© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
WOX13-like genes are required for reprogramming of leaf and
protoplast cells into stem cells in the moss Physcomitrella patens
ABSTRACT
Many differentiated plant cells can dedifferentiate into stem cells,
reflecting the remarkable developmental plasticity of plants. In the
moss Physcomitrella patens, cells at the wound margin of detached
leaves become reprogrammed into stem cells. Here, we report that
two paralogous P. patens WUSCHEL-related homeobox 13-like
(PpWOX13L) genes, homologs of stem cell regulators in flowering
plants, are transiently upregulated and required for the initiation of cell
growth during stem cell formation. Concordantly, Δppwox13l deletion
mutants fail to upregulate genes encoding homologs of cell wall
loosening factors during this process. During the moss life cycle, most
of the Δppwox13l mutant zygotes fail to expand and initiate an apical
stem cell to form the embryo. Our data show that PpWOX13L genes
are required for the initiation of cell growth specifically during stem cell
formation, in analogy to WOX stem cell functions in seed plants, but
using a different cellular mechanism.
KEY WORDS: Stem cell, WOX, Physcomitrella
INTRODUCTION
Differentiated cells can dedifferentiate under both natural and
artificial conditions. In mice and humans, artificial expression
of transcription factors, for example, can cause transformation of
differentiated somatic cells into pluripotent stem cells (reviewed by
Masip et al., 2010). Stem cell regeneration from differentiated cells
1
2
National Institute for Basic Biology, Okazaki 444-8585, Japan. ERATO, Japan
3
Science and Technology Agency, Okazaki 444-8585, Japan. Department of
Biological Science, Graduate School of Science, Hiroshima University, Higashi4
Hiroshima 739-8526, Japan. BIOSS Centre for Biological Signalling Studies,
Faculty of Biology, Albert-Ludwigs-Universitä t Freiburg, Freiburg 79104, Germany.
5
6
Institut fü r Entwicklungsbiologie, Universitä t Kö ln, Kö ln 50674, Germany. School
of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8585,
7
Japan. Department of Plant Sciences, University of Oxford, South Parks Road,
8
Oxford OX1 3RB, UK. Graduate School of Science, Nagoya University, Furo-cho,
9
Chikusa-ku, Nagoya, Aichi 464-8602, Japan. JST, ERATO, Higashiyama
Live-Holonics Project, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi
10
464-8602, Japan. Advanced Science Research Center, Kanazawa University,
11
Kanazawa 920-0934, Japan. National Plant Phenomics Centre, Institute of
Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth
12
SY23 3EB, UK. Plant Global Education Project, Graduate School of Biological
Sciences, Nara Institute of Science and Technology, Ikoma 630-0192, Japan.
13
Faculty of Biology, University of Marburg, Marburg, Germany.
*Present address: Graduate School of Science, University of Tokyo, 7-3-1 Hongo,
Tokyo 113-0033, Japan.
‡
These authors contributed equally to this work
§
Authors for correspondence ([email protected]; [email protected])
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
Received 11 April 2013; Accepted 13 February 2014
1660
occurs more readily in plants, reflecting the remarkable plasticity of
plant cell identity (Birnbaum and Sanchez Alvarado, 2008). It can
occur under natural conditions (Steeves and Sussex, 1989) but also
after wounding or addition of phytohormones (Skoog and Miller,
1957). In seed plants, experimentally induced regeneration can
involve the formation of a mass of disorganized tissue, called the
callus, from which a complete plant can subsequently be obtained,
but regeneration without an intermediate callus stage has also been
reported (Williams and Maheswaran, 1986; Vogel, 2005; De Smet
et al., 2006; Garces et al., 2007; Sena et al., 2009). In non-seed
plants, differentiated cells can generate stem cells without the
requirement of exogenously supplied hormones and without
forming a callus (Chopra and Kumra, 1988; Raghavan, 1989). In
the moss Physcomitrella patens (Physcomitrella), differentiated
cells at the wound margin of detached gametophore leaves are
reprogrammed into apical stem cells, which subsequently produce
the filamentous chloronemata by asymmetric divisions (Prigge and
Bezanilla, 2010; Ishikawa et al., 2011). Although several
transcription factors involved in the induction of reprogramming
have been characterized in flowering plants (Banno et al., 2001;
Gordon et al., 2007; Sugimoto et al., 2010; Iwase et al., 2011), no
regulators have been reported in non-seed plants.
Seed plants contain three subclades of WUS-related homeobox
(WOX) genes (Haecker et al., 2004). First, several members of the
WUSCHEL (WUS) subclade are involved in stem cell maintenance:
WUS in the shoot meristem (Laux et al., 1996), WOX5 in the root
meristem (Kamiya et al., 2003; Sarkar et al., 2007), WOX3 in leaf
marginal meristems (Shimizu et al., 2009; Nakata et al., 2012) and
WOX4 in the vascular meristem (Hirakawa et al., 2010; Ji et al.,
2010). Expression of WUS promotes pluripotent stem cell fate in the
shoot apical meristem of Arabidopsis thaliana (Arabidopsis) from a
small underlying group of cells, named the organizing center
(Mayer et al., 1998; Schoof et al., 2000). In addition, WUS and
WOX5 are involved in the de novo formation of meristems
and somatic embryos from differentiated cells (Zuo et al., 2002;
Gallois et al., 2004; Gordon et al., 2007; Chen et al., 2009; Sugimoto
et al., 2010). Second, two members of the WOX9 clade, WOX8 and
WOX9, redundantly regulate zygote development and embryo axis
formation (Breuninger et al., 2008; Ueda et al., 2011) and WOX9 has an
additional role in shoot meristem regulation (Wu et al., 2005). Finally,
recent reports show that members of the WOX13 clade affect replum
development, and dehiscence, flowering time, lateral root formation,
and fertility (Deveaux et al., 2008; Romera-Branchat et al., 2012).
The WOX13 clade appears to contain the evolutionarily basal
members, whereas the WUS and WOX9 clades diverged during
the seed plant lineage. Concordantly, all WOX genes found in the
Physcomitrella genome group into the WOX13 clade (Deveaux
et al., 2008; van der Graaff et al., 2009; Nardmann et al., 2009). The
DEVELOPMENT
Keiko Sakakibara1,2,3, *,‡, Pascal Reisewitz4,5,‡, Tsuyoshi Aoyama1,6, Thomas Friedrich4, Sayuri Ando1,2,7,
Yoshikatsu Sato1,2,8,9, Yosuke Tamada1,6, Tomoaki Nishiyama1,2,10, Yuji Hiwatashi1,6,11, Tetsuya Kurata1,2,12,
Masaki Ishikawa1,2,6, Hironori Deguchi3, Stefan A. Rensing4,13, Wolfgang Werr5, Takashi Murata1,6,
Mitsuyasu Hasebe1,2,6,§ and Thomas Laux4,§
absence of WUS-clade WOX genes in mosses raises the question of
how stem cells are regulated. In contrast to the complex meristems
of seed plants, growth of mosses depends on single, apical stem
cells that undergo asymmetric cell divisions (Kofuji and Hasebe,
2014). In the gametophytic phase of Physcomitrella, chloronema
and caulonema stem cells form filaments (Prigge and Bezanilla,
2010) and the gametophore stem cell gives rise to a leafy shoot
(Harrison et al., 2009). In the sporophytic phase, the division of the
zygote generates an apical stem cell that gives rise to the embryo by
several asymmetric divisions, and a basal cell that forms part of the
foot structure (Kofuji et al., 2009). Here, we report that loss-offunction mutants of the Physcomitrella WOX13-like genes show
aberrations during stem cell formation from the zygote, protoplasts
and detached leaves. Phenotypic and molecular analysis implies a
crucial role for moss WOX13-like genes in the activation of cellular
growth programs.
