The Moss Physcomitrium (Physcomitrella) Patens: A Model Organism For Non-Seed Plants

The Plant Cell, Vol. 32: 1361–1376, May 2020, www.plantcell.org ã 2020 ASPB.
REVIEW ARTICLE
The Moss Physcomitrium (Physcomitrella) patens: A Model

[ ]
Organism for Non-Seed Plants OPEN
Stefan A. Rensing,a,1 Bernard Goffinet,b Rabea Meyberg,a Shu-Zon Wu,c and Magdalena Bezanillac,1
a Facultyof Biology, Plant Cell Biology, Philipps University of Marburg, 35037 Marburg an der Lahn, Hesse, Germany
Downloaded from https://academic.oup.com/plcell/article/32/5/1361/6115584 by guest on 06 March 2022

b Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, Connecticut 06269
c Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755
ORCID IDs: 0000-0002-0225-873X (S.A.R.); 0000-0002-2754-3895 (B.G.); 0000-0002-9977-4000 (R.M.); 0000-0003-1387-2011

(S.-Z.W.); 0000-0001-6124-9916 (M.B.)
Since the discovery two decades ago that transgenes are efficiently integrated into the genome of Physcomitrella patens by
homologous recombination, this moss has been a premier model system to study evolutionary developmental biology
questions, stem cell reprogramming, and the biology of nonvascular plants. P. patens was the first non-seed plant to have its
genome sequenced. With this level of genomic information, together with increasing molecular genetic tools, a large number
of reverse genetic studies have propelled the use of this model system. A number of technological advances have recently
opened the door to forward genetics as well as extremely efficient and precise genome editing in P. patens. Additionally,
careful phylogenetic studies with increased resolution have suggested that P. patens emerged from within Physcomitrium.
Thus, rather than Physcomitrella patens, the species should be named Physcomitrium patens. Here we review these
advances and describe the areas where P. patens has had the most impact on plant biology.
Introduction: Development of P. patens as a Model Genetics and Genomics of P. patens) and destined to become
Species a model for evolutionary developmental and cell biological studies
(see Hot Topics in P. patens Research).
P. patens Gransden was established as a model species based on
cultures derived from a single spore of a sample collected by
H.L.K. Whitehouse from a site in Gransden Wood (Huntingdon- Systematics, Ecology, and Geography of P. patens
shire, UK) in 1962. From the 1960s to the 1980s, this moss was
Physcomitrella belongs to the Funariaceae within the Funariales,
primarily used as a genetic system to isolate and study mutants in
a diverse lineage of small, typically once subapically branched
a variety of processes including plant morphology, hormone bi-
plants with terminal sex organs, possessing a sporophyte with
ology, nutrition, and responses to gravity (Engel, 1968; Ashton and
a capsule dehiscing via the loss of a lid, and two opposite sets of
Cove, 1977; Ashton et al., 1979; Cove, 1983). Early studies on the
peristome teeth lining the capsule mouth (Vitt, 1984). Phys-
effects of phytohormones (Reski and Abel, 1985; Knight et al.,
comitrella is, however, somewhat atypical for the family, as it lacks
1995; Machuka et al., 1999) revealed that the action of abscisic
both a peristome and a differentiated line of sporangial de-
acid (ABA) was conserved throughout the evolutionary history of
hiscence. Physcomitrella is a rather derived member of the family
land plants (Khandelwal et al., 2010; Richardt et al., 2010). In 1997
(Figure 1), having arisen ;10 million years ago (Beike et al., 2014;
(Schaefer and Zrÿd, 1997) reported that gene targeting via ho-
Medina et al., 2019), and may be sister to a clade comprising
mologous recombination was feasible in P. patens, triggering an
Aphanorrhegma serratum (now Physcomitrium serratum), Phys-
immediate generation of mutants (Girke et al., 1998), which,
comitrium immersum (both with immersed capsules), and Phys-
following the development of a method to induce sexual re-
comitrium collenchymatum and Physcomitrium sphaericum (both
production (Hohe et al., 2002) and initial transcriptomic charac-
with capsules emerging on a stalk from among the leaves; Medina
terization (Rensing et al., 2002), raised this moss to functional
et al., 2019).
genomics model status (Reski and Reg, 2004; Cove, 2005; Frank
Physcomitrella patens was originally recognized by Hedwig
et al., 2005). Due to its phylogenetic position as a member of the
(1801) in the genus Phascum (now Tortula, Pottiaceae) due to its
sister linage to vascular plants and thus other established model
small and immersed capsules lacking a peristome. Subsequently,
species, P. patens was soon targeted for genome sequencing (see
the species was transferred to the genus Ephemerum (Hampe,
1837), a phenotypically similar genus long considered to be allied
1 Address
to the Funariaceae (Vitt, 1984) but subsequently recognized as
correspondence to stefan.rensing@biologie.uni-marburg.de
and magdalena.bezanilla@dartmouth.edu.
resulting from convergent evolution in the Pottiaceae (Goffinet and
[OPEN]
Articles can be viewed without a subscription. Cox, 2000). In 1849, Physcomitrella was established as a single
www.plantcell.org/cgi/doi/10.1105/tpc.19.00828 species, P. patens (Bruch and Schimper, 1849). Subsequently, in
1362 The Plant Cell
recent expansion (Beike et al., 2014). By contrast, P. magdalenae

is endemic to Africa, where it is known to occur in three localities at
equatorial latitudes (Figure 2; Beike et al., 2014). Physcomitrium
serratum (formerly Aphanorrhegma serratum) is endemic to
Eastern North America (Goffinet, 2007a). All these species grow in
moist open soil along paths or in fields, or in seasonally wet areas
such as flood plains of lakes and edges of rice fields, at low to
moderate elevations.
Anatomy and Morphology of P. patens

