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 carbonconcentrating 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.

SummaryHow genes shape diverse plant and animal body forms is a key question in biology. Unlike animal cells, plant cells are confined by rigid cell walls, and cell division plane orientation and growth rather than cell movement determine overall body form. The emergence of plants on land coincided with a new capacity to rotate stem cell divisions through multiple planes, and this enabled three-dimensional (3D) forms to arise from ancestral forms constrained to 2D growth. The genes involved in this evolutionary innovation are largely unknown. The evolution of 3D growth is recapitulated during the development of modern mosses when leafy shoots arise from a filamentous (2D) precursor tissue. Here, we show that a conserved, CLAVATA peptide and receptor-like kinase pathway originated with land plants and orients stem cell division planes during the transition from 2D to 3D growth in a moss, Physcomitrella. We find that this newly identified role for CLAVATA in regulating cell division plane orientation is shared between Physcomitrella and Arabidopsis. We report that roles for CLAVATA in regulating cell proliferation and cell fate are also shared and that CLAVATA-like peptides act via conserved receptor components in Physcomitrella. Our results suggest that CLAVATA was a genetic novelty enabling the morphological innovation of 3D growth in land plants.

Seeds of flowering plants can be formed sexually or asexually through apomixis. Apomixis occurs in about 400 species and is of great interest for agriculture as it produces clonal offspring. It differs from sexual reproduction in three major aspects: (1) While the sexual megaspore mother cell (MMC) undergoes meiosis, the apomictic initial cell (AIC) omits or aborts meiosis (apomeiosis); (2) the unreduced egg cell of apomicts forms an embryo without fertilization (parthenogenesis); and (3) the formation of functional endosperm requires specific developmental adaptations. Currently, our knowledge about the gene regulatory programs underlying apomixis is scarce. We used the apomict Boechera gunnisoniana, a close relative of Arabidopsis thaliana, to investigate the transcriptional basis underlying apomeiosis and parthenogenesis. Here, we present the first comprehensive reference transcriptome for reproductive development in an apomict. To compare sexual and apomictic development at the cellular level, we used laser-assisted microdissection combined with microarray and RNA-Seq analyses. Conservation of enriched gene ontologies between the AIC and the MMC likely reflects functions of importance to germline initiation, illustrating the close developmental relationship of sexuality and apomixis. However, several regulatory pathways differ between sexual and apomictic germlines, including cell cycle control, hormonal pathways, epigenetic and transcriptional regulation. Enrichment of specific signal transduction pathways are a feature of the apomictic germline, as is spermidine metabolism, which is associated with somatic embryogenesis in various plants. Our study provides a comprehensive reference dataset for apomictic development and yields important new insights into the transcriptional basis underlying apomixis in relation to sexual reproduction.

Background Activity and safety of the SARS-CoV2 BNT162b2 vaccine in actively treated patients with solid tumors is currently unknown. Methods We conducted a retrospective study of 326 patients with solid tumors treated with anti-cancer medications to determine the proportion of cancer patients with immunogenicity against SARS-CoV2, following two doses of the BNT162b2 vaccine. Control group was comprised of 164 vaccinated healthy adults. Anti-SARS-CoV-2 S IgG (Immunoglobulin G) antibodies (Abs) were measured, using level>50 AU/ml as cutoff for seropositivity. Adverse effects were collected using a questionnaire. All statistical tests were 2-sided. Results Most patients (205, 62.9%) were treated with chemotherapy, either alone or with additional therapy, 55 (16.9%) were treated with immune checkpoint inhibitors (ICI) and 38 (11.7%) with targeted therapy alone, 28 (8.6%) received other combinations. The vaccine was well tolerated and no severe side effects were reported. Among patients with cancer 39 (11.9%) were seronegative, compared to 5 (3.0%) of the control group (P=0.001). Median IgG titers were statistically significant lower among patients with cancer compared to control (931 AU/ml vs. 2817 AU/ml, P=0.003). Seronegativity proportions were higher in the chemotherapy treated group (19, 18.8%) compared to the ICI-treated patients (5, 9.1%) and to those treated with targeted therapy (1, 2.6%) (P=0.02. Titers were also statistically significant different among treatment types (P=0.002). Conclusion The BNT162b2 vaccine is safe and effective in actively treated patients with cancer. The relatively lower antibody titers and lower proportion of seropositive patients, especially among chemotherapy treated patients, call for continuing the use of personal protective measures in these patients, even following vaccination.

In this article, we characterized roles for CLAVATA in the development of a moss, Physcomitrella patens, focusing on the 2D to 3D growth transition. Ongoing work to further characterize mutant phenotypes identified some phenotype discrepancies among the Ppclv1a and Ppclv1b mutant lines published in the original paper. For this reason, we implemented further checks of the published manuscript and fully sequenced both PpCLV1a and PpCLV1b loci in all mutants originally reported in the Methods S1 figure ''CRISPR/Cas9 strategy for generating Ppclv1 mutants.'' Although the conclusions of the paper remain valid, our investigations revealed errors that we wish to correct. We found that Ppclv1a line 18 (Figures 4D and 4K; Methods S1, CRISPR figure, panel E) contained an 805 bp deletion at the PpCLV1a locus, but while Ppclv1a line 18 plants had phenotypes resembling WT plants, there was also a 9 bp deletion at PpCLV1b. We found no mutations at PpCLV1a or PpCLV1b in Ppclv1a line 29 or Ppclv1a line 32 (Methods S1, CRISPR figure, panels F and G), and these lines were indistinguishable from WT plants. The genotype of Ppclv1b line 2 (Figures 4E and 4L; Methods S1, CRISPR figure, panel H) was reported as a 2 bp deletion, but genome walking now confirms that there is a 4 bp deletion and >6 kb insertion at PpCLV1b, and the insertion comprises sequence integrated from the pACT::Cas9 expression vector used to engineer the lines [1]. The genotypes of Ppclv1b line 9 and Ppclv1b line 33 (Methods S1, CRISPR figure, panels I and J) were not previously reported, and while Ppclv1b line 9 has a 47 bp deletion at the PpCLV1b locus, Ppclv1b line 33 has no mutation at PpCLV1a or PpCLV1b, and an indistinguishable phenotype from WT plants. The reported genotypes of Ppclv1a1b lines 6, 8, and 12 were accurate (Methods S1, CRISPR figure, panels K-M). Consequently, we have re-engineered and fully sequenced PpCLV1a and PpCLV1b in three independent Ppclv1a and Ppclv1b mutant lines to verify the mutant phenotypes reported in Figure 4. The Ppclv1a and Ppclv1b mutant phenotypes previously reported hold true, and the genotypes and phenotypes of lines now in use are shown in Figure 1 below.

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 plant A. agrestis to highlight its potential in answering key questions of land plant biology and evolution.

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 βglucuronidase (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.

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