Compilation of key molecular reporter lines published in literature to study cell and developmental processes in
Mosses are a cosmopolitan group of land plants, sister to vascular plants, with a high potential for molecular and cell biological research. The species Physcomitrium patens has helped gaining better understanding of the biological processes of the plant cell, and it has become a central system to understand water-to-land plant transition through 2D-to-3D growth transition, regulation of asymmetric cell division, shoot apical cell establishment and maintenance, phyllotaxis and regeneration. P. patens was the first fully sequenced moss in 2008, with the latest annotated release in 2018. It has been shown that many gene functions and networks are conserved in mosses when compared to angiosperms. Importantly, this model organism has a simplified and accessible body structure that facilitates close tracking in time and space with the support of live cell imaging set-ups and multiple reporter lines. This has become possible thanks to its fully established molecular toolkit, with highly efficient PEG-assisted, CRISPR/Cas9 and RNAi transformation and silencing protocols, among others. Here we provide examples on how mosses exhibit advantages over vascular plants to study several processes and their future potential to answer some other outstanding questions in plant cell biology.
- model organism
- plant development
- cell polarity
- asymmetric division
- stem cell
1.1 Mosses in context
Mosses are plants that belong to the Bryophytes, a cosmopolitan sister group of vascular plants with the last common ancestor between 400 and 500 million years ago [1, 2]. As a mostly avascular lineage, Bryophytes, that include mosses, liverworts and hornworts, thrive in mostly moist niches near the surface and stay compact (<10 cm), with some neovascularised exceptions that grow up to 65 cm [3, 4, 5]. Their cosmopolitan distribution in a variety of biotopes including moist and arid environments, can be explained by unique adaptations like drought, freezing and salinity tolerance [4, 6, 7]. Mosses’ life cycle is dominantly gametophytic (the photosynthetic and growing phase is haploid), and the size and architecture of their organs is smaller and simpler than that of vascular plants, with leaf-like structures (phyllids) and sexual organs (antheridia and archegonia) of often only one cell of thickness, stem-like structures of circa ten cells and spore-bearing containers (sporangia) of single-cell spores [4, 8, 9, 10].
This miniaturised body renders mosses accessible systems for the study and dissection of cell and molecular aspects of plant biology that require close monitoring in time and space . Such studies greatly benefit of the accessibility to single cells in a multicellular context. For instance, asymmetric and directional cell divisions are key life developmental tools to build an organism, but we lack understanding on how these processes are exactly controlled and regulated . Importantly, these developmental drivers are shared between mosses and vascular plants, and to some extent with animals, and associated gene functions seem to be highly conserved despite the long period of independent evolution .
In the last two decades, mosses have gained high interest in plant research, with
Hereby, we present how mosses, thanks to their simplified body plan and genetic networks in development, and with special focus in
1.2 Moss morphology and life cycle
As most land plants, mosses have alternating generations between the haploid gametophyte and diploid sporophytes. However, unlike vascular plants, mosses spend most of their life cycle in the gametophytic stage, in which most of the organism asexual development, including photosynthesis and growth, occurs. The sexual organs eventually develop at this stage to give rise to the embryo after fertilisation, that produces the sporophyte over the gametophyte. This fruiting stage is diploid until haploidisation in spore formation takes place [11, 14].
Starting from a spore, the first developmental stage of the moss is the chloronema, a chloroplast-rich and single cell-wide filamentous tissue that serves for initial colony expansion, early photosynthesis and nutrient absorption. The cells are slightly elongated (~80 μm long), and the intercellular cell walls are oriented perpendicular to the growth direction . This filament eventually transitions to caulonema, a quick-growing filamentous cell type that have underdeveloped chloroplasts at early stage, with longer and narrower cells (~250–300 μm long) that grow twice as fast, and with oblique intercellular cell walls [15, 16, 17]. This tissue has exploratory purposes and is favoured in stressful, light-poor, and nutrient-poor conditions, possibly with the aim of finding more suitable conditions [18, 19]. Caulonema can transition again to (secondary) chloronema . The filamentous tissues are collectively referred to as protonemata and can laterally grow and divide to branch as new filaments. The protonemata grow in a mat-like fashion that shapes the two-dimensional (2D) developmental stage of mosses where the growth is confined in a plane of few millimetres of thickness.
