LETTERS
Conserved factors regulate signalling in Arabidopsis
thaliana shoot and root stem cell organizers
Ananda K. Sarkar1
*{, Marijn Luijten2
*, Shunsuke Miyashima3
, Michael Lenhard1
{, Takashi Hashimoto3
,
Keiji Nakajima3
, Ben Scheres2
, Renze Heidstra2
& Thomas Laux1
Throughout the lifespan of a plant, which in some cases can last
more than one thousand years, the stem cell niches in the root
and shoot apical meristems provide cells for the formation of
complete root and shoot systems, respectively. Both niches are
superficially different and it has remained unclear whether common
regulatory mechanisms exist. Here we address whether root
and shoot meristems use related factors for stem cell maintenance.
In the root niche the quiescent centre cells, surrounded by the stem
cells, express the homeobox gene WOX5 (WUSCHEL-RELATED
HOMEOBOX 5)1
, a homologue of the WUSCHEL (WUS) gene that
non-cell-autonomously maintains stem cells in the shoot meri-
stem2
. Loss of WOX5 function in the root meristem stem cell
niche causes terminal differentiation in distal stem cells and,
redundantly with other regulators, also provokes differentiation
of the proximal meristem. Conversely, gain of WOX5 function
blocks differentiation of distal stem cell descendents that normally
differentiate. Importantly, both WOX5 and WUS maintain stem
cells in either a root or shoot context. Together, our data indicate
that stem cell maintenance signalling in both meristems employs
related regulators.
Higher organisms evolved the ability to keep founder cells undifferentiated
and pluripotent by signals provided in specialized stem
cell niches3
. In the model plant Arabidopsis thaliana, root and shoot
meristem stem cell niches are organized differently compared with
each other4
. In the root meristem, the stem cells surround a small
group of rarely dividing cells, termed the quiescent centre (Fig. 1a),
and give rise to distal (columella), lateral (lateral root cap and epidermis)
and proximal (cortex, endodermis and stele) cell types.
Ablation studies show that short range signals from the quiescent
centre keep only the directly abutting stem cells undifferentiated5
,
similar to most animal stem cell niches studied so far3
. In contrast,
in the shoot meristem, a zone of three stem cell layers is maintained by
an underlying organizing centre2
(Supplementary Fig. 1a), which regulates
the stem cell pool as a whole. In addition, the regulatory genes
described so far for root and shoot stem cell niches are different6
.
In the shoot meristem, a feedback mechanism between the organizing
centre and stem cells dynamically regulates maintenance of the
stem cell pool: WUS activity in the organizing centre keeps stem cells
undifferentiated and induces expression of the signal peptide
CLAVATA3 (CLV3), which in turn restricts the size of the WUS
expression domain (Supplementary Fig. 1a)7,8
. We asked whether a
related stem cell maintenance mechanism might operate in the root
by analysing the WOX family for functions in the root meristem1
.
Expression of WOX5 messenger RNA1
and a WOX5-GUS reporter
gene initiates in the embryonic cell lineage that gives rise to the
quiescent centre (Supplementary Fig. 1b), and persists in the
quiescent centre (Fig. 1b; and Supplementary Fig. 1c) during postembryonic
root growth. This expression pattern is strikingly similar
to that of WUS in the shoot meristem2
(Supplementary Fig. 2), raising
the question of whether both genes might have similar functions
in the respective stem cell niches.
In seedlings of the putative null allele wox5-1 (Supplementary Fig.
1d, e), the cells at the quiescent centre position have abnormal shape
and are enlarged in comparison to wild type (Fig. 1c, d, arrows, and
Supplementary Table 1). Notably, adjacent columella stem cells are
even larger, suggesting that they have undergone differentiation
(Fig. 1d, bottom arrow). No other abnormality was observed in
wox5-1 mutants. Complementation of the mutant defects by a
WOX5 complementary DNA construct confirmed that the wox5-1
mutation causes the observed phenotype (Supplementary Fig. 3a).