Like all land plants, mosses possess a haplodiplontic life cycle in
which both generations, the haploid gametophyte and the diploid
sporophyte, are multicellular (reviewed in Rensing, 2016). How-
ever, unlike all extant polysporangiophytes (e.g., seed plants), the
dominant moss generation is the haploid phase, and the sporo-
phytic phase is monosporangiate (i.e., the sporophyte bears
a single terminal capsule). Starting from the germination of the
spore, i.e., the juvenile phase of the gametophyte, the protonema
develops (Figures 3 and 4). Protonemata are tip-growing filaments
that emerge and spread by branching and apical extension and are
composed of two major cell types: chloronema and caulonema.
Figure 1. Summarized Phylogeny of the Funariaceae.
Chloronema, the most basal cell type to emerge from the ger-
The phylogeny was inferred from variation in 648 target capture nuclear minating spore, are rich in chloroplasts and have cell plates that
exons, highlighting the polyphyletic distribution of Physcomitrella s. lat. are transverse to the long axis of the cell. The apical cell continues
(including Aphanorrhegma) within Physcomitrium (Medina et al., 2019). All
to divide, and several days after germination it transitions into
relationships between main lineages represented (i.e., genera), except
a caulonemal cell, which has obliquely positioned cell plates,
Entosthodon (i.e., bootstrap between 90 and 100%) are maximally sup-
ported under maximum likelihood analyses of a concatenated alignment of initially has fewer chloroplasts, and is typically thinner and grows
the nuclear loci, concordance among a majority of gene trees inferred from faster than the chloronema. In the absence of light but in the
exons and their flanking regions, and a coalescence-based gene tree presence of a carbon source, only caulonemal cells grow against
summary analysis (see Medina et al. [2019] for details). the gravity vector (Cove et al., 1978) and are then also referred to as
skotonema. Caulonemata are reminiscent of fungal runner hyphae
and may serve to cover long distances. Under the action of ABA,
1864, the species was moved to Aphanorrhegma (Lindberg, vegetative diaspores (brachycytes or brood cells) develop (Arif
1864), a then monospecific genus established by (Sullivant, 1848) et al., 2019), while the formation of specialized side branch initials
as an eastern North American endemic species resembling P. (so-called buds) represent the transition to the three-dimensional
patens except for its equatorial line of dehiscence of the spo- growth phase leading to the hormone-controlled development of
rangium. In 1851, an argument was made for accommodating P. gametophores, the leafy shoots of the moss plant (Reski and Abel,
patens in Physcomitrium (Mitten, 1851), a hypothesis congruent 1985; Harrison et al., 2009; Coudert et al., 2015).
with phylogenetic (Liu et al., 2012; Beike et al., 2014) and phy- Buds develop into erect, foliate gametophores. Gametophores
logenomic inferences (Medina et al., 2018, 2019), whereby the are structured into a stem and phyllids (also called leaflets;
species (Beike et al., 2014) or subspecies of Physcomitrella sensu nonvascular leaves). Both structures may possess specialized cell
(Tan, 1979) do not share a unique common ancestor (Liu et al., types for water and nutrient transport whose formation is con-
2012) but arose independently from within Physcomitrium (Fig- trolled by NAC transcription factors, a process similar to sporo-
ure 1; Beike et al., 2014; Medina et al., 2018, 2019). Consequently, phytic vasculature development in vascular plants (Xu et al., 2014).
all taxa of Physcomitrella were transferred to Physcomitrium, and The stem is short and considered to be unbranched (see Coudert
the correct name for Physcomitrella patens is thus Physcomitrium et al., 2017), and its inner cells are differentiated in an axial or
patens Mitten (Medina et al., 2019). The species is also known by central strand of long, narrow, thin-walled cells. The leaves are
its common name, the spreading earthmoss (Edwards, 2012). few, spirally inserted, and spreading when moist. They are typi-
cally ovate lanceolate to obovate, with a short, slender apex. The
Distribution blade is unistratose (comprising a single cell layer) except for the
median region, which is differentiated into a costa or midrib ex-
Physcomitrium (previously Physcomitrella) patens occurs in Eu- tending two-thirds up the leaf. The laminal cells vary from rect-
rope, North America, and East Asia (Medina et al., 2015; Higuchi angular to rhombic in shape, and are chlorophyllose throughout.
and Takahashi, 2012), whereas Physcomitrium readeri (including Under short day conditions (Hohe et al., 2002), gametangio-
Physcomitrium californica) is disjunct among Southern Australia, genesis occurs, yielding the male and female gametangia mixed in
Western North America, Japan, and Western Europe (Figure 2; a single cluster at the apex of now adult gametophores (Landberg
Medina et al., 2015). Such ranges are likely the result of a rather et al., 2013; Hiss et al., 2017). Individuals are bisexual, with male
The Moss P. patens 1363

Figure 2. P. patens.
(A) Geographic distribution (black triangle), contrasted to the distribution of P. readeri including P. californica (blue triangles), P. magdalenae (pink circles),
and P. serratum (orange area). Map adapted from Medina et al. (2015) based on specimens examined by Goffinet (2007a, 2007b) complemented by reports
by Faubert (2013) and Higuchi and Takahashi (2012).
(B) Example of an ecological habitat of Physcomitrium patens (i.e., lake floodplain in Yunnan China).
(C) A gametophore with a single terminal mature sporophyte. Scale bar 5 1 mm.
(antheridia) and female (archegonia) gametangia split between antheridia initially develop at the apex of the stem, which sub-
apical clusters (Goffinet, 2007b). In culture, mixed sex organs have sequently resume growth to develop archegonia, relegating the
rarely been observed (Nakosteen and Hughes, 1978). Instead, antheridia to an axillary position (Figure 4). Thus, under optimal
Figure 3. Life Cycle of P. patens.

Modified and reproduced from Strotbek et al. (2013), doi.org/10.1387/ijdb.130189wf with the permission of UPV/EHU Press.
1364 The Plant Cell

