Land plants share several core factors responsible for female gametophyte development, despite their differing structures and developmental programs. New work providing molecular dissection of reproductive phases in non-angiosperm plants is a powerful tool for elucidating the underlying genetic network.

The sexual reproductive organs of bryophytes - in which gametes necessary for fertilization are produced, namely, male antheridia and female archegonia - are formed from vegetative haploid gametophytes. In dioicous bryophytes such as Marchantia polymorpha, the genes within the sex-determining regions in distinct sexual strains have been identified. However, in monoicous bryophytes such as Physcomitrium patens, how the two sex fates are specified on the same gametophyte remained unknown. Here, we identified an RWP-RK domain-containing transcription factor in P. patens, PpRKD, as a factor required for the development of female organs, based on the absence of archegonia in loss-of-function Pprkd mutants and the specific expression of PpRKD in archegonia. When ectopically induced, the expression of PpRKD resulted in the repression of antheridial development and the emergence of archegonium-like organs. Furthermore, the young primordia inside the antheridial bundle displayed typical archegonial division patterns, suggesting that PpRKD confer female fate to antheridium primordia. This study represents the first instance where the function of sex determination has been identified among RKD orthologs in land plants. This finding should provide a new framework for the molecular evolutionary context of the genes in the RKD family, considering the recent elucidation of their roles in algae.

Background Land plants exhibit a haplodiplontic life cycle, whereby multicellular bodies develop in both the haploid and diploid generations. The early-diverging land plants, known as bryophytes, have a haploid-dominant life cycle, in which a short-lived multicellular body in the diploid generation, known as the sporophyte, develops on the maternal haploid gametophyte tissues. The moss Physcomitrium ( Physcomitrella ) patens has become one of the most powerful model systems in evolutionary plant developmental studies. To induce diploid sporophytes of P. paten s, several protocols are implemented. One of the conventional approaches is to grow approximately one-month-old gametophores for another month on Jiffy-7 pellets made from the peat moss that is difficult to fully sterilize. A more efficient method to obtain all tissues throughout the life cycle should accelerate studies of P. paten s. Results Here, we investigated the effect of nitrogen conditions on the growth and development of P. patens . We provide an improved protocol for the sporophyte induction of P. patens using a BCD-based solid culture medium without Jiffy-7 pellets, based on the finding that the formation of gametangia and subsequent sporophytes is promoted by nitrogen-free growth conditions. The protocol consists of two steps; first, culture the protonemata and gametophores on nitrogen-rich medium under continuous light at 25 °C, and then transfer the gametophores onto nitrogen-free medium under short-day and at 15 °C for sporophyte induction. The protocol enables to shorten the induction period and reduce the culture space. Conclusions Our more efficient and shortened protocol for inducing the formation of sporophytes will contribute to future studies into the fertilization or the diploid sporophyte generation of P. patens .

The hornworts are a small group of land plants, consisting of only 11 families and approximately 220 species. Despite their small size as a group, their phylogenetic position and unique biology are of great importance. Hornworts, together with mosses and liverworts, form the monophyletic group of bryophytes that is sister to all other land plants (Tracheophytes). It is only recently that hornworts became amenable to experimental investigation with the establishment of Anthoceros agrestis as a model system. In this perspective, we summarize the recent advances in the development of A. agrestis as an experimental system and compare it with other plant model systems. We also discuss how A. agrestis can help to further research in comparative developmental studies across land plants and to solve key questions of plant biology associated with the colonization of the terrestrial environment. Finally, we explore the significance of A. agrestis in crop improvement and synthetic biology applications in general.

In flowering plants, strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), an SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating its origin in the common ancestor of land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