*These authors contributed equally to this work.
1
Institute of Biology III, University of Freiburg, Scha¨nzlestraße 1, 79104 Freiburg, Germany. 2
Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht,
The Netherlands. 3
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. {Present addresses: Cold Spring
Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, New York 11724, USA (A.K.S.); John Innes Centre, Colney Lane, Norwich, N4 7UH, UK (M.K.).
col
vas
lrc
epc
en
qc
cscCSC
QC
Stem
cells
Wild type
Wild type
QC
CSC
dc
wox5-1
ba
Differentiation
Differentiation
Figure 1 | WOX5 expression in the quiescent centre is required for root
meristem development. a, Schematic of A. thaliana root meristem with
columella (col), columella stem cells (CSC), quiescent centre (QC), lateral
root cap (lrc), epidermis (ep), cortex (c), endodermis (en) and vascular
bundle (vas), redrawn with permission from ref. 4. b, WOX5 mRNA
expression in the quiescent centre. c–d, wox5-1 roots display enlarged cells at
the quiescent centre and columella stem cell (CSC) positions. Scale bars,
50 mm.
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Of several quiescent-centre-specific reporter genes9
, only QC184 is
not expressed in wox5-1 and expression is lacking as early as the heart
embryo stage (Fig. 2a, b, d, e; and Supplementary Table 2), indicating
that WOX5 functions in root development from early embryogenesis.
In contrast, QC25, QC46 and WOX5-GUS are expressed in
slightly expanded domains compared to wild type (Fig. 2c, f; Supplementary
Fig. 3c–h; and Supplementary Table 2). These data
indicate that WOX5 is required for some aspects of quiescentcentre-specific
gene expression, but seems not to be a major factor
in quiescent centre specification.
We asked whether in wox5-1 roots the distal stem cells might have
undergone premature differentiation, which in the columella can be
visualized by the accumulation of starch-granule-containing organelles
involved in gravitropic sensing. Indeed, in wox5-1 roots, the
cells at the stem cell position, but not the quiescent centre, accumulate
starch granules (Fig. 2e, f; and Supplementary Table 2). In
contrast, the proximal meristem appears normal. These findings
indicate that WOX5 gene expression in the quiescent centre is required
to non-cell-autonomously maintain the distal stem cells as
undifferentiated, similar to the role of WUS in the shoot2
.
We then investigated whether WOX5 activity is also sufficient to
repress columella cell differentiation, by ubiquitously expressing
a dexamethasone (DEX)-inducible WOX5 gene. DEX induction
results in small cells that lack starch granules in the place of differentiated
columella and lateral root cap cells (Fig. 2h, i; Supplementary
Fig. 4a; and Supplementary Table 1), indicating that they have
not undergone normal differentiation. Unlike quiescent centre cells,
the small cells divide, as shown by the continuously increasing cell
number and expression of the cell cycle marker CYCB1-GUS (Supplementary
Fig. 4b) and quiescent centre markers are not induced
(Fig. 2h). Consistent with a loss of differentiated columella cells,
35S[GVG]-WOX5 (35S-WOX5; Supplementary Methods) roots display
reduced gravitropism (Supplementary Fig. 4c). After withdrawal
of DEX, however, the small cells readily accumulate starch grains
typical for differentiated columella cells (Fig. 2l). Normally, laser ablation
of the quiescent centre causes differentiation of stem cells9,10
, but
this did not have any effect in 35S-WOX5 (Fig. 2j, k). Thus, ectopic
WOX5 expression is sufficient to block differentiation of columella
stem cell daughters without the requirement of any further quiescent
centre signalling.
