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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]

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.)