RESULTS
PpWOX13-like genes are upregulated during stem cell
formation in detached leaves
Three loci containing WOX-related sequences in the Physcomitrella
genome (Nardmann and Werr, 2007; Richardt et al., 2007) cluster
with the Arabidopsis WOX13 clade (Deveaux et al., 2008; van der
Graaff et al., 2009). Given that Arabidopsis WOX10 and WOX14 in
this clade represent Brassicaceae-specific duplications of an
ancestral WOX13-like gene (Deveaux et al., 2008), we named the
moss homologs PpWOX13-like (PpWOX13L) genes (Fig. 1A). We
detected transcripts of PpWOX13LA [DDBJ/EMBL/GenBank
accession number: AB699867, formerly PpWOXA (Nardmann
and Werr, 2007) and PpaWOX02 (Deveaux et al., 2008)] and
PpWOX13LB [accession number: AB699868, formerly PpWOXB
(Nardmann and Werr, 2007) and PpaWOX01 (Deveaux et al.,
2008)] by RT-PCR throughout the moss life cycle, whereas no
transcript for PpWOX13LC was detectable at any developmental
stage examined (Fig. 1B).
To investigate the spatiotemporal expression patterns of
PpWOX13LA and PpWOX13LB proteins, we generated several
knock-in reporter lines by inserting a fluorescent protein coding
sequence just before the stop codon of each gene (supplementary
material Fig. S1A-H). Three different plants each of at least two
independently generated reporter lines were studied. All different
PpWOX13LA and PpWOX13LB reporter lines displayed
indistinguishable expression patterns (Fig. 1D-M; supplementary
material Fig. S1I-R) and no aberrant phenotypes were detected,
suggesting that the observed expression patterns are authentic. For
brevity, we focus on the PpWOX13LA-Citrine and PpWOX13LBCitrine reporter lines. Fluorescent signals of both fusion proteins
were detected in nuclei of all tissues examined: protonemata, young
and mature gametophores, antheridia, archegonia, unfertilized egg
cells, zygotes, sporophyte cells, and spores (Fig. 1D-M;
supplementary material Fig. S1I-R). Notably, the signal was
stronger in the apical stem cells of chloronemata and caulonemata
compared with subapical cells (Fig. 1D,E; supplementary material
Fig. S1I,J, arrows), and in egg cells and zygotes compared with
surrounding cells (Fig. 1I,J; supplementary material Fig. S1N,O,
red arrows).
In order to investigate PpWOX13L expression during the induced
formation of stem cells, we used a detached leaf assay, in which
chloronema stem cells are established from reprogrammed
differentiated leaf cells (Ishikawa et al., 2011). We observed that
whole-leaf PpWOX13LA and PpWOX13LB transcript levels
transiently increased after detachment (Fig. 1C). To investigate
Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
protein levels at a cellular level, we used the reporter lines and found
that the expression of both reporters slightly increased in most leaf
cells until 12 h after detachment and then specifically in the cells
facing the cut from which stem cells were generated (Fig. 1N;
supplementary material Fig. S1S and Movie 1). During subsequent
chloronema growth, the signal remained stronger in the apical stem
cell than in its descendants.
PpWOX13-like genes are required for outgrowth and
development of the zygote
To investigate the role of PpWOX13L genes, we generated several
independent deletion mutants (supplementary material Fig. S2A-I),
replacing each gene via homologous recombination either with a
cassette of a GUS reporter gene and a selection marker
(Δppwox13la1 and Δppwox13lb1) or with only a selection marker
cassette (Δppwox13la2 and Δppwox13lb2). Both single mutants did
not display any robust differences compared with the wild type at any
stage of the life cycle (data not shown). Although we observed
attenuated fertilization, zygote expansion, and subsequent cell
divisions in the single deletion lines (Δppwox13la2 and
Δppwox13lb2; supplementary material Table S3), they eventually
exhibited normal sporangium formation rates and further
development compared with wild type (supplementary material
Table S2). In the double mutant (Δppwox13lab), chloronemata,
caulonemata and gametophores were also indistinguishable from
wild type (supplementary material Fig. S2J-O). Expression of
PpWOX13LC was not detected by RT-PCR in the double mutant as
in the wild type (supplementary material Fig. S3). In contrast to wild
type, we observed fewer sporophytes in two independent double
deletion mutant lines (Fig. 2A,B; supplementary material Table S2).
A detailed confocal microscopic analysis revealed that the double
mutant is able to form sperm and an egg cell, and that it displayed
sperm in the archegonium cavity (Fig. 2D), a brownish archegonium
neck (supplementary material Table S2) and stronger autofluorescence in the nucleus than in the cytoplasm (Tanahashi et al.,
2005), and in these respects was indistinguishable from the wild type
(Fig. 2C-F; supplementary material Table S3). The last three criteria
are indicative of fertilization in wild type (Tanahashi et al., 2005).
Therefore, although we cannot exclude alternative explanations,
these findings strongly suggest that fertilization has taken place in the
double mutant.
After fertilization, the wild-type zygote expands by dispersed
growth, reaching the size of the archegonial cavity before undergoing
an asymmetric cell division into an apical stem cell and a basal cell
(Fig. 2G,H). Subsequently, ∼60% of brown-necked archegonia
contain an embryo with more than two cells 4 weeks after induction
of gametangia (Fig. 2H; supplementary material Table S3). By
contrast, in the double mutant, we observed only unexpanded
zygotes of a size similar to an unfertilized egg (Fig. 2F), but did not
find expanded or divided zygotes as in the wild type (Fig. 2G,H).
After 2 months of incubation under gametangium-inducing
conditions, no normal sporangia were found (supplementary
material Table S2). Several gametophores of the double mutant
produced malformed sporangia without normal spores, reminiscent
of sporangia formed by parthenogenesis (Tanahashi et al., 2005)
(Fig. 2B, inset).
To confirm further that the double mutant can form normal, fertile
sperm and egg cells, it was crossed with the wild type. To this end,
gametophores were submerged with distilled water, which increased
the rate of sporophyte formation. Sporangia were formed on 92.5
±3.9% (mean±s.d.; n=10) of 100 gametophores of self-crossed wild
type, and on 5.6±5.0% of self-crossed double deletion line (n=10).
1661
DEVELOPMENT
RESEARCH ARTICLE
Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
Fig. 1. Amino acid sequences and expression of PpWOX13L. (A) ClustalW alignment of full-length amino acid sequences of the WOX13-clade proteins
in Arabidopsis thaliana (At), Oryza sativa (Os), Physcomitrella patens (Pp) and Ostreococcus tauri (Ot). The homeodomain and the WOX13-specific
N-terminal domain (Deveaux et al., 2008) are highlighted. (B) Semi-quantitative RT-PCR of PpWOX13LA and PpWOX13LB expression during the life cycle
of Physcomitrella. RanD (Phypa55364) is used as RNA control. 7d, 7-day-old tissue containing only protonemata; 14d, 14-day-old tissue containing
protonemata and gametophores; 14dai, gametophores 14 days after gametangia induction; 1mai, gametophores with sporophytes one month after
gametangia induction. PCR reactions ran for 28 cycles with identical template concentrations. (C) PpWOX13LA and PpWOX13LB transcript levels (tags per
million, TPM) after leaf detachment determined by 5′-DGE (Nishiyama et al., 2012). Abscissae indicate the time after leaf detachment. Results of three
independent experiments are shown. (D-N) Localization of PpWOX13LA-Citrine fusion protein. (D-G) Bright-field images (upper images in D-F and left-hand
image in G) and fluorescent images (lower images in D-F and right-hand image in G) of chloronema (D), caulonema (E), young gametophore (F) and
gametophore (G). Arrows in D and E indicate nuclei of apical stem cells. (H-M) Laser scanning microscopy images of antheridia (H), an archegonium with a
ventral canal cell (yellow arrow) and an unfertilized egg cell (red arrow) (I), an archegonium with an unfertilized egg cell (red arrow) after degeneration of the
ventral canal cell (J), an archegonium containing a young sporophyte (K,L), and a spore (M). (N) Snapshots of Citrine fluorescence at indicated time after
leaf excision taken from a time-lapse movie (supplementary material Movie 1). Scale bars: 100 μm (D,E,H,N); 50 μm (F,I-L); 200 μm (G); 25 μm (M).