Sometimes, the lateral cell outgrowth (the cell initial) gives rise to a bud cell instead of a branch cell, which is the beginning of gametophore development and the transition to three-dimensional (3D) growth . The identity of the cell initial can be predicted by the division plane angle, implying that identity is determined before division (Figure 1). Currently, the list of known genes involved in the path selection and division plane orientation is growing, but the early determinants of bud formation and branching remain unknown [18, 21]. The bud grows by well-defined asymmetrical and oriented divisions to form the gametophore, the leafy shoot-like plantlet of mosses that ultimately bears gamete-producing organs.
The bud basal cell gives rise to a new type of filamentous tissue, the rhizoids, with pigmented, caulonema-like morphology. They function as anchorage to the ground to stabilise the up-growing gametophore and contribute to nutrient and water uptake, similar to roots and root hairs of seed plants, but with the tissue complexity of root hairs [22, 23]. The bud apical cells divide in precise directions to give rise to oriented phyllid initial cells with a particular phyllotactic pattern (i.e. lateral organ organisation around the shoot; e.g. spiral) to develop the gametophore.
The apical cells eventually arrest their proliferation, or terminally give rise to sexual organs (firstly antheridia, and later archegonia) under autumnal/spring conditions: short day (8 h), low light (20 μmol/m2/s) and low temperature (15°C) [24, 25]. Despite the asynchronous development of male and female gametangia, this moss is self-fertilising, and thus tends to genetically self-isolate . Flagella-driven spermatozoids (male gametes) move towards the archegonial venter in liquid water and fertilise the egg cell to give rise to a diploid zygote. The zygote will subsequently develop, via an embryonic stage with a new 2D-to-3D transition, into the sporophyte, that consists of the foot (the interface with the gametophyte) and a short stalk (seta) with a terminal capsule. In the capsule, meiosis gives rise to up to few thousands of haploid spores [8, 26].
The first documented ecotype, known as ‘Gransden’ (United Kingdom, 1962), has reduced rates of sporophyte formation, probably due to long asexual propagation in laboratories . In many laboratory lineages, it has become self-sterile, rendering it unattractive for studies dependant on sexual reproduction. On the contrary, the more recently isolated ecotypes ‘Villersexel’ (France, 2003) and ‘Reute’ (Germany, 2006) have 15 times more sporophytes (77% of total gametophores), indicating a high fertility rate . Despite these differences, all ecotypes can be propagated asexually in identical conditions from any tissue thanks to the high regeneration rate of this moss, through which explants will redifferentiate into chloronema and initiate the life cycle from there [28, 29].
2. Outstanding developmental processes
Several essential developmental processes are shared between all land plants, including angiosperms and bryophytes. Strikingly, in mosses many shared processes take place with a simplified set of genes and sometimes in single cells. Therefore, the underlying genetic regulatory networks of development are easier to study. In this section, we highlight some of these processes and their unique ease of study in the moss
2.1 The 2D-to-3D transition
The ability to structure organs in three dimensions (3D) was an essential feature for water-to-land transition. Many aquatic plants develop in a homogeneous environment, whereas land plants faced a highly distinct environment at ground surface level (a plane) and at the air/soil axis (perpendicular to this plane). This required land plants to develop specific tissues to efficiently grow in each dimension and cope with new challenges .
From a physiological perspective, the transition from 2D-to-3D growth in mosses consists in the development of complex structures such as the gametophore that grows out of the surface plane where protonema thrive, exhibiting negative gravitropism and positive phototropism [30, 31, 32]. The development of the gametophore relies on the ability of the moss to define different organisation in each dimension of space. The basal units from which this spatial assembly takes place are ultimately single cell primordia, from which organs emanate . To this end, a cell must spatially sense intrinsic and extrinsic signals and transduce them to subcellular structures that pave the way to division plane positioning and subsequent asymmetric and oriented cell division. However, the molecular basis of spatial sensing and its transduction to organisation, action and maintenance of the division machinery has not been fully elucidated yet .
Cellularly, the 2D-to-3D transition in a moss occurs during bud formation (Figure 1). When a cell initial is formed in protonemata, in 5% of the cases it has a bud initial cell identity instead of a branch initial identity. Each bud initial swells and undergoes divisions oriented in all three axes of space . This transition is supported and maintained by a different genetic and molecular machinery than that of protonemal development, and is the molecular basis of 3D growth. These proteins are specifically present from the first cell of the bud and onwards (e.g. DEK1, NOG1), and remains active in subsequent proliferating organs (e.g. shoot apical meristem, phyllids, gametangia) [13, 34, 35, 36]. In general, gradients or local clusters of proteins, peptides, nucleic acids, and hormones can be signals, sensors and/or actuators in developmental processes, and what is upstream of the cascade of 2D-to-3D transition remains a mystery. Some actors that are already on the radar include oscillations of auxin and cytokinin concentrations, ROP and SOSEKI proteins and CLE/CLV peptides-receptors, and they have been pinned down to different moments of the process and at different locations [12, 37, 38]. However, how do they coordinate their activity remains elusive and an active area of study.