Previous studies highlighted auxin-dependent transcription of the
PLETHORA (PLT) genes and SHORTROOT/SCARECROW (SHR/
SCR) functions as essential components in root meristem maintenance
and we asked how WOX5 interacts with these pathways9,11–13
. In
mutants for the auxin response regulators BODENLOS (BDL) or
MONOPTEROS (MP; Fig. 3d–f; and Supplementary Table 3),
WOX5 expression is rarely detected, consistent with the notion that
BDL/MP-mediated auxin signalling is required for the embryonic
initiation of the quiescent centre13,14
. Auxin accumulation in the
quiescent centre region provides patterning information to the root
meristem mediated through PLT genes9,12,15
. In wox5-1 mutants,
auxin accumulation seems normal based on the expression of the
auxin response reporter gene DR5-GUS (Supplementary Fig. 5a,
b). In plt1 plt2 double mutants, WOX5 expression is occasionally
slightly expanded (Fig. 3g–i; and Supplementary Table 3), whereas
PLT1 expression is normal in wox5-1 (Supplementary Fig. 5c, d and
Supplementary Table 2).
The SHR protein moves from provascular cells into the quiescent
centre and activates transcription of the SCR gene16
, which is required
to specify quiescent centre identity and to maintain stem cells11
.
WOX5 expression is reduced or undetectable in shr and scr mutants
(Fig. 3j–l; Supplementary Fig. 5g–i; and Supplementary Table 3),
whereas an SCR reporter gene is correctly expressed in wox5-1 roots
(Supplementary Fig. 5e, f; and Supplementary Table 2). Collectively,
our data indicate that WOX5 expression depends on the induction in
the root pole by MP-mediated auxin signalling and on SHR/SCR
activity, whereas PLT1 and PLT2 have only a minor role in confining
WOX5 expression to the quiescent centre.
The requirement of WOX5 for only columella stem cell maintenance
suggests unidirectionality of this signalling pathway in stem cell
control, similar to WUS-mediated stem cell control in the shoot stem
cell niche. We investigated whether effects of the wox5-1 mutation on
the proximal meristem might be masked by redundant functions.
Indeed, in scr-4, shr-1 single mutants and plt1 plt2 double mutants,
the wox5-1 mutation promotes differentiation in the proximal region
of the root meristem in addition to its effects on distal stem cells
(Fig. 3m; and Supplementary Fig. 5j, k). This suggests that WOX5
gene expression in the quiescent centre redundantly contributes to
proximal stem cell activity or alternatively that WOX5 has a stemcell-independent
function in the proximal meristem, as has been
reported for its upstream regulator SCR11
(Supplementary Fig. 7).
The striking similarities of WOX5 and WUS expression patterns
and functions in root and shoot stem cell niche organizers raised the
ih
b
e
a
j
c
f
k
g
d
l
QC46/+DEX
QC184/WT
QC
CSC
COL
QC184/wox5-1
QC
(CSC)
COL
QC
(CSC)
COL
QC184/WT
}
WOX5-GUS/WT
QC
CSC
COL
WOX5-GUS/wox5-1
}
QC46/–DEX
QC184/wox5-1
}
+DEX –DEX
+DEX
}
QC
QC
QC QC
Figure 2 | WOX5 represses differentiation in the columella. a–f, wox5-1
heart-torpedo stage embryos (d) and root tips (e) lack QC184 expression,
express WOX5-GUS (f) in an enlarged domain and accumulate starch at the
CSC position (e, f). g, h, 35S-WOX5 expression gives small cells in the
columella, which lack QC46 expression and starch grains (h). j–l, Quiescent
centre ablation (arrow) does not induce differentiation (j, k), whereas
transfer to non-inducing medium does (l; control, i). Nomarski (a–i, k, l) and
confocal (j) median longitudinal images are shown. GUS staining, blue;
starch, violet; waved brackets, extra small cells; COL, columella; CSC,
columella stem cells; QC, quiescent centre; DEX, dexamethasone. Scale bars,
20 mm (a, d), 50 mm (b, c, e, f, i–l) and 10 mm (g, h).