We observed that 8.9±3.6% of 100 gametophores of the double
deletion line that had been crossed with the wild type developed
sporangia (n=10). Among the sporangia on the crossed mutant
gametophores, we arbitrarily selected three sporangia with normal
morphology and sowed the spores on plates. Spores germinated
1662
normally and we collected all germinated chloronemata from each
line for genotyping for the PpWOX13L genes by PCR. In one of the
three lines, we identified both the wild type and the mutant alleles of
PpWOX13LA and PpWOX13LB, indicating that the Δppwox13lab
egg cell was fertilized with a wild-type sperm. In another two lines,
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
the analyzed chloronemata contained only mutant alleles. This
indicates that the double mutants can fertilize, at least under the
submerged crossing conditions. Together, these results indicate that
PpWOX13L genes are redundantly required for the outgrowth of the
zygote and the initiation of embryogenesis.
PpWOX13-like genes are required for initiation of cell growth
specifically during stem cell formation
Because of the upregulation of PpWOX13L reporters during stem
cell formation from detached leaves, we analyzed whether this
process requires PpWOX13 activity. In the wild type, live imaging
of detached leaves revealed that the majority of leaf cells at the
wound margin that had undergone division subsequently initiated
tip growth, giving rise to stem cells that produced chloronema
filaments by further divisions (Fig. 3A,C, red arrowheads). A
proportion of the divided cells, however, failed to initiate tip growth
and to undergo any further divisions within 72 h after detachment
(Fig. 3A,C, yellow arrowheads).
In contrast to the wild type, the Δppwox13lab double mutant
displays a strongly decreased frequency of divided cells that
initiated tip growth (Fig. 3B,C; supplementary material Movie 2).
We observed the same phenotype albeit at low frequency in some
Δppwox13la single mutant lines (data not shown), whereas no
difference from the wild type was detected for Δppwox13lb. Fortyeight hours after leaf detachment, expression of the protonemaspecific reporters RM09 and RM55 (Ishikawa et al., 2011) was
present in all cells that underwent divisions in both the wild type and
the double deletion mutant line Δppwox13lab#186 (Fig. 3D),
regardless of whether tip growth was initiated or not. This suggests
that cells at the wound margin of detached leaves, which enter
proliferation, acquire at least some aspects of chloronema identity
even if they are unable to initiate cellular growth. In summary,
during reprogramming of leaf cells into chloronema stem cells,
PpWOX13LA and PpWOX13LB are redundantly required for the
initiation of cellular growth, but not for entry into cell division or
expression of two chloronema identity markers.
As an independent test for PpWOX13L function in stem cell
formation, we analyzed the initiation of chloronema stem cells
from protoplasts. In the wild type, protoplasts cultivated under
high osmolarity conditions to prevent rupture firstly initiate tip
growth and secondly divide, producing a primary chloronema
stem cell, along with synthesizing a new cell wall (Jenkins and
1663
DEVELOPMENT
Fig. 2. Deletion of PpWOX13L genes blocks zygote
outgrowth. (A-H) Gametophores able to form sporangia (A,B),
an unfertilized egg (red arrow) and sperms (yellow arrowheads)
(C,D), a zygote (red arrow) (E,F), an expanded zygote (G) and a
two-cell stage sporophyte (H) are shown. Inset in B: malformed
sporangium. Criteria for fertilization are a brownish
archegonium neck (supplementary material Table S2) and
stronger auto-fluorescence in the nucleus than the cytoplasm
(Tanahashi et al., 2005). Outlines of cells and nuclei are
indicated in the images at the right (C-H). Bright field images
(A,B) and fluorescent images taken with a confocal laser
scanning microscope (C-H) of the wild type (A,C,E,G,H) and
Δppwox13lab#186 line (B,D,F). Scale bars: 5 mm (A,B);
100 μm (B, inset); 25 μm (C-H).
RESEARCH ARTICLE
Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
Cove, 1983). To investigate this process, we scored a primary
stem cell as established when the protoplast had elongated and
divided (supplementary material Fig. S3A). Subsequently, the
protoplast-derived stem cells form chloronema filaments while
still cultured on high osmolarity medium (Fig. 3E,G). During
side branch formation, subapical cells of these filaments form
lateral bulges, which become new chloronema stem cells by a
division roughly aligned to the filament surface (Fig. 3E). In the
double mutant, the formation of the primary stem cells from
protoplasts and of side branch stem cells was delayed compared
with the wild type (Fig. 3G, red dashed lines). In addition, we
observed chloronema subapical cells that failed to form a side
bulge like the wild type but rather divided in oblique directions
without formation of a side branch (Fig. 3E,F), reminiscent of the
defective stem cell formation in the leaf detachment assay
(Fig. 3B). Notably, when shifted to normal medium, the double
mutant formed side branches that were indistinguishable from the
wild type (data not shown), suggesting that a higher turgor is
required for cell outgrowth in the absence of PpWOX13L
activity, exposing a potential role of PpWOX13L in cell wall
expansion.
PpWOX13-like genes are required for upregulation of cell
wall loosening genes
Cellular outgrowth in plants requires the coordination of several
activities. First, the cell must have sufficient turgor pressure to
1664
overcome the cell wall resistance (Cosgrove, 2005; Taiz and Zeiger,
2010). Second, the cell wall becomes loosened at the site of growth
by enzymatic activities that disrupt cross-linkages between cellulose
microfibrils (Cosgrove, 2005; Taiz and Zeiger, 2010). In order to
investigate whether insufficient turgor might contribute to the
impaired outgrowth of reprogrammed leaf cells in the double
mutant, we determined the osmolarity required for plasmolysis of
the cells at the wound margin. When using 15 (supplementary
material Fig. S4B,C) or 45 (data not shown) minutes of incubation,
complete plasmolysis of wild-type cells requires 0.55 M mannitol,
whereas 0.6 M mannitol is required for the double mutant (n=50),
suggesting an even higher turgor in the double-mutant cells. Thus,
a reduced turgor is not the reason for the blocked outgrowth during
stem cell formation in the double mutant.
To analyze whether cell wall loosening might require
PpWOX13L function during stem cell formation, we compared
transcriptomes of wild-type and the double-mutant lines after leaf
detachment. To this end, we performed digital gene expression
profiling with mRNA 5′-end tags (5′-DGE) (Nishiyama et al.,
2012). We analyzed the expression profiles in intact gametophore
leaves (t=0) and gametophore leaves at 1.5, 3, 6, 12 and 24 h after
detachment. Comparison of expression between wild-type and the
mutant lines revealed <60 genes differentially expressed at each
time point until 6 h, and 115 and 254 genes were differentially
expressed at 12 and 24 h, respectively (supplementary material
Table S4). Notably, we found that cell wall-related genes are
DEVELOPMENT
Fig. 3. PpWOX13L genes are required for initiation of
tip growth during stem cell formation. (A) A wild-type
leaf cell that divided giving rise to one daughter acquiring
tip growth (red arrowheads). A divided cell without tip
growth is also indicated (yellow arrowheads). (B) In the
Δppwox13lab mutant, most leaf cells that divided did not
acquire tip growth. Yellow arrowheads in A and B
indicate septa of cells that divided after leaf detachment.
(C) Frequency of cells initiating tip growth at the wound
margin. n=16, corresponding to 223 and 297 cells.
Asterisks indicate significant deviation from the wild type
(P<0.01, Student’s t-test). Standard deviation given in
square brackets. (D) Expression of protonema-specific
reporter genes RM09 and RM55 are unaffected in
Δppwox13lab. Arrows indicate examples of
corresponding cells expressing GFP signal. (E-G) Stem
cell formation from protoplasts. (E,F) Bright-field images
and DAPI-stained filaments (right-hand side, bottom) of
regenerated protoplasts 13 days after plating.
Arrowheads and arrows indicate initiated chloronema
apical stem cells and the original protoplast cell,
respectively. In Δppwox13lab#186, several chloronema
cells have divided but did not initiate outgrowth (red or
white arrows in F). (G) Time course of stem cell formation
from protoplasts (n=50). Circles, triangles and crosses
indicate percentages of regenerated chloronemata from
protoplasts with at least one primary chloronema apical
stem cell, with at least one primary chloronema apical
stem cell and one side branch stem cell, and with at least
one primary chloronema apical stem cell and two side
branch stem cells, respectively. Bars indicate s.d. of
three independent experiments. Blue solid lines
and red dashed lines indicate the wild type and
Δppwox13lab#186, respectively. Asterisks indicate
significant differences in the numbers of apical cells
between the wild type and Δppwox13lab#186
(Mann–Whitney U-test, **P<0.01 in all three
experiments; *P<0.05 in all the three experiments).