Figure 4. Anatomy of P. patens during Different Developmental Stages.
(A) and (B) Tip-growing filamentous protonemata eventually develop buds (A), which grow into leafy gametophores (B).
(C) and (D) Under cold- and short day conditions, sexual reproductive organs (gametangia) develop on the top (apex) of the gametophore.
(C) The male antheridia, which release motile biflagellate spermatozoids upon maturity, emerge in bundles comprising different developmental stages of
antheridia.
(D) The female archegonia arise next to the male organs and are flask-shaped.
(E) to (H) The egg is located in the archegonial venter and after fertilization, a sporophyte develops.
(E) Upon maturity, the brown sporophyte releases spores of the next generation.
(F) and (H) A heterozygote sporophyte of the fluorescent Reute-mCherry strain and the nonfluorescent Gransden strain.
(G) mCherry-fluorescent sporophyte is shown on top of the nonfluorescent gametophore, indicating a successful cross of both strains.
(H) Chloroplast autofluorescence is visible in the sporophyte as well as the leaflets.
Scale bars (A) and (C) 5 50 mm; (B), (F), (G), and (H) 200 mm; (D) 100 mm; and (E) 500 mm.
growth conditions, male and female sex organs are terminal, but sequence the P. patens genome. At the Seventh Annual Moss
when vegetative growth resumes after male gametangiogenesis, International Conference, MOSS 2004, in Freiburg, Germany,
female sex organs may appear to be present in a distinct cluster. a genome consortium was formed, and the P. patens genome was
Moss male gametes are biflagellate and require liquid water to subsequently sequenced by the U.S. Department of Energy Joint
swim into the archegonia (Renzaglia and Garbary, 2001). After Genome Institute. The nuclear genome was published in 2008
passing the archegonial neck canal cells and reaching the ar- (Rensing et al., 2008) and lived up to the expectations that its
chegonial venter (base), the sperm cells (also known as sper- comparison with the other available plant genomes would allow key
matozoids or antherozoids) fertilize the egg cell to form the diploid events in land plant evolution to be traced. The plastid genome was
zygote. The zygote develops into an embryo and then into published by Sugiura et al. (2003) and the mitochondrial genome by
a sporophyte, which possesses a single apical capsule in which Terasawa et al. (2007), completing the draft genomic resources.
meiosis eventually occurs, yielding haploid spores (Landberg Since 2010, P. patens has been one of the U.S. Department
et al., 2013; Hiss et al., 2017). A single sporophyte typically de- of Energy flagship genomes (https://jgi.doe.gov/our-science/
velops on a gametophore (Figure 4). The sporophyte is composed science-programs/plant-genomics/plant-flagship-genomes/), and
of a short stalk terminated by a spherical capsule covered by more genomic and transcriptomic resources continue to be
a small fleeting calyptra (thin hood) and bearing stomata defined developed. The genome assembly, previously consisting of
by a single guard cell in its lower portion. A differentiated line of ;2,000 scaffolds and containing ;5% gap regions, was re-
dehiscence is lacking, and the mature capsule opens irregularly to cently brought to the pseudo-chromosomal level, with most of
free the small unicellular spores. The sporophyte is anchored to the sequence data represented in 27 pseudochromosomes
the gametophore by a foot, with transfer cells at the intersection of (with 1% remaining gaps), as well as updated plastid and mi-
the two generations insuring the movement of nutrients (Regmi tochondrial genomes (Lang et al., 2018). Moreover, substantial
et al., 2017). The spores are covered in part by sporopollenin, transcriptomic evidence was generated using RNA sequenc-
a compound also found in male gametophytic pollen of seed ing (RNA-seq; Perroud et al., 2018; Meyberg et al., 2020),
plants (Daku et al., 2016). complementing microarray-based expression profiling data-
sets (Hiss et al., 2014; Ortiz-Ramírez et al., 2016) and epigenetic
data (Lang et al., 2018; Widiez et al., 2014). The most recent
Genetics and Genomics of P. patens
gene annotation (v3.3) used large-scale RNA-seq data from the
As outlined above, the phylogenetic position of the mosses be- P. patens gene atlas project as expression evidence. This re-
tween the (then already sequenced) genomes of Arabidopsis lease contains 32,458 gene models and 86,669 protein-coding
(Arabidopsis thaliana)/rice (Oryza sativa)/poplar (Populus tricho- transcripts and is available from different sources (Table 1), with
carpa) and Chlamydomonas reinhardtii prompted the idea to CoGe and Phytozome being the primary repositories.
The analysis of the most recent genome assembly (Figure 5)

revealed that, contrary to most flowering plants, the distribution of
genes and transposable elements (TE) is homogeneous along
the P. patens chromosomes (Lang et al., 2018). This is mirrored
by a homogeneous distribution of recombination rates, which
is potentially rooted in the observation that P. patens is pre-
dominantly selfing. Interestingly however, the purging of delete-
rious mutations works efficiently in this moss (Szövényi et al.,
2013). An analysis of the pseudochromosomal assembly also
suggested that giant viruses (nucleocytoplasmic large DNA vi-
ruses [NCLDV]) embedded into the genome become transcrip-

tionally active during gametogenesis and protect the gametes
from viral infection via siRNA-mediated silencing. In terms of
epigenetics, an analysis of histone modifications suggested that
protonemal tissue is epigenetically primed for the drought stress
response (a condition that likely affects the gametophore stage),
potentially representing an adaptation to the P. patens lifestyle
(Widiez et al., 2014). In contrast to flowering plants, where genes
carrying methylation in the gene body are expressed, most genes
showing DNA methylation within their gene bodies in P. patens are
not expressed (Lang et al., 2018).
Resources and Databases Figure 5. P. patens Chromosomes.
Visualization of the V3 pseudochromosomal nuclear genome assembly

and v3.3 annotation (Lang et al., 2018). Each chromosome is represented
Plant material
by a gray circle, the area of which corresponds to the length of the
For a long time, the P. patens Gransden accession going back to chromosome. Concentric circles reflect the relative amounts of TEs (yel-
the 1962 Whitehouse isolate was exclusively used for analysis. low) and genes (green), with the areas of each circle corresponding to the
total length. The 330 unordered scaffolds are represented as an extra green
This accession was mainly distributed vegetatively and spread to
and yellow circle. Figure courtesy of Fabian Haas.
laboratories around the world. However, many laboratories ob-
served a severe loss of fertility in their Gransden strains over time,
whereas others are still fertile. To overcome the problem of low 1:1 segregation of the F1 generation. Another ecotype, Villersexel,
fertility, a new ecotype, Reute, was recently introduced (Hiss et al., is genetically more divergent and has been used to generate the
2017). The use of Reute allows sexual reproduction to be analyzed genetic map used for the V3 genome assembly (Kamisugi et al.,
with ease. A multi-omic comparison of Reute versus Gransden 2008; McDaniel et al., 2010; Lang et al., 2018). The genetic var-
revealed the accumulation of (epi-)mutations that affect the fertility iability of Gransden, Reute, Villersexel and another accession,
of the male germline in Gransden (Meyberg et al., 2020). Moreover, Kaskaskia, was revealed using deep sequencing. Many more ac-
fluorescent mutant strains are available (Perroud et al., 2011, cessions have also been used in several studies (von Stackelberg
2019) that allow for routine crossing and make it easy to analyze et al., 2006; McDaniel et al., 2010; Liu et al., 2012; Szövényi et al.,
Table 1. Advances in Genome Editing
Technique Consequences References
Transient expression of Cas9 and guide Nonhomologous end joining (NHEJ) repair leads to indels and frame shifts (Collonnier et al., 2017)
RNA
Transient expression of Cas9 and guide NHEJ repair leads to indels and frame shifts (Lopez-Obando et al., 2016;
RNAs targeting multiple genomic sites Mallett et al., 2019)
Transient expression of Cas9 and guide HDR enables the insertion of sequences encoding fluorescent proteins and (Mallett et al., 2019)
RNA together with a homology plasmid small cassette containing stop codons in all possible frames to ensure that
the stop codon is close to the protospacer site
Transient expression of Cas9 and guide HDR enables the insertion of stop codons from the oligos to ensure that (Yi and Goshima, 2019)
RNA together with homology oligos the stop codon is close to the protospacer site
Transient expression of LbCas12A and Efficient method to multiplex target sites use the CRISPR RNAs; NHEJ (Pu et al., 2019)
multiple CRISPR RNAs results in diverse deletions
Transient expression of SpCas9-NG and Makes it possible to perform base editing by relaxing the stringency of the (Veillet et al., 2020)
guide RNA Protospacer Adjacent Motif sequence requirement
1366 The Plant Cell
2013; Beike et al., 2014; Medina et al., 2015, 2018, 2019). These modification). In terms of comparative genomics, Phytozome
accessions are available from the authors, as well as the In- allows cross-species gene families to be identified, PLAZA
ternational Moss Stock Center (http://www.moss-stock-center. allows synteny (gene order) analysis to be performed, and
org/). TAPscan lists transcription factors and transcriptional regu-
lators. Several tools make use of expression profiling data,
namely Genevestigator (Hiss et al., 2014), the P. patens eFP
Databases
browser (Ortiz-Ramírez et al., 2016), and most recently Phy-
The main repository for the most recent P. patens genome as- tozome (Perroud et al., 2018). The data present in these tools,
sembly is CoGe (Table 2). In addition to the genome sequence as well as novel data, were recently integrated into a common
(nuclear, plastid, and mitochondrion), this assembly contains web-based tool, PEATmoss (https://peatmoss.online.uni-
annotation (genes, TEs), genetic variability (single nucleotide marburg.de/ppatens_db/contact.php), which includes a gene

polymorphisms), and processed evidence from deep sequen- model lookup database that allows different versions of gene
cing (transcript evidence, DNA methylation, and chromatin annotations to be compared (Fernandez-Pozo et al., 2019).
Table 2. Key Features of P. patens and Online Resources
Category Feature/Resource
Lifestyle Haploid, gametophyte-dominant