In vascular plants, the 2D-to-3D transition occurs only once in their lifecycle, during embryogenesis, where orthologs of the essential moss 3D machinery genes are also expressed [34, 35]. This event is confined in the endosperm during embryogenesis and its observation in seed plants requires seed and ovule microdissection, which makes
These features provide a privileged seat for
2.2 The shoot apical meristem/cell (SAM/SAC) assembly
The protagonist of 2D-to-3D growth transition is the formation of the shoot apical meristem (SAM), a region at the apical growth side responsible for the continuous formation of the aerial organs of the plant, including leaves and reproductive organs [40, 41]. The histology of the SAM in angiosperms describes a central zone with stem cells that self-renew, divide and radially differentiate into peripheral cells that determine organ initiation at specific locations (e.g. by placement and establishment of new leaf primordia) . While angiosperms present multicellular stem cell centre SAMs during their sporophytic phase, bryophytes present an unicellular structure, the shoot apical cell (SAC), both in their gametophytic and sporophytic phase (Figure 1) . Despite the differences, both SAM/SAC share a common organisation, with stem cell(s) at the centre, surrounded by regularly differentiating tissue .
The mechanisms that establish and maintain these pluripotent stem cell(s) in the SAM/SAC is unknown. It involves spatial sensing, cell-to-cell communication, and asymmetric and oriented cell divisions, which render mosses attractive systems for their accessibility.
The developmental origin of the sporophytic SAC in
The genetic make-up in both taxa has shown to rely on auxin response through AUXIN RESPONSE FACTORS (ARFs), cytokinin signalling by ARABIDOPSIS RESPONSE REGULATORS (ARRs), CHASE domain-containing histidine kinases (CHKs) and CYTOKININ OXIDASE/DEHYDROGENASE (CKX), local coordination through several transcription factors families like CLAVATAs (CLVs), CUP-SHAPED COTELYDONS (CUC), LATERAL ORGAN BOUNDARY (LOB) and signalling peptides like CLAVATA3/EMBRYO SURROUNDING REGIONRELATED (CLE) and chromatin modification by Polycomb Repressive Complex 1 and 2 (PRC1,2), among others . Although many key factors have conserved roles in SAM formation and maintenance in seed plants and mosses, some important factors in angiosperms, like the key regulator of stem cell maintenance WUSCHEL, are not found in
2.3 Phyllotaxis from a cell
The most noticeable outcome of the shoot apical meristem/cell (SAM/SAC) activity is the organised and oriented initiation of leaf primordia along the stem, which leads to a unique geometric pattern of leaves and shoot branches named
However, the pattern arises from essentially different SAMs/SACs: in angiosperms, phyllotaxis derives from a multicellular system with well-reported oscillating auxin peaks around the SAM growth axis, whereas
The limited understanding of the origin and underlying molecular mechanisms of this rotating pattern and derived phyllotactic pattern is largely confined to the sporophyte of the evolutionarily recent group of flowering plants (angiosperms). The available transcriptomic data of bud and tip cells and gametophores (or 3D shoots) may provide more insight in the transition from uniplanar to triplanar meristematic growth in moss .
Aligned with the phenotypic similarities of moss and angiosperm phyllotactic patterns, several factors known from Arabidopsis have also been found in mosses, including receptor signalling genes involved in shoot meristem size and patterning, hormone biosynthesis genes, transcription factors that control cell-specific mechanism of developmental pattering, chromatin remodelling complexes and cell cycle . Many of these factors are essentially executive and likely controlled by some spatial sensing machinery. Comparing them with new contributors or absent members in the minimal regulatory network of mosses may help unravelling the fundamental elements that trigger the orientation-specification machinery that greatly impacts plant architecture in all land plants, including relevant crops.