Scale bars: 50 μm (A,B,D); 100 μm (E,F).
RESEARCH ARTICLE
Expression of cell wall loosening genes enhances stem cell
formation in chloronemata
We hypothesized that cell wall loosening genes might be involved
in stem cell formation. Because transformation of the double
mutant was not successful for unknown reasons, we tested this
hypothesis in wild-type protoplasts. To this end, we expressed
three predicted β-expansin genes (Fig. 4) driven by the rice actin
promoter (Zhang et al., 1991), which provides high expression
levels in protoplasts and protonemata (Horstmann et al., 2004).
We analyzed stem cell formation from protoplasts comparing
chloronemata with similar expression intensities based on a
co-transformed p35S:GFP reporter. Transformation with each of
two different β-expansin genes, denoted as 146965 and 96876,
resulted in a significant increase of stem cell formation (Fig. 4N)
compared with the untransformed control, whereas transformation
with β-expansin 150257 had no significant effect. We conclude
that in the wild type, β-expansin activity is a limiting factor for
stem cell formation, at least in chloronemata regenerated from
protoplasts.
Arabidopsis WOX13 and WOX14 are not essential during
regeneration of root meristem stem cells
The flowering plant Arabidopsis encodes three members in the
WOX13 clade, namely WOX13, WOX14 and the putative pseudogene WOX10 (Haecker et al., 2004; Deveaux et al., 2008). We found
that WOX13 and WOX14 promoters have transcriptional activity in
root meristems (Fig. 5E,F). In order to analyze whether Arabidopsis
WOX13 clade genes might play a role in regeneration of stem cells
after wounding, in similarity to stem cell regeneration from detached
leaves in moss, we performed root regeneration assays using
T-DNA insertion mutants (Fig. 5A-D). wox13-2 carries an insertion
in exon 1, and transcript could not be detected by RT-PCR
(Romera-Branchat et al., 2012), indicating that this mutant is a
strong loss-of-function or a null allele. wox14-1 was previously
described as pst13645 and has the homeodomain deleted (Deveaux
et al., 2008). We found that wox13-2 and wox14-1 single mutants
and the wox13-2 wox14-1 double mutant regenerated root
meristems at a similar efficiency as the wild type (Fig. 5G). Thus,
in contrast to Physcomitrella, we did not detect a regenerationrelated function for Arabidopsis WOX13-clade genes.
DISCUSSION
Stem cell formation in seed plants is tightly associated with the
function of WOX genes. Here, we addressed whether this association
might be conserved outside of the seed plants by studying WOX
functions in the moss Physcomitrella. We show that both
PpWOX13L proteins accumulate in all cells examined (Fig. 1), and
that the double knockout mutants had defects in stem cell formation
from cut leaves and in protonemata derived from protoplasts as well
as in zygotes (Figs 2, 3). In all cases, cell wall expansion was arrested
(Figs 2, 3). Expression of cell wall loosening genes, including βexpansin genes, was reduced compared with wild type in the cut
leaves of the double mutant (Fig. 4), and β-expansin induction in
wild-type protoplasts enhanced the stem cell formation.
Together, these results indicate that the two moss PpWOX13L
genes are redundantly required for the formation of stem cells in cut
leaves and from protoplasts. The failure to initiate cell growth is the
first phenotypic manifestation during stem cell formation in the
double mutant, and the upregulation of genes encoding cell wall
loosening enzymes is reduced. Because normal growth of
chloronema and caulonema is not impaired, we propose that
moss WOX13L activity is involved in cellular growth specifically in
the process of stem cell formation at least in protoplasts and
dissected leaves.
The role of WOX13L in Physcomitrella
During stem cell formation from differentiated leaf cells, several
cellular changes, including the re-entry into cell cycle and the
initiation of tip growth, occur (Ishikawa et al., 2011). Our results
indicate that during this process: (1) the expression levels of
PpWOX13LA and PpWOX13LB genes are upregulated, (2) both
genes are redundantly required to initiate cellular outgrowth but not
for entry into the cell cycle, and (3) both genes are required for
upregulation of genes predicted to encode cell wall loosening
factors. Likewise, stem cell formation from protoplasts was delayed
in the double mutant. Furthermore, the initial outgrowth of a lateral
bulge during establishment of side-branch forming stem cells from
chloronema subapical cells was impaired. Curiously, this defect was
seen only on high but not on low osmolarity medium. We reason
that owing to the reduced cell wall loosening in the double mutant, a
higher turgor pressure is required for cell expansion. Finally, most of
the zygotes in the Δppwox13l double mutant neither elongated nor
entered cell division to form an apical stem cell, unlike the wild
type. Although we cannot exclude that in the zygote, PpWOX13L
genes are involved in cell outgrowth and division separately, a
plausible hypothesis in analogy to the effects in induced stem cell
regeneration is that the initiation of cell growth is primarily regulated
by WOX13L function in the zygote, and that the failure to undergo
cell division is a consequence thereof.
Is there a common function of PpWOX13L underlying all the
observed defects? We propose that the phenotypes observed in
the leaves, protoplasts and zygotes of the double mutant can be
explained by an impaired activation of cell growth due to insufficient
upregulation of cell wall loosening activities. Although zygotes and
stem cells employ different growth modes (diffuse cell growth
and tip growth, respectively) the requirement of cell wall loosening
enzymes is likely to be key in both processes. Further studies on
gene regulatory networks in reprogramming leaf cells and
protoplasts as well as on zygotes are necessary to examine
whether PpWOX13L genes function similarly in these cells.
1665
DEVELOPMENT
enriched in the set of genes with reduced expression in the mutant
compared with the wild type at 12 and 24 h after leaf detachment
(supplementary material Table S5). Among them, we found that 12
mRNAs predicted to encode cell wall loosening enzymes,
including expansins, xyloglucan-specific endo-β-1,4-glucanases
(XEG), xyloglucan endotransglucosylases/hydrolase (XET) and
pectin methylesterases (PME), are upregulated in the wild type
after leaf detachment, whereas this is not the case to the same extent
in the double mutant (Fig. 4A-L). For eight of these genes, the
lower expression levels in the mutant compared with the wild type
were confirmed by quantitative RT-PCR (supplementary material
Table S6). Furthermore, we found that during stem cell formation
from protoplasts, transcript levels of three β-expansin genes are
significantly lower in the double mutant than in the wild type,
although those of one α-expansin and XET are not (supplementary
material Table S6). By contrast, we did not detect different
transcript levels between the wild type and the double mutant for
genes associated with cellular tip growth, such as actin
cytoskeleton- or polar membrane transport-related functions
(supplementary material Table S4).
In summary, PpWOX13L activity is required for the upregulation
of specific cell wall loosening enzymes in protoplasts and cells of
detached leaves that regenerate stem cells.
Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
RESEARCH ARTICLE
Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
Fig. 4. PpWOX13 L activity regulates cell wall
loosening genes. (A-L) Accumulation patterns
of transcripts encoding cell wall loosening genes
after leaf excision in 5′-DGE analysis. Protein IDs
are shown above each graph. Horizontal axes
indicate the time after leaf detachment and
vertical axes indicate tags per million (TPM)
values. Blue and red lines indicate expression
levels in the wild type and the Δppwox13lab#186
double mutant, respectively. Results of three
independent experiments are shown.
(M,N) Transient expression of β-expansins
(146965 and 96876) increases apical stem cell
formation during protoplast regeneration in the
wild type, whereas β-expansin 150257 had no
significant effect. Primary chloronemal apical cell
and secondary chloronemal apical cells derived
from subapical cells are indicated by arrows and
arrowheads, respectively, and the number of
secondary apical cells is shown in the upper right
(M). The total number of secondary chloronema
apical stem cells was counted at the fifth day after
protoplast regeneration on the high osmolarity
medium. The total number of observed protoplast
regenerations is 60 (divided among three
biological replicates) for every construct.
Significantly different distributions from the
control are shown with P-values (Mann–Whitney
U-test). Scale bars: 50 μm.
