefore, is probably related to a nonprotonemal structure, such as the cuticle.

Stem cells self-renew and produce precursor cells that differentiate to become specialized cell types. Land plants generate several types of stem cells that give rise to most organs of the plant body and whose characters determine the body organization. The moss Physcomitrella patens forms eight types of stem cells throughout its life cycle. Under gametangium-inducing conditions, multiple antheridium apical stem cells are formed at the tip of the gametophore and each antheridium apical stem cell divides to form an antheridium. We found that the gametophore apical stem cell, which typically forms leaf and stem tissues, changes to become a new type of stem cell, which we term the antheridium initial stem cell. This antheridium initial stem cell produces multiple antheridium apical stem cells, resulting in a cluster of antheridia at the tip of gametophore. This is the first report of a land plant stem cell directly producing another type of stem cell during normal development. Notably, the antheridium apical stem cells are distally produced from the antheridium initial stem cell, similar to the root cap stem cells of vascular plants, suggesting the use of similar molecular mechanisms and a possible evolutionary relationship. This article is part of a discussion meeting issue ‘The Rhynie cherts: our earliest terrestrial ecosystem revisited’.

Membrane trafficking to the protein storage vacuole (PSV) is a specialized process in seed plants. However, this trafficking mechanism to PSV is poorly understood. Here, we show that three types of Beige and Chediak-Higashi (BEACH)-domain proteins contribute to both vacuolar protein transport and effector-triggered immunity (ETI). We screened a green fluorescent seed (GFS) library of Arabidopsis mutants with defects in vesicle trafficking and isolated two allelic mutants gfs3 and gfs12 with a defect in seed protein transport to PSV. The gene responsible for the mutant phenotype was found to encode a putative protein belonging to group D of BEACH-domain proteins, which possess kinase domains. Disruption of other BEACH-encoding loci in the gfs12 mutant showed that BEACH homologs acted in a cascading manner for PSV trafficking. The epistatic genetic interactions observed among BEACH homologs were also found in the ETI responses of the gfs12 and gfs12 bchb-1 mutants, which showed elevated avirulent bacterial growth. The GFS12 kinase domain interacted specifically with the pleckstrin homology domain of BchC1. These results suggest that a cascade of multiple BEACH-domain proteins contributes to vacuolar protein transport and plant defense.