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question of whether both genes are functionally equivalent. Indeed,
expression of the WUS cDNA from the WOX5 promoter completely
restores quiescent centre and stem cells in the wox5-1 root meristem
(Fig. 4a, b; and Supplementary Fig. 6a, b). Interestingly, ectopic shoot
tissue was never observed when WUS was expressed in the quiescent
centre. This contrasts to previous studies expressing WUS in a
broader range of cell types17
and favours the interpretation that
organizer signalling only maintains stem cells as undifferentiated,
whereas the fate of differentiating daughters is determined by the
tissue context. Conversely, expression of a WUS-WOX5 transgene
rescues premature termination of inflorescence meristems and
occasionally of floral meristems in the putative null allele wus-1
(Fig. 4c–f; and Supplementary Fig. 6d–g). The rescued wus-1 mutants
resemble the weak wus-6/jam allele18
, suggesting that the WUSWOX5
transgene complements WUS function but provides a
reduced level of organizing centre signalling. Defective ovule development
in wus mutants is not rescued by WUS-WOX5 (Supplementary
Fig. 6c), indicating that both regulators are interchangeable
in only stem cell maintenance. Significantly, the early embryo
genes WOX8 and WOX9 are not able to rescue wus-1 defects (H.
Breuninger and T.L., unpublished observations), showing that the
ability to regulate stem cell maintenance is not a general property of
WOX proteins.
0
5
10
15
20
25
30
j k
ih
l
g
m
RootHeart embryoGlobular embryo
*
scr-4
*
bdl
*
plt1 plt2
*
Wild type
bdl mp
Columbia
wox5-1
scr-4
wox5-1 scr-4
shr-1
wox5-1 shr-1
plt1 plt2
wox5-1 plt1 plt2
Meristematiccortexcells
ca b
d e f
scr-4 wox5-1 scr-4
3 4 6 3 4 6 3 4 6 3 4 6 days
Figure 3 | WOX5 interaction with SHR/SCR and auxin pathways.
a–l, Hybridization with WOX5 antisense probe. WOX5 expression was
detected in wild type (a–c), absent in bdl and mp (d–f, inset shows sporadic
exceptions), deregulated in plt1 plt2 (g–i), and undetectable in scr-4
(j–l, insets show sporadic exceptions). m, wox5-1 enhances differentiation in
the proximal meristem in scr-4 (inset), shr-1 and plt1 plt2 mutants. Error
bars, s.d. Longitudinal median histological (a, b, d, e, g, h, j, k) and
optical (c, f, i, l, m) sections are shown. Four-day-old roots (c, i, l) or middle
stage embryos (f) were used. Hybridization signal is red–blue; asterisk,
reddish colour is staining independent of hybridization reaction.
Arrows, quiescent centre position. Scale bars, 25 mm (a, b, d–f, g, h, j, k) and
50 mm (c, i, l).
ba
wus-1 WUS-WOX5/wus-1
wus-1 WUS-WOX5/wus-1
st
pe pe
st
WOX5-WUS/wox5-1wox5-1
c d
e f
Figure 4 | WOX5 and WUS are interchangeable in stem cell control.
a, b, WOX5-WUS expression restores quiescent centre and columella stem
cells (white arrows) in wox5-1. Violet, starch grains. c–f, Rescue of the shoot
meristem stem cell niche. wus-1 shoot (c) and floral meristem (e) fail to
maintain stem cells and terminate prematurely. WUS-WOX5 expression
restores an indeterminate inflorescence meristem (d) and complete flowers
(f). Twenty-six-day-old plants (c, d) and flowers (e, f) are shown. White
arrows indicate shoot apex (c, d). pe, petal; st, stamen. Scale bars, 1 cm
(c, d), 1 mm (e, f) and 50 mm (a, b).