1666
Comparison with Arabidopsis
The significance of cell growth regulation in stem cells has not been
studied yet in seed plants, where the formation of stem cells and
zygote development are also regulated by WOX genes. First, several
members of the WUS clade in seed plants function in maintaining
pluripotent stem cells (Aichinger et al., 2012). In Arabidopsis,
these include WUS in the shoot meristem (Laux et al., 1996), WOX5
in the root meristem (Kamiya et al., 2003; Sarkar et al., 2007),
WOX4 in the cambium (Hirakawa et al., 2010; Ji et al., 2010) and
WOX1 together with PRS (WOX3) in the leaf margins (Shimizu
et al., 2009; Nakata et al., 2012). Notably, it is the formation of the
primary shoot meristem in the embryo that is most sensitive to
reduction of WUS activity, whereas maintenance of stem cells
DEVELOPMENT
Notably, all mutant defects are restricted to formation of apical
stem cells, whereas growth of the established filaments by their
apical stem cells is unaffected. The broad expression of PpWOX13L
genes suggest yet unknown functions in non-stem cells.
Alternatively, the global expression of PpWOX13L genes could
contribute to the general competence for stem cell formation. Stem
cell regulators that are not restricted to the stem cell niche include,
for example, WUS and WOX5 in Arabidopsis, which, in addition to
their stem cell niches, are expressed in ovules and cotyledons,
respectively (Groß-Hardt et al., 2002; Sarkar et al., 2007). In
conclusion, we propose that PpWOX13L activity is required for the
onset of cell growth programs specifically during stem cell
formation, but not for maintaining them.
RESEARCH ARTICLE
Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
during inflorescence meristem growth is less sensitive (Graf et al.,
2010). Furthermore, both WUS and WOX5 are essential in
regenerating stem cells via tissue culture (Su et al., 2009;
Sugimoto et al., 2010). The requirement for WUS and WOX5
specifically during formation of stem cells but to a weaker extent
during further development is similar to that for PpWOX13L in
moss stem cell formation reported here. However, there are also
notable differences. Whereas in moss, we found that the activation
of the cell growth program is an essential part of PpWOX13L
function, in Arabidopsis, WUS and WOX5 genes may have a
repressive effect on cell enlargement based on their cellular mutant
phenotypes (Laux et al., 1996; Sarkar et al., 2007). One interesting
possibility to be tested in the future is whether these opposite
effects could be explained by the acquisition of the transcriptional
repressive WUS box in the WUS-clade genes (Lin et al., 2013).
Second, several WOX genes are required for normal development
in the Arabidopsis zygote (Breuninger et al., 2008; Ueda et al.,
2011), which superficially parallels the function of PpWOX13L
genes in Physcomitrella. However, also here there are marked
differences: in Arabidopsis, it is the asymmetry of the zygotic
division that is under control of WOX2, 8 and 9 genes, whereas an
effect on zygote expansion, as seen in the moss for PpWOX13L
genes, has not been reported. Zygote expansion in Arabidopsis is
rather under the control of the YODA pathway (Lukowitz et al.,
2004), which acts in parallel to WOX genes at later stages of embryo
patterning. Expression of a moss YODA ortholog is induced after
leaf detachment independently of PpWOX13L (Nishiyama et al.,
2012), but its function remains to be investigated.
Our mutant analysis of the only two expressed members of the
WOX13-clade in Arabidopsis, WOX13 and WOX14, did not reveal
an obvious stem cell regeneration defect. Of course, possible stem
cell- or regeneration-related functions of WOX13 and WOX14
could be masked by the existence of yet unknown redundant
factors. We consider it unlikely, however, that Arabidopsis
WOX13 genes function redundantly with WUS clade genes,
given the sequence differences in their recognition helix of the
homeodomain that mediates DNA contact (Deveaux et al., 2008;
Nardmann and Werr, 2012). Thus, the function of WOX13 clade
genes in Arabidopsis appears to be distinct from that of WUS clade
genes. WOX13-type genes evolved in an ancestor of green algae
and land plants (Deveaux et al., 2008) and in light of the early
separation of the two lineages in plant evolution, PpWOX13L
function in moss could be either close to the ancestral trait or a
derived character. The Δppwox13l deficiencies in activating cell
outgrowth are consistent with ancestry, as cell expansion is a sine
qua non condition for cell divisions in uni- or multicellular
organisms, and this view is compatible with the PpWOX13L
phylogenetic position in the ancestral branch of the WOX family
(Deveaux et al., 2008; van der Graaff et al., 2009; Nardmann
et al., 2009). However, the Δppwox13l phenotypes associate the
PpWOX13L function with stem cell formation, which is
reminiscent of the function of WUS clade members that evolved
after the separation of moss and vascular plant lineages
(Nardmann and Werr, 2012). The PpWOX13L function in moss
thus exemplifies either convergent evolution or recruitment of
WOX13 genes to the interface of cell enlargement/division and
cellular differentiation in the last common ancestor of mosses and
vascular plants.
It is noteworthy in this regard that expression of WOX13-clade
genes in Physcomitrella and Arabidopsis is not restricted to
1667
DEVELOPMENT
Fig. 5. Root tip regeneration in
Arabidopsis thaliana. (A-D) Regeneration
assay with wild-type roots carrying the
quiescent center (QC) specific marker
QC184 stained for GUS and with Lugol’s
solution that stains starch grains specific for
differentiated columella cells. Five days
after germination, roots were cut at a
distance of 100 μm from the root tip (black
bar in A). One day after cutting, cells with
columella identity are detected by Lugol
staining (B, arrow). Two days after cutting,
the roots form a typical root morphology (C),
and 3 days after, restore expression of
QC184 (D, arrowhead). (E,F) pWOX13:
erYFP (E) and pWOX14:erYFP (F) were
expressed in the root meristem. Inset in E:
enlarged view of boxed area. (G) Efficiency
of root tip regeneration 2 days after cutting:
no significant differences between the wild
type and mutants are apparent. Values
shown represent averages of two sibling
lines and four biological replicates, error
bars represent s.d.
RESEARCH ARTICLE
small signaling centers, and there is no indication of a non-cellautonomous function. By contrast, WUS clade function in stem
cell regulation appears to involve spatially restricted expression
in stem cell organizers and the organization of multicellular
niches via non-cell-autonomous mechanisms (Schoof et al.,
2000; Sarkar et al., 2007; Hirakawa et al., 2010), which
plausibly could have been an acquisition during evolution of
increasingly complex stem cell systems typical for angiosperms.
Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
described (Nishiyama et al., 2012). Gene Ontology enrichment analyses
were performed using the BinGO plugin (version 2.44) on Cytoscape
version 2.8.3 (Maere et al., 2005) with the table (Rensing et al., 2008)
available at (ftp://ftp.jgi-psf.org/pub/JGI_data/Physcomitrella_patens/v1.1/
Phypa1_1_goinfo_FilteredModels3.tab.gz). For each set of up- or
downregulated genes at each time point or the union of such sets for any
time point, overrepresented GO categories were searched. Hypergeometric
tests with Benjamini and Hochberg FDR were carried out with the
significance level of 0.01. Corrected P-values for significantly
overrepresented GO terms were obtained.
MATERIALS AND METHODS
Physcomitrella patens Cove-NIBB (Nishiyama et al., 2000) or Gransden 2004
(Rensing et al., 2008) lines were used as a wild-type strain and cultured on
BCDAT medium supplemented with 1 mM CaCl2 and 0.8% (w/v) agar
(BCDAT agar medium) at 25°C in continuous light or long-day conditions
(16 h/8 h) (Nishiyama et al., 2000). Gametophore preparation for transcriptome
analysis, quantitative RT-PCR analysis, and plasmolysis experiments (Ishikawa
et al., 2011), protoplast preparation (Nishiyama et al., 2000), and gametangia/
sporophyte observation (Sakakibara et al., 2008) were previously described.
Transformation and genetic crosses
The primer sequences used for plasmid construction are provided in
supplementary material Table S1. The constructs used for transformation are
shown in supplementary material Fig. S1A-D and Fig. S2A-D. Transformation
was performed as described previously (Nishiyama et al., 2000; Knight et al.,
2002). Stably transformed lines were screened by DNA gel-blot analyses. To
exclude polyploid lines, flow cytometry analysis was performed (Okano et al.,
2009). Crosses were performed as previously described (Tanahashi et al., 2005).
