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SUMMARY: Plants colonized the land approximately 470 million years ago, coinciding with the development of apical cells that divide in three planes. The molecular mechanisms that underly the development of the 3D growth pattern are poorly understood, mainly because 3D growth in seed plants starts during embryo development. In contrast, the transition from 2D to 3D growth in the moss Physcomitrium patens has been widely studied, and it involves a large turnover of the transcriptome to allow the establishment of stage‐specific transcripts that facilitate this developmental transition. N6‐Methyladenosine (m6A) is the most abundant, dynamic and conserved internal nucleotide modification present on eukaryotic mRNA and serves as a layer of post‐transcriptional regulation directly affecting several cellular processes and developmental pathways in many organisms. In Arabidopsis, m6A has been reported to be essential for organ growth and determination, embryo development and responses to environmental signals. In this study, we identified the main genes of the m6A methyltransferase complex (MTC), MTA, MTB and FIP37, in P. patens and demonstrate that their inactivation leads to the loss of m6A in mRNA, a delay in the formation of gametophore buds and defects in spore development. Genome‐wide analysis revealed several transcripts affected in the Ppmta background. We demonstrate that the PpAPB1–PpAPB4 transcripts, encoding central factors orchestrating the transition from 2D to 3D growth in P. patens, are modified by m6A, whereas in the Ppmta mutant the lack of the m6A marker is associated with a corresponding decrease in transcript accumulation. Overall, we suggest that m6A is essential to enable the proper accumulation of these and other bud‐specific transcripts directing the turnover of stage‐specific transcriptomes, and thus promoting the transition from protonema to gametophore buds in P. patens. Significance Statement: The developmental transition from 2D to 3D growth in Physcomitrium patens involves transcriptome rearrangements. The N6‐methyladenosine (m6A) modification of mRNA is a post‐transcriptional regulator affecting cellular and developmental pathways in eukaryotes. The inactivation of genes encoding for m6A methyltransferase complex proteins leads to developmental defects in gametophore bud and spore. The PpAPB1–PpAPB4 transcripts, encoding for central regulators of bud formation, are modified by m6A, among many others, suggesting that m6A enables the proper accumulation of specific transcripts, promoting bud development. [ABSTRACT FROM AUTHOR]

The ability to address the CRISPR-Cas9 nuclease complex to any target DNA using customizable single-guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single-guide RNAs (sg RNAs) were designed to target an endogenous reporter gene, Pp APT, whose inactivation confers resistance to 2-fluoroadenine. Transformation of moss protoplasts with these sg RNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the Pp APT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end-joining (alt- EJ)-driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the Pp APT gene, most transgene integration events occur by homology-driven repair ( HDR) at the target locus but also that Cas9-induced double-strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR-mediated gene targeting events (30%) is still possible in the absence of Pp RAD51 protein, indicating that CRISPR-induced HDR is only partially mediated by the classical homologous recombination pathway. [ABSTRACT FROM AUTHOR]

Root hairs are a major site for the uptake of water and nutrients into plants, and they form an increasingly important model system for the study of development in higher plants. We now report on the molecular genetic analysis of thesrh1mutant inArabidopsis thalianaimpaired in root hair tip growth. We show thatsrh1is a new allele ofcow1(can of worms1) and we identified theCOW1gene using a positional cloning strategy. The N-terminus of the COW1 protein is 32% identical to an essential phosphatidylinositol transfer protein (PITP), the yeast Sec14 protein (sec14p) while the C-terminus is 34.5% identical to a late nodulin ofLotus japonicus, Nlj16. We show that expression of the COW1 lipid-binding domain complements the growth defect associated with Sec14p dysfunction in yeast. In addition, we show that GFP fused to the COW1 protein specifically accumulates at the site of root hair outgrowth. We conclude that the COW1 protein is a PITP, essential for proper root hair growth. [ABSTRACT FROM AUTHOR]

Plants colonized the land approximately 470 million years ago, coinciding with the development of apical cells that divide in three planes. The molecular mechanisms that underly the development of the 3D growth pattern are poorly understood, mainly because 3D growth in seed plants starts during embryo development. In contrast, the transition from 2D to 3D growth in the moss Physcomitrium patens has been widely studied, and it involves a large turnover of the transcriptome to allow the establishment of stage-specific transcripts that facilitate this developmental transition. N 6 -Methyladenosine (m 6 A) is the most abundant, dynamic and conserved internal nucleotide modification present on eukaryotic mRNA and serves as a layer of post-transcriptional regulation directly affecting several cellular processes and developmental pathways in many organisms. In Arabidopsis, m 6 A has been reported to be essential for organ growth and determination, embryo development and responses to environmental signals. In this study, we identified the main genes of the m 6 A methyltransferase complex (MTC), MTA, MTB and FIP37, in P. patens and demonstrate that their inactivation leads to the loss of m 6 A in mRNA, a delay in the formation of gametophore buds and defects in spore development. Genome-wide analysis revealed several transcripts affected in the Ppmta background. We demonstrate that the PpAPB1-PpAPB4 transcripts, encoding central factors orchestrating the transition from 2D to 3D growth in P. patens, are modified by m 6 A, whereas in the Ppmta mutant the lack of the m 6 A marker is associated with a corresponding decrease in transcript accumulation. Overall, we suggest that m 6 A is essential to enable the proper accumulation of these and other bud-specific transcripts directing the turnover of stage-specific transcriptomes, and thus promoting the transition from protonema to gametophore buds in P. patens., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Protoplast production with the moss Physcomitrium (Physcomitrella) patens has a long and successful history. As a tool, it has not only been the base of reverse genetic studies covering research fields as diverse as development, metabolism, or gene network regulation but also allowed its development as a bioengineering platform for protein production. We present here a standardized protocol for protoplast production from Physcomitrium (Physcomitrella) patens protonemata. Additionally, we detail procedures for their transfection, their plating for optimal regeneration, and three alternative selection approaches. To improve the consistency of protoplast regeneration, we describe a new option for protoplast embedding. The use of an alginate matrix to regenerate moss protoplast alleviates the use of warm agarized medium. Thus, it optimizes transformed protoplast survival without any morphological detrimental effect or impact on transfection efficiency., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)