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The extent of mechanistic similarity and evolutionary relationship
between root and shoot stem cell niches has remained unclear thus
far. Here we show that the organizers of both niches employ related
regulators to provide stem cell maintaining signals. However, there
also seem to be differences between both niches. For example, CLV3
homologues promote meristem differentiation but do not influence
stem cell maintenance in the root on overexpression and it is thus
unknown whether roots, similar to shoots, employ a WUS–CLV3
related feedback mechanism19,20
.
Our results suggest that WOX5 in the root stem cell niche has a
more direct function in stem cell signalling, rather than in specifying
quiescent centre identity (Supplementary Fig. 7). The nature of the
signals that are induced by WUS/WOX5 expression in the stem cell
organizers is presently unresolved. WOX5 protein itself or a downstream
factor might move to stem cells as the long-postulated shortrange
factor5
. To date, WOX5 protein has not been robustly detected,
presumably owing to very low expression levels. However, on the
basis of localization of a functional WUS–GUS fusion protein, movement
of WUS protein out of the organizing centre seems not to be
necessary for its function in shoot meristem stem cell maintenance,
indicating that factors downstream of WUS act as signals (Supplementary
Fig. 6h, i).
Palaeobotanical evidence for early root structures is controversial,
but the current view is that roots have evolved independently at least
twice in Lycophytina and Euphyllophytina21,22
. It remains to be
shown whether WUS and WOX5 shared an ancestral function in stem
cell control since root and shoot separation, or whether they have
been recruited for this function after the diversification of stem cell
niches.
METHODS
Plant work. wox5-1 (SALK038262) and wox5-3 (SALK147644) are transfer (T)DNA
insertion alleles in the Columbia (Col) accession and were obtained from
the Arabidopsis Biological Resource Center (ABRC, USA) and the Nottingham
Arabidopsis Stock Center (NASC, UK). 73DR5-GUS23
was kindly provided by
J. Marfett, and mpU51
(ref. 14) by G. Ju¨rgens (Tu¨bingen). Root length and
meristem size were measured as described10
. The number of meristematic cells
was obtained by counting cortex cells showing no signs of rapid elongation.
Quiescent centre laser ablations were performed in 4-day-old roots using a
Leica SP2 inverted confocal laser scanning microscope5
. Roots were stained
for amyloplasts using Lugol (Sigma) 24-h after ablation24
. For confocal microscopy,
root tips were mounted in 10 mg ml21
propidium iodide solution.
Microscope settings were as described5
.
Expression analysis. Starch granules24
and b-glucuronidase activity7
were visualized
as described. 35S-WOX5 seedlings were cultured on standard 0.53 MS
medium supplemented with or without 1 mM dexamethasone. In situ hybridization
of sections, whole-mount embryos, and 4-day-old seedlings were performed
as described1,24
. The PLT1 riboprobe has been described previously12
. The WOX5
riboprobe was prepared from a cDNA without the homeobox to avoid crosshybridization
with related mRNAs.
Further experimental details are provided as Supplementary Information.
Received 9 January; accepted 23 February 2007.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We are grateful to E. van der Graaff for the data shown in
Supplementary Fig. 4c, and E. Tucker, T. Demlow and B. Geiges for experimental
help. We thank G. Ju¨rgens for mp seeds, E. Kiegle and J. Haseloff for mGFP5ER and
UAS vectors, M. Terlou for software development for root and meristem
measurements, P. Graf for photography and E. Tucker and M. Tucker for comments
on the manuscript. This work was sponsored by grants from the European Union
(REGIA) and the Deutsche Forschungsgemeinschaft (to T.L.), a Netherlands
Genomics Initiative grant (to R.H.), and grants from the Japan Society for the
Promotion of Science and the Novartis Foundation (to K.N.).
Author Information Sequence of WOX5 mRNA is available in GenBank under
accession numbers NM_111961 and AY251398. Reprints and permissions
information is available at www.nature.com/reprints. The authors declare no
competing financial interests. Correspondence and requests for materials should
be addressed to T.L. (laux@biologie.uni-freiburg.de).
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