Microscopy
Images were captured using SZX16 and BX51 (Olympus). Citrine
fluorescent images were obtained using fluorescence microscopes with a
U-MNIBA3 filter unit or using confocal laser scanning microscope LSM
510 confocal system (Carl Zeiss) with a detecting fluorescence emission
between 530-600 nm under excitation with 514 nm.
To quantify reprogramming of leaf cells in wild-type and the Δppwox13l
deletion lines, 10-15 leaves of Δppwox13la1 and Δppwox13lb1 lines were
placed on a layer of BCDAT medium with 0.8% agar on a microscope slide.
Pictures were taken every 24 h and subsequently evaluated for division and
elongation events at the wound margin, with all cells directly adjacent to a
dead cell scored as margin cells. Three repetitions were carried out. For
observation of reprogramming in the wild-type and Δppwox13lab double
deletion lines, the third to fifth leaves from a gametophore tip were cut in half
and the detached leaves were laid on a glass-base Petri dish (Iwaki 3911-035,
Asahi Techno Glass, Shizuoka, Japan) and were then overlaid with BCDAT
agar medium. Images of leaf reprogramming were recorded at 10- or 20-min
intervals with a digital camera ORCA-AG (Hamamatsu Photonics K. K.)
using an inverted microscope IX81 (Olympus). The images were
reconstructed to create a movie with Metamorph software (Molecular
Devices).
For observation of nuclei, regenerated protoplasts at 6 days after plating
were fixed with 1% formaldehyde in 50 mM sodium phosphate buffer ( pH
7.0) and stained with 1 μg/ml DAPI (4′,6-diamidino-2-phenylindole). To
observe egg cells and embryos, shoot apices with gametangia and several
young leaves were collected 3-4 weeks after the induction of gametangia
and fixed in a solution of 4% (v/v) glutaraldehyde and 1 μg/ml DAPI in
12.5 mM sodium phosphate buffer ( pH 7.0) overnight at 4°C. The fixed
materials were then dehydrated in a graded ethanol series and cleared in a 2:1
mixture of benzyl benzoate and benzyl alcohol for observation on a LSM
510 confocal system. Fluorescence between 420 and 480 nm was detected
under excitation with 405 nm beams.
Digital gene expression profiling with mRNA 5′-end tags (5′-DGE)
analysis
Transcriptome comparisons between the wild-type and the
Δppwox13lab#186 double deletion lines were performed as previously
1668
Transient expression assay of β-expansins
Plasmids for transient expression assay of β-expansins were constructed using
pTFH22.4 (accession number: AB758445), which contains a rice actin
promoter (McElroy et al., 1990), a multiple-cloning site (SalI-AscI-SmaI-ApaISfiI), the pea rbcS-3A polyadenylation signal, the cauliflower mosaic virus
(CaMV) 35S promoter, sGFP, and the polyadenylation signal of the nopaline
synthase gene. cDNA of each β-expansin coding region was amplified by PCR
with primers shown in supplementary material Table S1 and cloned into the
AscI-ApaI site of pTFH22.4. The original pTFH22.4 was used as a control.
Transient transformation was performed using 10 μg of circular plasmid DNA
(Nishiyama et al., 2000). The protoplasts were left on high osmolarity plates
[6% (w/v) mannitol] after transformation. Twenty protoplasts or regenerating
chloronemata with GFP signal were arbitrarily chosen on the second, third,
fourth and fifth days after plating using the Olympus BX51 microscope with the
GFP filter set. Each regenerating plant was scored for the number of apical cells
showing tip growth, and the total number for each class was obtained from three
repeat experiments. The difference in the number of the acquired apical stem
cells was tested by the non-parametric Mann–Whitney U-test, because the
number of apical stem cells is not considered to follow the normal distribution
but is an ordered variable. The calculations were performed using the R
program (R Development Core Team, 2012).
Observation of plasmolysis
To examine plasmolysis in cut leaves, ten leaves were cut and incubated in
10 mM MES buffer ( pH 6.5) for 24 h, and then incubated in mannitol
solutions of different concentrations for 15 min or 45 min. Five arbitrarily
selected wound margin cells in each leaf were examined for plasmolysis.
Three biological replicates were analyzed for each experiment. Statistical
analysis was performed by Fisher’s exact test using the R program
(R Development Core Team, 2012).
RNA preparation and quantitative RT-PCR analysis
Excised leaves for quantitative RT-PCR analysis were prepared in the same
manner as samples for 5′-DGE analysis and collected 24 h after leaf excision.
Regenerated protoplasts for quantitative RT-PCR analysis were prepared
following protoplast regeneration. After overnight incubation, protoplasts
were collected, suspended in PRM/T medium without agar (BCDAT medium
supplemented with 10 mM CaCl2 and 0.044 M mannitol), and plated on the
PPM/B plate (BCDAT medium supplemented with 10 mM CaCl2, 6%
mannitol and 0.8% agar) covered with sterile cellophane. Regenerated
protoplasts were collected 4 days after plating.
Total RNA preparation and cDNA synthesis were described previously
(Ishikawa et al., 2011). Quantitative RT-PCR (qRT-PCR) was performed
using an ABI PRISM 7500 (Life Technologies) with the SYBR GreenER
qPCR Super mix Universal (Life Technologies) and appropriate primers
shown in supplementary material Table S1. Results were analyzed using the
comparative critical threshold method (Livak and Schmittgen, 2001). The
transcript levels were normalized against α-tubulin gene (TUA1) transcript
levels using TUA1 primers (Ishikawa et al., 2011). The quantification of
each sample was performed in triplicate. Three biological replicates were
analyzed for transcript accumulation.
Generation of Arabidopsis reporters
Promoter fragments including 4.4 kb ( pWOX13) and 1.4 kb ( pWOX14)
were amplified with primers containing adapter sequences for ligation
independent cloning (LIC) (Li and Elledge, 2007; Eschenfeldt et al., 2009),
DEVELOPMENT
Plant materials and growth conditions
and were subsequently inserted by the LIC procedure in front of an erYFP
and 35S-terminator within a binary vector. Plants were transformed using
the floral-dip method. Primer sequences are provided in supplementary
material Table S1.
Arabidopsis root regeneration assay
The QC184 marker (Sabatini et al., 1999) was introduced by crossing with
the wox13-2 mutant, and the selected wox13-2 QC184 plants were
subsequently crossed to wox14-1. Because these crosses generated a
mixture of three ecotypes (QC184: WS; wox13-2: Ler; wox14-1: No), wildtype controls were selected from the progeny of these crosses. The
regeneration assay was carried out as previously described (Sena et al.,
2009) and regeneration was scored two days after cutting (four biological
replicates for each background, n=22-48 for each replicate).
Acknowledgements
We thank Yohei Higuchi for the technical support to establish 5′-DGE indexing;
Nagisa Sugimoto for the time-lapse observation; Kazuhiko Nishitani for suggestions
on physiological experiments; Tomomichi Fujita for providing the plasmid pTFH22.4
and technical advice for the experiment; Tomoko Masuoka, Mika Hiramatsu and
Ikumi Kajikawa for genetic crosses and Physcomitrella genotyping; Thomas
Mü nster for providing plasmids and plant material; Eric van der Graaff for providing
plasmids; and Minako Ueda, Miyuki Nakata, Masaki Shimamura, Edwin Groot and
Judith Nardmann for helpful discussions.
Competing interests
The authors declare no competing financial interests.
Author contributions
K.S., P.R., T.N., W.W., S.A.R., H.D., M.H. and T.L. designed and discussed
experiments. K.S. and P.R. performed Physcomitrella mutant and expression
analysis. T.A. established the wox13l single and double mutant lines. T.F. performed
Arabidopsis experiments. S.A. performed Southern hybridization of wox13l lines
and confocal microscopy. Y.S. performed the time-lapse observation analysis. Y.T.
crossed Physcomitrella. Y.H. transformed Physcomitrella. T.N. and T.K. performed
transcriptome analysis. M.I. performed the protoplast transient assay. T.M.
conducted plasmolysis experiments. K.S., P.R., M.H. and T.L. wrote the paper.