Annual
Grows axenically in simple mineral medium
Self-fertile (predominantly selfing)
Stress-tolerant
Genetics and genomics Can be crossed for genetic studies
Gene targeting by homologous recombination
Genome editing by CRISPR/Cas9
Transfection of protoplasts and Agrobacterium-mediated transformation
Complete genomes, relatively small nuclear genome (500 Mbp, 27 chromosomes)
Transcriptome (expression profiling) data
Many mutants published
Genomes and primary Nuclear genome assembly V3 for browsing at CoGe (including gene models v3.3, single nucleotide polymorphisms, RNA-
annotation seq expression evidence, TE and noncoding RNA annotation, microarray probes, DNA methylation, and histone
modification)
https://genomevolution.org/coge/GenomeInfo.pl?gid533928
Nuclear, plastid, and mitochondrial genomes at CoGe
https://genomevolution.org/coge/OrganismView.pl?gid533928
Nuclear genome and gene models at Phytozome (including gene family assignment and RNA-seq expression evidence)
https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias5Org_Ppatens
P. patens gene model lookup database (for converting between different annotation versions)
https://peatmoss.online.uni-marburg.de/ppatens_db/pp_search_input.php
Online tools P. patens transcription factors and transcriptional regulators in TAPscan
https://plantcode.online.uni-marburg.de/tapscan/TAPs_for_species.php?species5Physcomitrella%20patens&id598
P. patens expression atlas PEATmoss (including microarray and RNA-seq dataset)
https://peatmoss.online.uni-marburg.de/expression_viewer/input
Older expression atlas tools include Genevestigator (https://genevestigator.com/gv/) and an eFP browser (http://www.
bar.utoronto.ca/efp_physcomitrella/cgi-bin/efpWeb.cgi)
P. patens in the PLAZA comparative genomics environment
https://bioinformatics.psb.ugent.be/plaza/versions/plaza_v4_dicots/organism/view/Physcomitrella1patens
https://bioinformatics.psb.ugent.be/plaza/versions/plaza_v4_monocots/organism/view/Physcomitrella1patens
P. patens in ENSEMBL plants
http://plants.ensembl.org/Physcomitrella_patens/Info/Index
Protocols and stocks Cold Spring Harbor protocols on cultivation of P. patens
http://cshprotocols.cshlp.org/content/2009/2/pdb.prot5136.abstract
Protonema cultivation at Bio-protocol
https://bio-protocol.org/e1556
PHYSCObase protocols
http://moss.nibb.ac.jp/protocol.html
International Moss Stock Center
http://www.moss-stock-center.org/
Protocols on the Bezanilla Laboratory website
https://sites.dartmouth.edu/bezanillalab/moss-methods/
Available Tools and Technologies for stably integrated RNAi constructs (Nakaoka et al., 2012)
have been employed. However, while gene silencing approaches
are often very effective, they can suffer from variable levels of
Transformation silencing.
Recent advances in genome editing technologies (Table 1)
To perform genetic manipulation, it is critical to be able to transfer capitalizing on CRISPR-Cas9–mediated double-stranded break
DNA molecules of choice into the organism. P. patens pro- formation are quickly overcoming the limitations of traditional
tonemata can be transformed with DNA by particle bombardment. homologous recombination and gene silencing approaches. The
This method allows for the stable incorporation of DNA (Cho et al., transient expression of a guide RNA and the Cas9 enzyme readily
1999) as well as transient expression assays (Bezanilla et al., 2003; produces double-stranded breaks in P. patens, which are repaired
Marella et al., 2006). However, early on in the development of by nonhomologous end-joining, invariably incorporating muta-