Most described processes in this chapter require cell identity acquisition and maintenance. In certain circumstances (e.g. wounding), differentiated plant cells can reprogram to become new stem cells, divide and redifferentiate for organ
In most tissue cultures of other plant species, exogenous hormones (e.g. auxin and cytokinin) are required to induce callus formation and plant regeneration . However, in moss, cells are capable of regenerating from protoplasts or excised phyllids into new protonema filaments in the absence of exogenous hormones (Figure 1) . This implies that the whole regeneration toolkit is present in mosses and can be endogenously activated on demand, which makes them different from other taxa (e.g. angiosperms) .
When a phyllid is excised, cells neighbouring the cutting edge can reprogram from somatic cells to protonemal stem cells, which can then start a new life cycle . This regeneration process is easy to study in mosses for several reasons: firstly, cell identity conversion can be easily tracked with protonema stem cell reporters ; secondly, aside of the simplicity of
The mechanistic studies of the cell fate acquisition can benefit from this simple cell type conversion in comparison to other model systems used in cell reprogramming investigation (e.g. regenerative callus or Arabidopsis roots that consist of multiple cell types that possess different tissue identity) for its minimality and event frequency [65, 66]. In angiosperms, regeneration is often reduced to localised stem cell pools (e.g. the base of leaves), takes longer to establish, and it is multicellular and asynchronous at the explant level [58, 67].
Previous studies have taken advantage of the abovementioned features to investigate gene expression profile during phyllid cell reprogramming, which revealed that genes involved in stress, proteolysis, and hormone signalling pathways are induced from 6 to 24 h after cutting . Some genes have been demonstrated to play essential roles in moss leaf reprogramming, including
One interesting feature of moss regeneration is inhibition of neighbouring cells. The necessary cell–cell (apoplastic e.g. Ca2+-mediated) or cell-to-cell (plasmodesmata-mediated) communication makes regeneration an attractive developmental process to study this cell crosstalk [71, 72, 73]. The phytohormone ABA is a key responsible of the dynamic regulation of the permeability of plasmodesmata in response to changing environments, such as wounding. Control in plasmodesmata pore size can influence the signalling molecules that can pass through or can be blocked in particular cells, which can have a direct effect in development of the processes mentioned until now [74, 75].
2.5 Hormone regulation
The signalling pathways and functions of plant hormones are substantially conserved in
Three plant hormones—auxin, cytokinin and strigolactone—have shown to regulate shoot branching patterns (phyllotaxis) and activation in angiosperms. Auxin moves down the main shoot of angiosperms to inhibit branch development, while cytokinin promotes branching. In addition to branching, auxin is the key molecule in the control of plant growth and development, and promotes organ differentiation [76, 77]. Exogenous application of auxin or its inhibitors results in irregular cell shapes and inhibit lateral organ formation, for instance in shoot apical meristem (SAM) maintenance. The understanding of hormone regulation and signalling in angiosperms progresses slowly due to tissue and gene network complexity. In
Cytokinins and strigolactones also influence plant architecture, both in angiosperms and mosses. The levels of cytokinin are high and precisely distributed in the central stem cell region of SAM in angiosperms to maintain stemcellness . In the root apical meristem, auxin and cytokinin act antagonistically in meristem size control, but its levels, distribution and interaction in
As shown, many processes that define plant architecture are regulated in similar ways both in angiosperms and mosses. However, mosses offer a reduced gene network and regulation complexity that facilitates the analysis of hormone functions in the related developmental processes. Furthermore, subcellular and tissue-level transport and distribution of hormones can be best visualised in their simple plant bodies. In such plant models, new hormone functions will prove to be easier to study and translate to agronomically relevant plants.
3. Protocols and tools
Many valuable online resources with information on protocols, stocks, tools and genetic information have been exhaustively compiled elsewhere . Hereby, we provide some additional information and summary of the essentials of research in
The small size and simple architecture of moss organs allows detailed microscopic visualisation easy to accomplish in almost all tissues. The strings of cells in protonema and their branching is trivial to closely visualise, and the transition to gametophores can be well tracked until the stem-like centre becomes slightly thicker than a dozen of cells and grows out of the plane. From it, the leaf-like structures (phyllids) have only one cell of thickness except in the midrib and can be tore apart for up-close visualisation. The terminal sexual organs (antheridia and archegonia) have a 3D structure that is easy to fully dissect or directly visualise due to their monolayered sack structures. The subsequently developed spore-bearing containers (sporangia) are full of single-cell spores .