Funding
This work was funded by the Young Researcher Overseas Visits Program for Vitalizing
Brain Circulation (K.S., Y.H., M.I., Y.T. and M.H.); the Japan Science and Technology
Agency ERATO program (M.H.); the Ministry of Education, Culture, Sports, Science
and Technology [grant no. 24113521 to Y.T., no. 25291067 and 22128002 to M.H.]; the
Deutsche Forschungsgemeinschaft [WE1262/7-1, SFB 680 to W.W.]; the European
Research Area in Plant Genomics program (T.L.); the EU-cofunded INTERREG IV
Upper Rhine Project 692 A17 (T.L.); the Excellence Initiative of the German Federal
and State Governments [BIOSS, EXC 294 to T.L. and S.A.R.]; and the German Federal
Ministry of Education and Research (Freiburg Initiative for Systems Biology) [FKZ
0313921 to S.A.R. and T.L.]. Deposited in PMC for immediate release.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.097444/-/DC1
References
Aichinger, E., Kornet, N., Friedrich, T. and Laux, T. (2012). Plant stem cell niches.
Annu. Rev. Plant Biol. 63, 615-636.
Banno, H., Ikeda, Y., Niu, Q. W. and Chua, N. H. (2001). Overexpression of
Arabidopsis ESR1 induces initiation of shoot regeneration. Plant Cell 13,
2609-2618.
Birnbaum, K. D. and Sanchez Alvarado, A. (2008). Slicing across kingdoms:
regeneration in plants and animals. Cell 132, 697-710.
Breuninger, H., Rikirsch, E., Hermann, M., Ueda, M. and Laux, T. (2008).
Differential expression of WOX genes mediates apical-basal axis formation in the
Arabidopsis embryo. Dev. Cell 14, 867-876.
Chen, S.-K., Kurdyukov, S., Kereszt, A. and Wang, X.-D. (2009). The association
of homeobox gene expression with stem cell formation and morphogenesis in
cultured Medicago trunculata. Planta 230, 827-840.
Chopra, R. N. and Kumra, P. K. (1988). Biology of Bryophytes. New Delhi: Wiley
Eastern.
Cosgrove, D. J. (2005). Growth of the plant cell wall. Nat. Rev. Mol. Cell Biol. 6,
850-861.
De Smet, I., Vanneste, S., Inzé , D. and Beeckman, T. (2006). Lateral root initiation
or the birth of a new meristem. Plant Mol. Biol. 60, 871-887.
Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
Deveaux, Y., Toffano-Nioche, C., Claisse, G., Thareau, V., Morin, H., Laufs, P.,
Moreau, H., Kreis, M. and Lecharny, A. (2008). Genes of the most conserved
WOX clade in plants affect root and flower development in Arabidopsis. BMC Evol.
Biol. 8, 291.
Eschenfeldt, W. H., Lucy, S., Millard, C. S., Joachimiak, A. and Mark, I. D. (2009).
A family of LIC vectors for high-throughput cloning and purification of proteins.
Methods Mol. Biol. 498, 105-115.
Gallois, J.-L., Nora, F. R., Mizukami, Y. and Sablowski, R. (2004). WUSCHEL
induces shoot stem cell activity and developmental plasticity in the root meristem.
Genes Dev. 18, 375-380.
Garces, H. M. P., Champagne, C. E. M., Townsley, B. T., Park, S., Malho, R.,
Pedroso, M. C., Harada, J. J. and Sinha, N. R. (2007). Evolution of asexual
reproduction in leaves of the genus Kalanchoë . Proc. Natl. Acad. Sci. U.S.A. 104,
15578-15583.
Gordon, S. P., Heisler, M. G., Reddy, G. V., Ohno, C., Das, P. and Meyerowitz, E. M.
(2007). Pattern formation during de novo assembly of the Arabidopsis shoot
meristem. Development 134, 3539-3548.
van der Graaff, E., Laux, T. and Rensing, S. A. (2009). The WUS homeoboxcontaining (WOX) protein family. Genome Biol. 10, 248.
Graf, P., Dolzblasz, A., Wurschum, T., Lenhard, M., Pfreundt, U. and Laux, T.
(2010). MGOUN1 encodes an Arabidopsis type IB DNA topoisomerase required
in stem cell regulation and to maintain developmentally regulated gene silencing.
Plant Cell 22, 716-728.
Groß-Hardt, R., Lenhard, M. and Laux, T. (2002). WUSCHEL signaling functions
in interregional communication during Arabidopsis ovule development. Genes
Dev. 16, 1129-1138.
Haecker, A., Groß-Hardt, R., Geiges, B., Sarkar, A., Breuninger, H., Herrmann, M.
and Laux, T. (2004). Expression dynamics of WOX genes mark cell fate decisions
during early embryonic patterning in Arabidopsis thaliana. Development 131,
657-668.
Harrison, C. J., Roeder, A. H. K., Meyerowitz, E. M. and Langdale, J. A. (2009).
Local cues and asymmetric cell divisions underpin body plan transitions in the
moss Physcomitrella patens Curr. Biol. 19, 461-471.
Hirakawa, Y., Kondo, Y. and Fukuda, H. (2010). TDIF peptide signaling regulates
vascular stem cell proliferation via the WOX4 homeobox gene in Arabidopsis.
Plant Cell 22, 2618-2629.
Horstmann, V., Huether, C. M., Jost, W., Reski, R. and Decker, E. L. (2004).
Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors
activating gene expression within three orders of magnitude. BMC Biotechnol. 4,
13.
Ishikawa, M., Murata, T., Sato, Y., Nishiyama, T., Hiwatashi, Y., Imai, A., Kimura,
M., Sugimoto, N., Akita, A., Oguri, Y. et al. (2011). Physcomitrella cyclindependent kinase A links cell cycle reactivation to other cellular changes during
reprogramming of leaf cells. Plant Cell 23, 2924-2938.
Iwase, A., Mitsuda, N., Koyama, T., Hiratsu, K., Kojima, M., Arai, T., Inoue, Y.,
Seki, M., Sakakibara, H., Sugimoto, K. et al. (2011). The AP2/ERF transcription
factor WIND1 controls cell dedifferentiation in Arabidopsis. Curr. Biol. 21,
508-514.
Jenkins, G. and Cove, D. (1983). Light requirements for regeneration of protoplasts
of the moss Physcomitrella patens. Planta 157, 39-45.
Ji, J., Shimizu, R., Sinha, N. and Scanlon, M. J. (2010). Analyses of WOX4
transgenics provide further evidence for the evolution of the WOX gene family
during the regulation of diverse stem cell functions. Plant Signal. Behav. 5,
916-920.
Kamiya, N., Nagasaki, H., Morikami, A., Sato, Y. and Matsuoka, M. (2003).
Isolation and characterization of a rice WUSCHEL-type homeobox gene that is
specifically expressed in the central cells of a quiescent center in the root apical
meristem. Plant J. 35, 429-441.
Knight, C. D., Cove, D., Cuming, A. C. and Quatrano, R. S. (2002). Moss gene
technology. In Molecular Plant Biology (ed. P. M. Gilmartin and C. Bowler) pp.
285-301. Oxford: Oxford University Press.
Kofuji, R. and Hasebe, M. (2014). Eight types of stem cells in the life cycle of the
moss Physcomitrella patens. Curr. Opin. Plant Biol. 17, 13-21.
Kofuji, R., Yoshimura, T., Inoue, H., Sakakibara, K., Hiwatashi, Y., Kurata, T.,
Aoyama, T., Ueda, K. and Hasebe, M. (2009). Gametangia development
in the moss Physcomitrella patens. In The moss Physcomitrella (ed. D. Cove,
P. F. Perroud and C. D. Knight), pp. 167-181. West Sussex: Blackwell.
Laux, T., Mayer, K. F., Berger, J. and Jurgens, G. (1996). The WUSCHEL gene is
required for shoot and floral meristem integrity in Arabidopsis. Development 122,
87-96.
Li, M. Z. and Elledge, S. J. (2007). Harnessing homologous recombination in vitro to
generate recombinant DNA via SLIC. Nat. Methods 4, 251-256.
Lin, H., Niua, L., McHale, N. A., Ohme-Takagi, M., Mysore, K. S. and Tadege, M.