molecular genetics tools for P. patens, gene targeting events tions that generate frame-shift mutations (Collonnier et al., 2017).
were achieved using polyethylene-glycol (PEG)-mediated trans- Thus, by transforming P. patens with multiple guide RNAs and
formation of protoplasts (Kammerer and Cove, 1996; Schaefer Cas9, multiple genes can be targeted simultaneously to generate
and Zrÿd, 1997; Strepp et al., 1998). Given the proper osmoticum, higher-order knockouts in a single transformation (Lopez-Obando
P. patens protoplasts can be efficiently regenerated into whole et al., 2016). To ensure that regenerating plants have been
plants, allowing plants derived from a single protoplast harboring transformed with the plasmid containing Cas9 and/or the guide
DNA molecules of interest to be isolated. Thus, PEG-mediated RNA, the plants can be subjected to selection for ;7 d (Mallett
transformation has become the most widely used transformation et al., 2019). The selection pressure is then removed, and the
method. plasmid is lost. After the plants have grown to a sufficient size, DNA
is isolated from the plants to identify plants that have been edited.
Editing efficiencies can be as high as 90% depending on the
Reverse genetic approaches
efficiency of the protospacer and the expression levels of the guide
For over two decades, researchers have taken advantage of the RNA and Cas9 (Mallett et al., 2019). Various techniques, such as
innately high frequencies of homologous recombination in P. T7 endonuclease assays (Mashal et al., 1995) and competition
patens to insert DNA molecules into specific sites in the genome. PCR (Harayama and Riezman, 2017), can be used to rapidly
The insertion of sequences encoding fluorescent proteins has identify the edited sites.
opened the door to analyzing the localizations of fusion proteins When a homologous template is provided in addition to the
expressed from the native genomic context, which is critically guide RNA and Cas9, the double-stranded breaks are repaired
important, as the overexpression of fusion proteins often leads to from the homologous template, a process known as homology-
aberrant localization. Furthermore, the generation of gene dele- directed repair (HDR), allowing precise modifications to be in-
tions in P. patens has allowed gene functional analysis to be corporated at the cut site. In P. patens, the homologous template
performed throughout development. can be delivered as super-coiled plasmid (Collonnier et al., 2017;
However, because of gene family expansion due to recent Mallett et al., 2019) or as double-stranded oligos (Yi and Goshima,
genome duplications (Lang et al., 2018), which is common in 2019). Using HDR, it is possible to seamlessly integrate any
plants, the functions of medium to large gene families have been number of modifications, from single bp changes (Yi and Goshima,
difficult to study using traditional knock-out approaches due to 2019) to the integration of sequences encoding fluorescent
a large degree of functional redundancy. Furthermore, due to the proteins (Mallett et al., 2019). The overexpression or inducible
predominant haploid state of P. patens, it is impossible to isolate expression of a particular gene could be accomplished easily by
knock-outs of essential genes by transforming haploid proto- changing the promoter sequence.
plasts. To overcome these limitations, researchers have em- CRISPR-Cas9-mediated genome manipulation adds a power-
ployed gene silencing mediated by RNA interference (RNAi; ful set of genome editing tools to an already genetically tractable
Bezanilla et al., 2003, 2005; Nakaoka et al., 2012). The most system. In particular, CRISPR-Cas9 genome editing occurs
common approach is to transiently express a DNA construct without incorporating a selectable marker into the genome. The
containing sequences targeting specific genes arranged in in- selection is only used to ensure that the plants have taken up the
verted repeats. Using this approach, multiple genes can be tar- super-coiled plasmid enabling expression of the guide RNA and
geted simultaneously (Vidali et al., 2007). Transient expression of Cas9. After a week on selection, untransformed plants have died,
RNAi constructs in 7-d–old protonemata is a powerful approach to and the selection pressure is no longer needed. Using HDR
studying members of gene families (Vidali et al., 2007, 2009a) and coupled to CRISPR-Cas9, the genome can be altered seamlessly,
essential genes (Augustine et al., 2008) involved in protonemal making tagging a gene with a fluorescent protein at the 59 end
tissue development, particularly genes required for polarized trivial. In addition, super-coiled plasmids, which are easier to
growth. However, the stable integration of inverted repeat se- generate than linear DNA templates, act as efficient HDR tem-
quences does not occur at high frequency and thus, it can be plates. Finally, CRISPR-Cas9 editing provides an unbiased ap-
challenging to obtain stable transformants containing func- proach to uncovering hypomorphs of essential genes. With high
tional silencing constructs. Thus, to analyze later stages of de- rates of editing, null mutations in essential genes will result in very
velopment, the expression of artificial microRNA constructs few transformants. However, by targeting the gene at multiple
(Khraiwesh et al., 2010) and inducible expression using the sites with several protospacers, it may be possible to identify
estradiol-inducible system (Zuo et al., 2000; Kubo et al., 2013) viable edited plants with lesions that reduce the function of the
1368 The Plant Cell
protein. In addition to Cas9, a recent study demonstrated that robust forward genetic approaches by greatly expanding the
CRISPR using the programmable DNA endonuclease LbCas12a available tools and resources, making it possible to readily per-
efficiently edited the genome, expanding the tools available for form a variety of screens, such as enhancer and suppressor
gene editing in P. patens (Pu et al., 2019). screens.
Forward genetic approaches Imaging
In 1968, Engel used treatment with X-rays, as well as chemical Due to its small stature and anatomical simplicity, most P. patens
mutagens such as ethyl methanesulfonate, to isolate auxotrophic tissues are amenable to microscopic observation (Figures 4 and
and morphological mutants in P. patens (Engel, 1968). Sub- 6). Protonemal tissue is a two-dimensional network of filaments
sequent studies identified P. patens auxotrophs (Ashton and comprising single cells arranged in linear branching arrays. The

Cove, 1977), hormone-resistant mutants (Ashton et al., 1979), and developing gametophore emerges from a protonemal filament as
mutants with altered responses to gravity (Cove et al., 1978). a single cell that undergoes a series of stereotypic cell divisions
However, due to a lack of genetic mapping resources and the switching from polarized expansion to three-dimensional diffuse
inability to clone by complementation, the causal lesions for many expansion (Harrison et al., 2009). Expanded gametophore phyllids
of these mutants were never identified. Prigge et al. (2010) took (or leaflets) are only a single-cell-layer–thick, except for their
a candidate gene approach and identified the causal lesions in midribs. Given the simplicity of these tissues, microscopic ob-
a number of mutants resistant to the synthetic auxin, 1- servation readily occurs without the need for dissection or sec-
Naphthaleneacetic acid (NAA). More recently, the availability of tioning. In fact, with the recent advent of microfluidic imaging
a well-annotated genome coupled with marker lines in ecotypes devices, it is now possible to continuously observe development
that enable rapid identification of crossed sporophytes (Figure 4; from juvenile to adult tissues under a microscope at high temporal
Perroud et al., 2011, 2019) has paved the way for new genetic and spatial resolution (Bascom et al., 2016). These microfluidic
screens to identify causal mutations (Tables 3 and 4; Stevenson imaging devices are constructed from polydimethylsiloxane,
et al., 2016). In one such screen, Ding et al. (2018) mutagenized which is highly permeable to gases, allowing for continuous plant
protoplasts with UV light and isolated conditional loss of growth growth in a small dish filled with liquid medium in the presence of
mutants. Moody et al. (2018b) also used UV light mutagenesis to light. With designs that allow for protoplast trapping (Sakai et al.,
identify mutants no longer able to form gametophores. Without 2019) in addition to careful control of osmolarity (Bascom et al.,
gametophores, however, it would not be possible to cross the 2016), it is also possible to observe protoplast regeneration.
mutant strain to a different ecotype to obtain a mapping pop- Using these systems, coupled with versatile molecular manip-
ulation. To overcome this problem, Moody et al. (2018b) took ulation, numerous subcellular processes can be observed at high
advantage of somatic diploidization and performed a cross be- resolution. For example, by imaging the P. patens cytoskeleton
tween two somatic diploids. Both of these groups then suc- using a variety of imaging modalities, detailed studies of polarized
cessfully identified the causal mutations using whole-genome growth and cell division have been performed (Figure 6; Vidali et al.,
sequencing approaches on plants derived from their mapping 2009b, 2010; Furt et al., 2013; Wu and Bezanilla, 2014; Kosetsu
population (Ding et al., 2018; Moody et al., 2018b). In addition to et al., 2017; van Gisbergen et al., 2018; Wu and Bezanilla, 2018;
mutagenic agents, the tobacco (Nicotiana tabacum) Tnt1 retro- Yamada and Goshima, 2018; Kozgunova et al., 2019). Imaging
transposon can be introduced into P. patens using PEG-mediated fluorescent proteins in subcellular compartments has provided
protoplast transformation (Vives et al., 2016) or Agrobacterium-) insights into their dynamics (Figure 6; Furt et al., 2012). Imaging
mediated mutagenesis (Mohanasundaram et al., 2019), which using a recently developed P. patens ratiometric auxin-sensing
produced insertional mutations with a preference for genes and reporter revealed subtle differences between individual cells
GC-rich regions in the genome (Vives et al., 2016; Mohana- (Thelander et al., 2019). A ratio-metric calcium reporter was used to
sundaram et al., 2019). These studies have laid the foundation for monitor subcellular calcium levels during polarized growth (Bascom
Table 3. Forward Genetic Approaches Using UV Light as a Mutagen
Screen Identification of Lesion References
Isolated ABA mutants Crossed mutant plants to a distinct ecotype and performed (Stevenson
whole-genome sequencing on pooled mutants derived from the et al., 2016)
cross
Temperature-sensitive mutant screen identifying mutants that are Crossed mutant plants to a distinct ecotype and performed (Ding et al.,
unable to grow protonemata at the restrictive temperature whole-genome sequencing on pooled mutants derived from the 2018)
cross
Isolated mutants that were unable to form gametophores in an Took advantage of the ability of moss to form somatic hybrids; (Moody et al.,
effort to find genes involved in the transition from two- crossed two somatic hybrids to generate a mapping population 2018a)
dimensional to three-dimensional growth and performed whole-genome sequencing on mutants derived
from the cross
Table 4. Forward Genetic Approaches Using the Tobacco Tnt1 Retrotransposon as a Mutagen
Genomic Regions Showing