This miniaturised body renders mosses accessible systems for the study and dissection of cell and molecular biology that require close monitoring in time and space. Such studies greatly benefit of accessibility to single cells in their context for observation of subcellular responses
3.1.2 Reporter or marker lines
|Endomembrane system||Endoplasmic reticulum|||
|α-1,2-mannosidase||Golgi apparatus imaging|||
|Targeting signal type 1 (SKL)||Peroxisome imaging|||
|Mitochondria||Cytochrome c oxidase||Mitochondria imaging|||
|Tubulin α||Microtubule cytoskeleton|||
|MAP65||Antiparallel Microtubule-microtubule contacts|||
|Plasma membrane||SNAP-TM-mCherry||Membrane tracking|||
|Auxin||pDR5v2:GFP-GUS||Aux. induced fluor.|||
|GH3:GFP-GUS||Aux. induced fluor.|
|Protonema||pRM09:NLS4-GFP-GUS||Protonema identity reporting|||
|pRM55:NLS4-GFP-GUS||Protonema identity reporting|
Despite the advantageous physiological features of
Microfluidic devices are transparent and flexible structures commonly produced using the biocompatible and air-permeable polydimethylsiloxane (PDMS) polymer. In biology, they have been used for study and imaging of cell and tissue development
3.2.1 Forward genetics
Some groups carried out forward genetic screening by X-ray or chemical mutagens that generated mutants with hormone resistance or abnormal tropic responses [85, 98]. However, due to the lack of genomic information, the disrupted genes that caused the phenotypes were never identified.
Recently, the completion of genome sequencing and the establishment of its genetic mapping tools removed the obstacles in the forward genetic screening of
With this genetic mapping resource, in the past few years, researchers started to perform forward genetic screening by treating protoplast with UV light. In such screenings, mutants with phenotypic defects were successfully obtained and the causal lesions were identified by outcrossing and whole genome sequencing. Notably, essential genes that are crucial for growth may not be identified due to the lethality of their knock-outs; therefore, conditional screening was performed to overcome this problem. After the UV light treatment, plants were cultured under different temperatures and in such conditions, plants showed growth defect only in high temperature were selected as a temperature-sensitive mutant .
Another screening aimed to discover genes that are essential for the 2D-to-3D transition is also limited by developmental defects, given that mutants in this process cannot produce gametophores necessary for sexual crossing. To overcome this problem, instead of crossing, researchers generated somatic hybrids between Villersexel mutants and Gransden wildtype, which produced diploid sporophytes . Spores released from this hybrid sporophyte exhibited consistent phenotypic segregation ratio with meiosis. Mutant plants generated from these diploid spores were sequenced and genomically mapped to achieve the identification of new crucial genes for moss 2D-to-3D transition (e.g. NO GAMETOPHORES 1 and 2, or NOG1, NOG2).
In addition to UV light, tobacco Tnt1 retrotransposon was used to produce insertional mutations in genic and GC-rich regions . Both PEG- or Agrobacterium-mediated transformations were applied and successfully produced mutants.
3.2.2 Gene identification
3.2.3 Neutral locus integration
The integration of DNA constructs (including promoter, gene of interest and selection cassettes) necessary to produce transformants with stable expression and non-disrupting phenotypes requires targeting of neutral loci that do not intrinsically produce a phenotype when disrupted, often due to gene redundancy. In Table 2, there are several standard neutral loci indicated which reportedly showed no visible phenotypes or morphological defects when it is replaced by an entire gene expression cassette.
Currently there are several vector sets released to specifically target neutral loci, that contain their flanking regions homologous to parts of the locus at start and end of the vector. Cloning the gene of interest in between readily allows replacement of the targeted locus with the entire cassette via homologous recombination (see section
|XVE inducible overexpression|
XVE inducible RNAi expression
|Pp108 locus||pUGGi||Constitutive RNAi expression|||
|Pp108 locus||pTH-Ubi-Gate||Constitutive expression by the maize ubiquitin promoter|||
|Redundant copy of the ARPC2 gene||pTK-Ubi-Gate|
|Redundant copy of the ARPC3 gene||pTZ-Ubi-Gate|
|PTA1 locus||pT10G||Overexpression by EF1α promoter|||
3.2.4 Homologous recombination
3.2.5 Targeted double strand break and directed repair (CRISPR/Cas9)
The game-changing CRISPR/Cas9 method has proven an efficient and effective tool in
In Nogué’s lab, a co-delivery method was developed where each element of the system (Cas9, sgRNA and selection cassettes) was present in a separate plasmid. In this method, Cas9 expression is driven by an actin promoter and ready to use as is. The selection strategy can be chosen freely due to the lack of integration, minding the presence of resistance in the background lines. This method is also suitable for multiple mutations in different genes at once, given that more than one sgRNA plasmid can be simultaneously delivered in one transformation with still sufficiently high efficiencies of transformation .