(2013). Evolutionarily conserved repressive activity of WOX proteins mediates leaf
blade outgrowth and floral organ development in plants. Proc. Natl. Acad. Sci.
U.S.A. 110, 366-371.
Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data
using real-time quantitative PCR and the 2-DDCT method. Methods 25, 402-408.
Lukowitz, W., Roeder, A., Parmenter, D. and Somerville, C. (2004). A MAPKK
kinase gene regulates extra-embryonic cell fate in Arabidopsis. Cell 116, 109-119.
1669
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root.
Cell 99, 463-472.
Sakakibara, K., Nishiyama, T., Deguchi, H. and Hasebe, M. (2008). Class 1
KNOX genes are not involved in shoot development in the moss
Physcomitrella patens but do function in sporophyte development. Evol. Dev.
10, 555-566.
Sarkar, A. K., Luijten, M., Miyashima, S., Lenhard, M., Hashimoto, T., Nakajima,
K., Scheres, B., Heidstra, R. and Laux, T. (2007). Conserved factors regulate
signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature 446,
811-814.
Schoof, H., Lenhard, M., Haecker, A., Mayer, K. F. X., Jü rgens, G. and Laux, T.
(2000). The stem cell population of Arabidopsis shoot meristems is maintained by
a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100,
635-644.
Sena, G., Wang, X., Liu, H.-Y., Hofhuis, H. and Birnbaum, K. D. (2009). Organ
regeneration does not require a functional stem cell niche in plants. Nature 457,
1150-1153.
Shimizu, R., Ji, J., Kelsey, E., Ohtsu, K., Schnable, P. S. and Scanlon, M. J.
(2009). Tissue specificity and evolution of meristematic WOX3 function. Plant
Physiol. 149, 841-850.
Skoog, F. and Miller, C. O. (1957). Chemical regulation of growth and organ
formation in plant tissues cultured in vitro. Soc. Exp. Biol. Symp. 11, 118-131.
Steeves, T. A. and Sussex, I. M. (1989). Patterns in Plant Development.
Cambridge: Cambridge University Press.
Su, Y. H., Zhao, X. Y., Liu, Y. B., Zhang, C. L., O’Neill, S. D. and Zhang, X. S.
(2009). Auxin-induced WUS expression is essential for embryonic stem cell
renewal during somatic embryogenesis in Arabidopsis. Plant J. 59, 448-460.
Sugimoto, K., Jiao, Y. and Meyerowitz, E. M. (2010). Arabidopsis regeneration
from multiple tissues occurs via a root development pathway. Dev. Cell 18,
463-471.
Taiz, L. and Zeiger, E. (2010). Plant Physiology. Sunderland, Massachusetts:
Sinauer Associates.
Tanahashi, T., Sumikawa, N., Kato, M. and Hasebe, M. (2005). Diversification of
gene function: homologs of the floral regulator FLO/LFY control the first zygotic
cell division in the moss Physcomitrella patens. Development 132, 1727-1736.
Ueda, M., Zhang, Z. and Laux, T. (2011). Transcriptional activation of Arabidopsis
axis patterning genes WOX8/9 links zygote polarity to embryo development. Dev.
Cell 20, 264-270.
Vogel, G. (2005). How does a single somatic cell become a whole plant. Science
309, 86.
Williams, E. G. and Maheswaran, G. (1986). Somatic embryogenesis: factors
influencing coordinated behaviour of cells as an embryogenic group. Ann. Bot. 57,
443-462.
Wu, X., Dabi, T. and Weigel, D. (2005). Requirement of homeobox gene STIMPY/
WOX9 for Arabidopsis meristem growth and maintenance. Curr. Biol. 15,
436-440.
Zhang, W., McElroy, D. and Wu, R. (1991). Analysis of rice Act1 5′ region activity in
transgenic rice plants. Plant Cell 3, 1155-1165.
Zuo, J., Niu, Q.-W., Frugis, G. and Chua, N.-H. (2002). The WUSCHEL gene
promotes vegetative-to-embryonic transition in Arabidopsis. Plant J. 30, 349-359.
DEVELOPMENT
Maere, S., Heymans, K. and Kuiper, M. (2005). BiNGO: a Cytoscape plugin to
assess overrepresentation of Gene Ontology categories in biological networks.
Bioinformatics 21, 3448-3449.
Masip, M., Veiga, A., Izpisú a Belmonte, J. C. and Simó n, C. (2010).
Reprogramming with defined factors: from induced pluripotency to induced
transdifferentiation. Mol. Hum. Reprod. 16, 856-868.
Mayer, K. F. X., Schoof, H., Haecker, A., Lenhard, M., Jü rgens, G. and Laux, T.
(1998). Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot
meristem. Cell 95, 805-815.
McElroy, D., Zhang, W., Cao, J. and Wu, R. (1990). Isolation of an efficient actin
promoter for use in rice transformation. Plant Cell 2, 163-171.
Nakata, M., Matsumoto, N., Tsugeki, R., Rikirsch, E., Laux, T. and Okada, K.
(2012). Roles of the middle domain-specific WUSCHEL-RELATED HOMEOBOX
genes in early development of leaves in Arabidopsis. Plant Cell 24, 519-535.
Nardmann, J. and Werr, W. (2007). The evolution of plant regulatory networks:
what Arabidopsis cannot say for itself. Curr. Opin. Plant Biol. 10, 653-659.
Nardmann, J. and Werr, W. (2012). The invention of WUS-like stem cell-promoting
functions in plants predates leptosporangiate ferns. Plant Mol. Biol. 78, 123-134.
Nardmann, J., Reisewitz, P. and Werr, W. (2009). Discrete shoot and root stem
cell-promoting WUS/WOX5 functions are an evolutionary innovation of
angiosperms. Mol. Biol. Evol. 26, 1745-1755.
Nishiyama, T., Hiwatashi, Y., Sakakibara, K., Kato, M. and Hasebe, M. (2000).
Tagged mutagenesis and gene-trap in the moss Physcomitrella patens by shuttle
mutagenesis. DNA Res. 7, 9-17.
Nishiyama, T., Miyawaki, K., Ohshima, M., Thompson, K., Nagashima, A.,
Hasebe, M. and Kurata, T. (2012). Digital gene expression profiling by 5′-end
sequencing of cDNAs during reprogramming in the moss Physcomitrella patens.
PLoS ONE 7, e36471.
Okano, Y., Aono, N., Hiwatashi, Y., Murata, T., Nishiyama, T., Ishikawa, T.,
Kubo, M. and Hasebe, M. (2009). A polycomb repressive complex 2 gene
regulates apogamy and gives evolutionary insights into early land plant evolution.
Proc. Natl. Acad. Sci. U.S.A. 106, 16321-16326.
Prigge, M. J. and Bezanilla, M. (2010). Evolutionary crossroads in developmental
biology: Physcomitrella patens. Development 137, 3535-3543.
R Development Core Team (2012). R: A Language and Environment for Statistical
Computing. Vienna, Austria: R Foundation for Statistical Computing.
Raghavan, V. (1989). Developmental Biology of Fern Gametophytes. Cambridge:
Cambridge University Press.
Rensing, S. A., Lang, D., Zimmer, A. D., Terry, A., Salamov, A., Shapiro, H.,
Nishiyama, T., Perroud, P.-F., Lindquist, E. A., Kamisugi, Y. et al. (2008). The
Physcomitrella genome reveals evolutionary insights into the conquest of land by
plants. Science 319, 64-69.
Richardt, S., Lang, D., Reski, R., Frank, W. and Rensing, S. A. (2007).
PlanTAPDB, a phylogeny-based resource of plant transcription-associated
proteins. Plant Physiol. 143, 1452-1466.
Romera-Branchat, M., Ripoll, J. J., Yanofsky, M. F. and Pelaz, S. (2012). The
WOX13 homeobox gene promotes replum formation in the Arabidopsis thaliana
fruit. Plant J. 73, 37-49.
Sabatini, S., Beis, D., Wolkenfelt, H., Murfett, J., Guilfoyle, T., Malamy, J.,
Benfey, P., Leyser, O., Bechtold, N., Weisbeek, P. et al. (1999). An
Development (2014) 141, 1660-1670 doi:10.1242/dev.097444
1670