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

Data from: Phylogenetic distribution and expression pattern analyses identified a divergent basal body assembly protein involved in land plant spermatogenesis Author information Shizuka Koshimizu1, Naoki Minamino2, Tomoaki Nishiyama3, Emiko Yoro4, Mayuko Sato5, Mayumi Wakazaki5, Kiminori Toyooka5, Kazuo Ebine2,6, Keiko Sakakibara4, Takashi Ueda2,6, and Kentaro Yano1 1 School of Agriculture, Meiji University, Kawasaki 214-8571, Japan 2 Division of Cellular Dynamics, National Institute for Basic Biology, Okazaki 444-8585, Japan 3 Research Center for Experimental Modeling of Human Disease, Kanazawa University, Kanazawa 920-0934, Japan 4 Department of Life Science, Rikkyo University, Tokyo 171‐8501, Japan 5 RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan 6 Department of Basic Biology, SOKENDAI (The Graduate University for Advanced Studies), Okazaki 444-8585, Japan This directory contains 0_README (this file) 1_Marchantia_FL_data 2_Physcomitrella_FL_data 3_Physcomitrella_TEM_images 4_PAML 1_Marchantia_FL_data Raw image data using quantification of spermatid phenotypes in Marchantia. quantification.xlsx: Summary of quantification data. 211111 and 211210: Observation date. ∟Mpbld10-1, Mpbld10-2, and Tak-1: Observed lines. ∟raw data: *.lsm files are raw image data. ∟binary image: *.tif files are binarization images of the Hoechst33342 data. ∟DIC: *.tif files are maximum intensity projection images of the C2 channels (detection of DIC images) of the raw data. ∟Hoechst33342: *.tif files are maximum intensity projection images of the C1 channels (detection of Hoechst33342 signals) of the raw data. 2_Physcomitrella_FL_data Merged data of DIC and Hoechst33342 signal images using quantification of spermatid phenotypes in Physcomitrella. line22-*.png: The images of Ppbld10-22 mutant. line30-*.png: The images of Ppbld10-30 mutant. wt.png: The images of wild type. Number in the images 1: With flagella 2: Without flagella 3_Physcomitrella_TEM_images TEM images of spermatids in the Ppbld10-30 mutant, except for images shown in Supporting Information Fig. S11. 4_PAML Files using analysis by PALM. input.fasta: Input multi fasta file. species_tree.nwk: The gene tree file for the global clock model (rooted using chlorophytes as an outgroup). species_tree_marked.nwk: A gene tree file with marks specifying land plant stem and crown branches as category #1 (for a local clock model). clock1: A directory containing output files of a PAML run with the global clock model (clock = 1) clock2: A directory containing output files of a PAML run with a local clock model (clock = 2). The branches assumed to have a different rate (r1) than the default rate r0 are specified with #1 in species_tree_marked.nwk.

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

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

We have developed a simple Agrobacterium-mediated method for the stable transformation of the hornwort Anthoceros agrestis , the fifth bryophyte species for which a genetic manipulation technique becomes available. High transformation efficiency was achieved by using thallus tissue grown under low-light conditions. We generated a total of 216 transgenic A. agrestis lines expressing the β-Glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under the control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing, and land plant evolution in general.

Oogamy is a form of sexual reproduction and evolved independently in animals, fungi, and plants. In streptophyte plants, Charophyceae, Coleochaetophyceae, bryophytes, lycophytes, ferns (monilophytes), and some gymnosperms (Cycads and Ginkgo) utilize spermatozoids as the male gamete. Plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning plant spermatogenesis remain to be elucidated. To identify the genes involved in plant spermatogenesis, we performed computational analyses and successfully found deeply divergent BLD10s by combining multiple methods and omics-data. We then validated the functions of candidate genes in the liverwort Marchantia polymorpha and the moss Physcomitrium patens and found that MpBLD10 and PpBLD10 are required for normal basal body and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. Streptophyte BLD10s are orthologous to BLD10/CEP135 family proteins, which function in basal body assembly, but we found that BLD10s evolved especially fast in land plants and MpBLD10 might obtain additional functions in spermatozoid formation through the fast molecular evolution. This study provides a successful example of combinatorial study from evolutionary and molecular genetic perspectives that elucidated a function of the key protein of the basal body formation that fast evolved in land plants.

Hornworts comprise a bryophyte lineage that diverged from other extant land plants >400 million years ago and bears unique biological features, including a distinct sporophyte architecture, cyanobacterial symbiosis and a pyrenoid-based carbon-concentrating mechanism (CCM). Here, we provide three high-quality genomes of Anthoceros hornworts. Phylogenomic analyses place hornworts as a sister clade to liverworts plus mosses with high support. The Anthoceros genomes lack repeat-dense centromeres as well as whole-genome duplication, and contain a limited transcription factor repertoire. Several genes involved in angiosperm meristem and stomatal function are conserved in Anthoceros and upregulated during sporophyte development, suggesting possible homologies at the genetic level. We identified candidate genes involved in cyanobacterial symbiosis and found that LCIB , a Chlamydomonas CCM gene, is present in hornworts but absent in other plant lineages, implying a possible conserved role in CCM function. We anticipate that these hornwort genomes will serve as essential references for future hornwort research and comparative studies across land plants.

Extant land plants consist of two deeply divergent groups, tracheophytes and bryophytes, which shared a common ancestor some 500 million years ago. While information about vascular plants and the two of the three lineages of bryophytes, the mosses and liverworts, is steadily accumulating, the biology of hornworts remains poorly explored. Yet, as the sister group to liverworts and mosses, hornworts are critical in understanding the evolution of key land plant traits. Until recently, there was no hornwort model species amenable to systematic experimental investigation, which hampered detailed insight into the molecular biology and genetics of this unique group of land plants. The emerging hornwort model species,Anthoceros agrestis, is instrumental in our efforts to better understand not only hornwort biology but also fundamental questions of land plant evolution. To this end, here we provide an overview of hornwort biology and current research on the model plantA. agrestisto highlight its potential in answering key questions of land plant biology and evolution.