Transformation Method Insertions References
PEG-mediated transformation of protoplasts Generated mutations in genic regions (Vives et al., 2016)
Agrobacterium-mediated transformation Generated mutations in genic regions and GC-rich regions (Mohanasundaram et al., 2019)
et al., 2018) and plant-wide responses to dehydration in gametophore advances in our understanding of detailed cellular and subcellular
tissue (Storti et al., 2018). With the numerous approaches available to processes at high temporal and spatial resolution in a phylogenetically

image P. patens, this model system is poised to yield fundamental informative lineage of plants.
Figure 6. Fluorescence Imaging of P. patens.

(A) Confocal images of cells expressing fluorescent proteins targeted to different organelles. From left to right: Endoplasmic reticulum labeled with signal
peptide-mEGFP-KDEL, Golgi bodies labeled with the first 49 amino acids of the soybean a-1,2-mannosidase fused to YFP, vacuolar membrane (tonoplast)
labeled with mEGFP fused to PpVam3 (Pp3c2_35310), and the mitochondria labeled with the first 76 amino acids of the protein encoded by
Pp3c18_16000V3.1 fused to mCherry.
(B) Time-lapse confocal images of a cell expressing a mitochondria protein fused to mCherry. Mitochondria became fragmented when the cell enters cell
division (12 min). Scale bar 5 10 mm.
(C) Time-lapse confocal images of the endoplasmic reticulum (ER) and microtubules (MT) during cell division. The ER is labeled with signal peptide-mCherry-
KDEL and microtubules are labeled with mEGFP-tubulin.
(A), (B), and (C) Images are maximum projections of Z-stacks. Scale bars 5 10 mm.
(D) Images of Lifeact-mEGFP labeling the actin cytoskeleton, mEGFP-tubulin labeling microtubules, and endoplasmic reticulum acquired by variable angle
epifluorescence microscopy, which produces images of low background and high contrast, allowing fine structures to be observed at high temporal
resolution. Scale bars 5 2 mm.
1370 The Plant Cell
Missing Tools and Resources involved a particular species of Physcomitrium as one of the
parents (McDaniel et al., 2010; Beike et al., 2014; Medina et al.,
Furthering our understanding of genetic processes in P. patens 2018). Physcomitrium patens itself served as one parent for hy-
would benefit from a larger collection of accessions/ecotypes bridization with species of Physcomitrium (Britton, 1895) and even
encompassing broader genetic variation. Currently, we are with the more distantly related Funaria hygrometrica (von Wettstein,
considering Gransden, Reute, and Villersexel to be ecotypes, 1924). The ability to trigger somatic hybridization (Moody et al.,
because they show different phenotypes and genetic variability, 2018a) and to visualize hybridization events involving P. patens
and can be crossed (Lang et al., 2018). However, studies of (Perroud et al., 2011) provide unique tools to explore the genomic
genetic variability within populations are underway and will be consequences of hybridization in the Funariaceae. Autopolyploidy
needed to answer questions about genetic and epigenetic may be more common than allopolyploidy considering the large
variability. Also, while P. patens is comparatively well repre- number of species, such as P. patens, known to harbor more than
sented in terms of available accessions, this is not the case for P.