In Bezanilla’s lab, a whole set of gateway destination vectors for CRISPR/Cas9 system was developed. The strategy was to design a vector set to finally put all the three essential components (Cas9, sgRNA and selection cassettes) in a single expression plasmid. In both protocols, the sgRNA can be designed and optimised using the online design tool CRISPOR V1 against
To increase the accuracy of mutations, the CRISPR/Cas9 system is applied with a homology-directed repair (HDR), which allows for seamless knock-in or point mutation in desired sites. The template DNA can be a donor plasmid that harbours homologous fragments or oligodeoxynucleotides (ODNs) [111, 113]. By co-transforming the plasmid or ODNs together with CRISPR/Cas9 and sgRNA vectors, both methods show high accuracy to generate a desired point mutation or scarless insertion with a fluorescence tag at any suitable location of the gene.
3.2.6 RNA interference
Given that moss possesses a relatively big gene family, arguably due to its double genome duplication, the employment of gene deletion strategies might be inefficient to investigate gene functions due to the high redundancy rate (e.g. there are four ROP genes with highly homologous or identical sequences) . For this, RNA interference (RNAi) strategies offer an alternative to overcome this problem, and the procedure has been well-established.
To generate an RNAi construct for a gene of interest, a DNA sequence of 300 to 1000 bp is subcloned in a destination vector. After standard PEG-mediated transformation, the silencing effect can be detected after 24 h and last up to 3 weeks . To avoid lethal effects when constitutively expressing interfering RNA, an oestradiol-inducible RNAi system is available . Coupling RNAi silencing activation with fluorescent reporters facilitates screenings of loss of function phenotypes .
Standard transformation protocols have been applicable to
|PEG/Mannitol||One to two round selection, 10% of transformants, 3–4 weeks for stable transformants||[114, 115, 116, 117]|
|Four-round selection, 100% positive transformants, 12–16 weeks|||
|Particle bombardment||Easy to conduct; less used.|
Transient and stable DNA integration.
Due to their accessibility, tractability and close yet independent phylogenetic position, the interest in Bryophytes has increased dramatically in the last decade . Beyond
Ceratodon purpureus(Hedw.) Brid.
The fire moss
4.2 Hypnales W.R. Buck & Vitt
The Hypnales (Bryopsida) are the biggest and most diverse order of mosses with varied morphology, and mostly exhibit pleurocarpous (i.e. non-erect) growth fashion. It includes
Remarkably, the common aquatic moss
4.4 Polytrichopsida Doweld
The moss class Polytrichopsida is the second biggest (~200 species) after Bryopsida (~11500 species), and its species have unique morphological characteristics that make them uniquely interesting for developmental biology . Despite mosses being regarded as avascular plants, some exhibit hydroids and leptoids, a functionally analogous tissue to tracheids and sieve elements of vascular plants that slightly differs morphologically and developmentally. Polytrichopsida has several genera with such structures, in some cases underdeveloped and, in others, completely functional, like in
Despite its potential to provide valuable insight, genetic tools have barely been developed for this taxon, but full mitochondrial and plastid genomes have recently been published for
4.5 Other mosses
The desert moss
The heavy metal-tolerant moss
We have shown how mosses are an increasingly relevant model group to plant developmental biology due to their distinct accessibility at physiological and molecular level. Their evolutionary distance with agronomically relevant plants does not diminish their potential to help to understand fundamental questions of development that remain unsolved, given that most essential regulatory networks are conserved. This has been shown in the hormonal regulation of branching and shoot and root development, regeneration, the establishment of shoot apical cells and the genetic make-up in 2D-to-3D transition. The utility of mosses has convinced the scientific community to the point of promoting the sequencing of eight new species to explore other physiological processes in the last few years. We have also shown how
The authors would like to thank Dr. Jeroen de Keijzer and Dr. Tijs Ketelaar for their thoughtful and detailed review of the manuscript. Also, the funding agencies Technology, Knowledge and Innovation, division Horticulture and Propagating Material (TKI T&U) and the Dutch Research Council (NWO) (reference number: TKILWV20.390) for funding JFC and the ERC grant to Prof. J. Friml (reference number: PR1023ERC02) for funding HT. The authors would like to sincerely apologise for the literature not cited that may be relevant for this chapter and is not present due to space constraints.