Evolutionary mechanisms underlying innovation of cell types have remained largely unclear. In multicellular eukaryotes, the evolutionary molecular origin of sperm differentiation is unknown in most lineages. Here, we report that in algal ancestors of land plants, changes in the DNA-binding domain of the ancestor of the MYB transcription factor DUO1 enabled the recognition of a new cis-regulatory element. This event led to the differentiation of motile sperm. After neo-functionalization, DUO1 acquired sperm lineage-specific expression in the common ancestor of land plants. Subsequently the downstream network of DUO1 was rewired leading to sperm with distinct morphologies. Conjugating green algae, a sister group of land plants, accumulated mutations in the DNA-binding domain of DUO1 and lost sperm differentiation. Our findings suggest that the emergence of DUO1 was the defining event in the evolution of sperm differentiation and the varied modes of sexual reproduction in the land plant lineage.

............................................................................................................................ 5 General Introduction.............................................................................................................. 6 Chapter 1: Isolation of Homeodomain-Leucine Zipper Genes from the Moss Physcomitrella patens and the Evolution of Homeodomain-Leucine Zipper Genes in Land Plants ............... 10 1-1. Summary 10 1-2. Introduction 11 1-3. Materials and Methods 12 Strain and culture conditions 12 Cloning the HD-Zip genes 12 Phylogenetic analysis 18 Difference of evolutionary rates between the HD-Zip I and II subfamilies 19 RNA gel blot analyses 20

Land plants evolved from freshwater algaelike ancestors approximately 480 million years ago. Land plants developed many new morphological features during the evolution including the origin of a multicellular diploid sporophyte, a sporophytic apical meristem that produces complex body architecture, stomata and the production of lateral organs (leaves), vascular tissue, roots, seeds and flowers. Genetic changes drove the development of these morphological features and recent technological innovations in genomics and genetic modification technologies have allowed us to investigate these genetic changes. Here, I describe recent research on several transcription factors that contributed to the morphological evolution of land plants, including the homeodomain proteins KNOX, BELL and WOX, as well as transcription factors of the bHLH, NAC and FLO/LFY families. I also discuss the conservation of plant hormone signalling, which acts to coordinate the multicellular body plan.

Land plant bodies develop from meristems, groups of pluripotent stem cells, which may persist throughout the life of a plant or, alternatively, have a transitory existence. Early diverging land plants exhibit indeterminate (persistent) growth in their haploid gametophytic generation, whereas later diverging lineages exhibit indeterminate growth in their diploid sporophytic generation, raising the question of whether genetic machinery directing meristematic functions was co-opted between generations. Class III HD-Zip (C3HDZ) genes are required for the establishment and maintenance of shoot apical meristems in flowering plants. We demonstrate that in the moss Physcomitrella patens, C3HDZ genes are expressed in transitory meristems in both the gametophytic and sporophytic generations, but not in the persistent shoot meristem of the gametyphyte. Loss-of-function of P. patens C3HDZ was engineered using ectopic expression of miR166, an endogenous regulator of C3HDZ gene activity. Loss of C3HDZ gene function impaired the function of gametophytic transitory meristematic activity but did not compromise the functioning of the persistent shoot apical meristem during the gametophyte generation. These results argue against a wholesale co-option of meristematic gene regulatory networks from the gametophyte to the sporophyte during land plant evolution, instead suggesting that persistent meristems with a single apical cell in P. patens and persistent complex meristems in flowering plants are regulated by different genetic programs.

Land plants evolved from freshwater algaelike ancestors approximately 480 million years ago. Land plants developed many new morphological features during the evolution including the origin of a multicellular diploid sporophyte, a sporophytic apical meristem that produces complex body architecture, stomata and the production of lateral organs (leaves), vascular tissue, roots, seeds and flowers. Genetic changes drove the development of these morphological features and recent technological innovations in genomics and genetic modification technologies have allowed us to investigate these genetic changes. Here, I describe recent research on several transcription factors that contributed to the morphological evolution of land plants, including the homeodomain proteins KNOX, BELL and WOX, as well as transcription factors of the bHLH, NAC and FLO/LFY families. I also discuss the conservation of plant hormone signalling, which acts to coordinate the multicellular body plan.

The life cycles of eukaryotes alternate between haploid and diploid phases, which are initiated by meiosis and gamete fusion, respectively. In both ascomycete and basidiomycete fungi and chlorophyte algae, the haploid-to-diploid transition is regulated by a pair of paralogous homeodomain protein encoding genes. That a common genetic program controls the haploid-to-diploid transition in phylogenetically disparate eukaryotic lineages suggests this may be the ancestral function for homeodomain proteins. Multicellularity has evolved independently in many eukaryotic lineages in either one or both phases of the life cycle. Organisms, such as land plants, exhibiting a life cycle whereby multicellular bodies develop in both the haploid and diploid phases are often referred to as possessing an alternation of generations. We review recent progress on understanding the genetic basis for the land plant alternation of generations and highlight the roles that homeodomain-encoding genes may have played in the evolution of complex multicellularity in this lineage.