one karyotype (Fritsch, 1991). How autopolyploidy triggers spe-
magdalenae and P. readeri, for which additional accessions ciation in plants is a wide-open area of research and remains
would be helpful to study convergent trait evolution (see Hot completely unexplored in bryophytes. Yet, artificial autopolyploids
Topics in P. patens Research). While the above-mentioned In- are easily created in mosses through apospory, i.e., the de-
ternational Moss Stock Center makes it possible to store mu- velopment of a diploid gametophyte from (injured) sporophytic
tants and have them available to the community, there is no tissue. Furthermore, intragametophytic selfing, which may be
resource yet like the Arabidopsis Salk lines, i.e., no collection of preponderant in the Funariaceae, would result in a perfectly ho-
mutants that the community can draw upon. mozygous sporophyte. Consequently, the aposporous, diploid
In terms of databases, more unity of web-based tools and more gametophyte would in theory bear two identical copies of the
cross links would aid in finding and comparing information. It genome of the original haploid gametophyte, providing a unique
would be useful to develop a unified expression profile interface system among land plants for investigating the genomic or epi-
and gene model lookup database, as outlined above. genetic changes and their transcriptomic consequences in the
first generation after autopolyploidy. Ongoing studies in F. hy-
Hot Topics in P. patens Research grometrica, for which aposporous gametophyte are more readily
developed, reveal that autopolyploidy is immediately character-
ized by significant shifts in gene expression (Rahmatpour, 2019).
Polyploidy and hybridization
Polyploidy and hybridization have long been studied in mosses, Evolutionary developmental approaches
including Physcomitrella/Physcomitrium (von Wettstein, 1924;
Pettet, 1964; Fritsch, 1991; reviewed by Rensing et al., 2012). As outlined above, P. patens is an excellent moss for evolutionary
Engel (1968) described 14 chromosomes for P. patens Gransden. developmental biology (evo-devo) studies due to its phylogenetic
Approximately 26 chromosomes were subsequently identified position, its propensity for gene targeting via homologous re-
in this moss (Fritsch, 1991), while most recently, 27 chromo- combination, its relatively simple morphology, and the dominant
somes were identified (Reski et al., 1994). Due to their small size, haploid phase that is the hallmark of all bryophytes. The latter
it is difficult to observe mitotic chromosomes in P. patens. Even allows for the analysis of various mutants such as those affected in
before the genome was sequenced, an ancestral whole ge- sexual reproduction: in flowering plants, such mutants are often
nome duplication was evident in this species (Rensing et al., embryo-lethal. In such cases, the P. patens gametophytic gen-
2007). The genetic map used for the recent V3 assembly (Lang eration can nevertheless be grown and propagated. On top of that,
et al., 2018) is ordered into 27 linkage groups thought to rep- both generations can easily be tracked and accessed. Prominent
resent the chromosomes. Indeed, the most recent genome evo-devo studies over the past decade include studies revealing
assembly suggests that at least two rounds of genome dupli- the conservation of the polycomb group complex in repressing the
cation might have brought the ancestral chromosome number sporophytic body plan (Mosquna et al., 2009; Okano et al., 2009)
of seven to 14 and then to 27 (potentially by the fusion of two and of HD-TALE transcription factors in repressing the gameto-
chromosomes). phytic or promoting the sporophytic developmental program
Inferences from transcriptomic data reveal signatures of past (Sakakibara et al., 2003; Horst et al., 2016; Ortiz-Ramírez et al.,
whole-genome duplications across the phylogeny of mosses 2017). Another hot topic is identifying and analyzing the con-
(Devos et al., 2016; Johnson et al., 2016; Lang et al., 2018) unlike servation of regulators of plant three-dimensional and tip growth
the other two bryophyte groups (Lang et al., 2018), and karyo- (Spinner et al., 2010; Pires et al., 2013; Perroud et al., 2014, 2020;
logical data from extant populations suggest the widespread Proust et al., 2016; Moody et al., 2018b; Tang et al., 2020). The
occurrence of neopolyploidization within many species (Fritsch, relatively simple body plan of protonema and the observation that
1991). In mosses, as in other plants, polyploidy results from either they undergo tip growth and cell division enable them to be an-
hybridization or autopolyploidy. The former mechanism is not alyzed using approaches not feasible in many other plants. These
uncommon in mosses (Natcheva and Cronberg, 2004; Shaw, and other approaches have greatly increased our understanding
2009), although its significance during the diversification of of early land plant evolution. P. patens is in an informative phy-
mosses remains ambiguous. Hybridization may account for the logenetic position, facilitating trait inference during plant terres-
origin of several species in the Funariaceae, which might have trialization (Puttick et al., 2018; Rensing, 2018; Delaux et al., 2019).
The analysis of P. patens, together with other model non-seed Cell biology
plants and streptophyte algae (Bowman et al., 2017; Rensing,
2017; Nishiyama et al., 2018), furthers our understanding of how In the past 10 years, P. patens has become a key model system for
plants conquered land via the analysis of gene presence (com- studying plant cell biology. Due to the ease of genetically trans-
parative genomics) and conservation (evo-devo). forming plants and the ability to tag genes at their loci with DNA
encoding fluorescent proteins, it has become standard to analyze
the localization of proteins expressed from their native genomic
Reprogramming context. It is also routine to demonstrate that the tagged protein
Early on, P. patens was recognized as a unique system for is functional. With these tools in hand, a number of actin-
studying the molecular basis of stem cell reprogramming due to associated proteins have been identified that are required to
the ability of differentiated cells to re-enter the cell cycle (Kofuji and carry out tip growth (Vidali et al., 2007, 2009a, 2010; Augustine

Hasebe, 2014), de-differentiate, and form new protonemal cells in et al., 2008, 2011; Wu et al., 2011; van Gisbergen et al., 2012,
response to wounding and in the absence of any exogenously 2018). A tour-de-force study tagged every single kinesin gene
applied phytohormones. In fact, P. patens protoplasts can re- (encoding microtubule-based motors) in the moss genome and
generate into whole plants without exogenous hormones, which is analyzed their localization, discovering that some kinesins have
an extreme example of reprogramming in response to wounding. conserved functions, while others have surprisingly divergent
These attributes have been used to study the regulatory networks functions (Miki et al., 2014). Careful quantitative analysis of
involved in cellular reprogramming. An analysis of gene expres- organellar dynamics in moss protonemata demonstrated that
sion during reprogramming revealed that initially, genes involved most organelles in P. patens move significantly more slowly in
in stress responses and proteolysis were upregulated while genes protonemata than in pollen tubes or root hairs (Furt et al., 2012).
involved in metabolic processes, particularly photosynthesis, Analysis of organellar dynamics also demonstrated that moss
were downregulated. As reprogramming progressed, metabolic protonemata do not exhibit cytoplasmic streaming (Furt et al.,
gene expression including genes involved in photosynthesis and 2012). Thus, protonemata can be used to analyze the intracellular
biosynthetic processes recovered. Auxin and cytokinin signaling motility of organelles without the complexity of actively streaming
pathways were also activated (Nishiyama et al., 2012). Quanti- cytoplasm. Taking advantage of this feature, cell biological ob-
tative proteomic analyses of protoplast regeneration yielded servations coupled with loss-of-function studies have identified
similar results (Wang et al., 2017). Stemming from gene expression cargos for kinesin motors, such as the nucleus and chloroplasts
studies (Nishiyama et al., 2012), cyclin-dependent kinase A was (Suetsugu et al., 2012; Miki et al., 2015; Yamada et al., 2017; Yamada
found to be essential for coordinating cell cycle re-entry and and Goshima, 2018). These studies suggest that microtubules
protonemal gene expression required to establish a new stem might play important roles in organelle transport in plants, potentially
cell (Ishikawa et al., 2011). In addition, two WUSCHEL-related shifting the long-standing paradigm that plants predominantly use
homeobox13-like genes were shown to be required for cell growth actin, not microtubules, for long-distance intracellular transport.
during reprogramming and the establishment of stem cell fate, as Protonemal cells are an excellent system for studying both cell
genes for cell wall loosening factors were no longer upregulated division and polarized growth. Using these cells, the actin-based
in WUSCHEL-related homeobox13-like gene knockout lines molecular motor myosin VIII was found to couple the actin cy-
(Sakakibara et al., 2014). Intriguingly, Cold-Shock Domain Protein1 toskeleton to microtubules, effectively guiding phragmoplast
(PpCsp1), which shows sequence similarity and shared domains expansion. This study answered a long-standing question re-
with Lin28, a gene required for induced pluripotent stem cell garding the role of actin in cell division (Wu and Bezanilla, 2014).
generation in humans, is upregulated in P. patens cells Subsequently, myosin VIII was also shown to link actin to mi-
during reprogramming. The upregulation of PpCsp1 promoted crotubules at the apex of protonemal cells, serving to ensure
reprogramming, while deleting PpCsp1 and its closely related persistent polarized growth (Wu and Bezanilla, 2018). A number of
genes inhibited reprogramming. This study identified a potentially studies have begun to piece together the role of interdigitating
conserved stem cell factor functioning in both plants and animals microtubules at the phragmoplast equator in P. patens. The
(Li et al., 2017). Finally, by screening for factors involved in stem overlap zone, which requires the function of the microtubule
cell reprogramming, Ishikawa et al. (2019) discovered a tran- cross-linking protein Map65, is spatially confined by the micro-
scription factor from the AP2/ERF subgroup that, when overex- tubule motor Kinesin-4 and recruits components of the exocyst
pressed, altered the epigenetic landscape, decreasing repressive complex, thereby defining the region where vesicles are delivered
chromatin marks to a level sufficient to induce reprogramming in to build the new cell plate (Kosetsu et al., 2013; de Keijzer et al.,
undamaged differentiated cells. By isolating and examining the 2017; Tang et al., 2019). Relatively few studies have focused on
reprogramming ability of one, two, or three cells from game- mitosis in P. patens. However, a recent study (Kozgunova et al.,
tophores, Sato et al. (2017) discovered that there is an inhibitory 2019) demonstrated that the depletion of kinetochore proteins
signal that diffuses from the cell that alters the stem cell fate of leads to cytokinesis failures, resulting in polyploidy, which is in
neighboring cells, such that if two cells are isolated, 80% of the contrast to animal cells, where depletion leads to aneuploidy. This
time only one of the cells successfully reprograms. These studies study raises the intriguing possibility that cytokinesis failure may
have provided unprecedented insights into cellular reprogram- be a route to polyploidy in plants.
ming. Future studies are sure to mechanistically dissect the With respect to polarized growth, key factors essential for cell
signaling pathways involved in stem cell fate specification and polarity have been identified in P. patens, including the small
reprogramming. GTPase ROP (Burkart et al., 2015) and actin-associated proteins
1372 The Plant Cell
such as actin depolymerizing factor (Augustine et al., 2008), Ashton, N.W., Grimsley, N.H., and Cove, D.J. (1979). Analysis of
profilin (Vidali et al., 2007), and myosin XI (Vidali et al., 2010). Using gametophytic development in the moss, Physcomitrella patens,
rapid RNAi approaches, the functions of whole gene families using auxin and cytokinin resistant mutants. Planta 144: 427–435.
in polarized growth have been analyzed. The actin filament- Augustine, R.C., Vidali, L., Kleinman, K.P., and Bezanilla, M.
(2008). Actin depolymerizing factor is essential for viability in
promoting factors formins have class-specific functions, with
plants, and its phosphoregulation is important for tip growth. Plant
class-II rather than class-I formins essential for polarity (Vidali
J. 54: 863–875.
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of GTPase-activating proteins, exchange factors, and dissocia- M. (2011). Actin interacting protein1 and actin depolymerizing factor
tion inhibitors, all of which are encoded by small gene families. A drive rapid actin dynamics in Physcomitrella patens. Plant Cell 23:
systematic RNAi approach was used to identify regulators in- 3696–3710.
volved in tip growth (Bascom et al., 2019). Together, these studies Bascom, C.S., Jr., Wu, S.-Z., Nelson, K., Oakey, J., and Bezanilla,

have elucidated the framework for the molecular regulation of the M. (2016). Long-term growth of moss in microfluidic devices en-
polarized secretion of cell wall materials in P. patens, with many ables subcellular studies in development. Plant Physiol. 172: 28–37.
factors conserved across plant lineages. Bascom, C.S., Jr., Winship, L.J., and Bezanilla, M. (2018). Simul-
taneous imaging and functional studies reveal a tight correlation
between calcium and actin networks. Proc. Natl. Acad. Sci. USA
Outlook 115: E2869–E2878.
Bascom, C., Jr., Burkart, G.M., Mallett, D.R., O’Sullivan, J.E.,
P. patens represents only one lineage out of the many in mosses Tomaszewski, A.J., Walsh, K., and Bezanilla, M. (2019). Systematic
(e.g., orders in Liu et al., 2019). While more flowering plant models survey of the function of ROP regulators and effectors during tip growth
are being developed, the non-seed (or flagellated) plants are in the moss Physcomitrella patens. J. Exp. Bot. 70: 447–457.
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represent the monophyletic Setaphyta (Renzaglia et al., 2018), and Bezanilla, M., Perroud, P.-F., Pan, A., Klueh, P., and Quatrano, R.S.
it is highly probable that while the Marchantia lineage sub- (2005). An RNAi system in Physcomitrella patens with an internal
sequently lost genes and regulatory complexity, the Physcomi- marker for silencing allows for rapid identification of loss of function
trium lineage gained them (Puttick et al., 2018). Hence, we need to phenotypes. Plant Biol (Stuttg) 7: 251–257.
study both organisms, as well as additional species. The hornwort Bowman, J.L., et al. (2017). Insights into land plant evolution garnered
Anthoceros is currently being established as a model system, from the Marchantia polymorpha genome. Cell 171: 287–304.e15.
representing the third lineage of bryophytes (Szövényi et al., 2015). Britton, E. (1895). Contributions to American Bryology—IX. Bull.
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ACKNOWLEDGMENTS larized growth and cell adhesion. J. Cell Sci. 128: 2553–2564.
Cho, S.H., Chung, Y.S., Cho, S.K., Rim, Y.W., and Shin, J.S. (1999).
We thank Fabian Haas for contributing to Figure 5, and we thank members
Particle bombardment mediated transformation and GFP expres-
of the Bezanilla lab for carefully reading the article. This work was supported
sion in the moss Physcomitrella patens. Mol. Cells 9: 14–19.
by the National Science Foundation (grant DEB-1753811 to B.G.).
Collonnier, C., Epert, A., Mara, K., Maclot, F., Guyon-Debast, A.,
Charlot, F., White, C., Schaefer, D.G., and Nogué, F. (2017).
CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-
Received October 24, 2019; revised February 17, 2020; accepted March 5,
dependent and RAD51-independent gene targeting in the moss Phys-
2020; published March 9, 2020.
comitrella patens. Plant Biotechnol. J. 15: 122–131.
Coudert, Y., Palubicki, W., Ljung, K., Novak, O., Leyser, O., and
Harrison, C.J. (2015). Three ancient hormonal cues co-ordinate
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The Moss Physcomitrium (Physcomitrella) Patens: A Model Organism For Non-Seed Plants

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The Plant Cell, Vol. 32: 1361–1376, May 2020, www.plantcell.org ã 2020 ASPB.

The Moss Physcomitrium (Physcomitrella) patens: A Model

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ORCID IDs: 0000-0002-0225-873X (S.A.R.); 0000-0002-2754-3895 (B.G.); 0000-0002-9977-4000 (R.M.); 0000-0003-1387-2011

recent expansion (Beike et al., 2014). By contrast, P. magdalenae

Anatomy and Morphology of P. patens

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Figure 3. Life Cycle of P. patens.

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The analysis of the most recent genome assembly (Figure 5)

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Resources and Databases Figure 5. P. patens Chromosomes.

Visualization of the V3 pseudochromosomal nuclear genome assembly

Table 1. Advances in Genome Editing

Technique Consequences References

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Table 2. Key Features of P. patens and Online Resources

Lifestyle Haploid, gametophyte-dominant

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Forward genetic approaches Imaging

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Table 3. Forward Genetic Approaches Using UV Light as a Mutagen

Screen Identiﬁcation of Lesion References

Genomic Regions Showing

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Figure 6. Fluorescence Imaging of P. patens.

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