scispace - formally typeset

Journal ArticleDOI

PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development

25 Oct 2007-Nature (Nature Publishing Group)-Vol. 449, Iss: 7165, pp 1053-1057

TL;DR: These findings indicate that PLT protein dosage is translated into distinct cellular responses and high levels of PLT activity promote stem cell identity and maintenance; lower levels promote mitotic activity of stem cell daughters; and further reduction in levels is required for cell differentiation.
Abstract: Factors with a graded distribution can program fields of cells in a dose-dependent manner, but no evidence has hitherto surfaced for such mechanisms in plants. In the Arabidopsis thaliana root, two PLETHORA (PLT) genes encoding AP2-domain transcription factors have been shown to maintain the activity of stem cells. Here we show that a clade of four PLT homologues is necessary for root formation. Promoter activity and protein fusions of PLT homologues display gradient distributions with maxima in the stem cell area. PLT activities are largely additive and dosage dependent. High levels of PLT activity promote stem cell identity and maintenance; lower levels promote mitotic activity of stem cell daughters; and further reduction in levels is required for cell differentiation. Our findings indicate that PLT protein dosage is translated into distinct cellular responses.
Topics: Root meristem growth (57%), Cellular differentiation (55%), Stem cell (53%), Arabidopsis (52%), Developmental biology (51%)

Summary (1 min read)

Jump to:  and [Summary]

Summary

  • Factors with a graded distribution can program fields of cells in a dose-dependent manner, but no evidence has hitherto surfaced for such mechanisms in plants.
  • The authors findings indicate that PLT protein dosage is translated into distinct cellular responses.
  • Whether similar mechanisms occur in plants has been controversial; dosage-sensitive action of plant hormones has been inferred only after external application.
  • Stem cells are maintained in local micro-environments, which are similar to animal stem cell niches.
  • Stem cell daughters undergo additional divisions in transit-amplifying cell compartments called meristems; when cells leave the meristem they rapidly expand and differentiate.
  • Plt1;plt2 mutants display stem cell loss, loss of transit-amplifying cells and reduced cell expansion.
  • At the heart-stage of embryo development, BBM is expressed in provascular cells and in the lens-shaped quiescent centre progenitor cell (Fig. 1c).
  • The plt3-1 mutant allele carries a T-DNA insertion interrupting the first AP2 domain (Supplementary Fig. 2).
  • Homozygous plt3 single mutants have slightly shorter roots and meristems compared to wild type, but plt1 plt2plt3 triple homozygotes are rootless (Fig. 1e, upper inset).
  • The embryonic root pole of triple homozygous seedlings is fully differentiated at 3 days post germination (d.p.g.) and adventitious root primordia arrest at 6 d.p.g. (Supplementary Fig. 3).
  • Therefore, PLT1, PLT2 and PLT3 redundantly control expression of multiple PIN genes in the embryonic and postembryonic root.
  • These defects initiate in the early basal embryo (Supplementary Fig. 5) and resemble those in mutants of the auxin response factor MONOPTEROS and the auxin perception machinery.

Did you find this useful? Give us your feedback

Content maybe subject to copyright    Report

LETTERS
PLETHORA proteins as dose-dependent master
regulators of Arabidopsis root development
Carla Galinha
1
*{, Hugo Hofhuis
1
*, Marijn Luijten
1
, Viola Willemsen
1
, Ikram Blilou
1
, Renze Heidstra
1
& Ben Scheres
1
Factors with a graded distribution can program fields of cells in a
dose-dependent manner
1,2
, but no evidence has hitherto surfaced
for such mechanisms in plants. In the Arabidopsis thaliana root,
two PLETHORA (PLT) genes encoding AP2-domain transcription
factors have been shown to maintain the activity of stem cells
3
.
Here we show that a clade of four PLT homologues is necessary for
root formation. Promoter activity and protein fusions of PLT
homologues display gradient distributions with maxima in the
stem cell area. PLT activities are largely additive and dosage
dependent. High levels of PLT activity promote stem cell identity
and maintenance; lower levels promote mitotic activity of stem cell
daughters; and further reduction in levels is required for cell dif-
ferentiation. Our findings indicate that PLT protein dosage is
translated into distinct cellular responses.
During animal development, instructive molecules acquire a
graded distribution and induce distinct cellular responses in a con-
centration-dependent manner. Whether similar mechanisms occur
in plants has been controversial; dosage-sensitive action of plant
hormones has been inferred only after external application
4
. Plant
stem cell regions, which supply cells for the growing root and shoot
systems
5
, are potential sites of action for instructive gradients. Stem
cells are maintained in local micro-environments, which are similar
to animal stem cell niches
6
. Stem cell daughters undergo additional
divisions in transit-amplifying cell compartments called meristems;
when cells leave the meristem they rapidly expand and differentiate.
The PLETHORA1 (PLT1, At3g20840) and PLT2 (At1g51190) genes
encode AP2-domain transcription factor family members essential
for defining the root stem cell niche
3
. plt1;plt2 mutants display stem
cell loss, loss of transit-amplifying cells and reduced cell expansion.
PLT1 and PLT2 expression strongly correlates with a transcriptional
response maximum to the plant hormone auxin in the root tip
3,7
and this maximum has been shown to have profound organizing
activity
8
—a property often associated with sources of instructive
gradients. Here, we reveal that the PLT gene family controls distinct
aspects of root development in a dose-dependent manner through
PLT expression gradients that culminate in the stem cell niche.
The proteins encoded by At5g10510/AINTEGUMENTA-LIKE6
(AIL6)/PLT3 and At5g17430/BABY BOOM(BBM) group with
PLT1 and PLT2 in the AP2/ERF transcription factor family (Sup-
plementary Fig. 1)
9
, and these candidate redundant factors are pre-
dicted to be expressed in the root
10
.
From the heart-stage of embryogenesis onward, PLT3 is expressed
in provascular cells, the quiescent centre and columella progenitor
cells (Fig. 1a). Post-embryonically, PLT3 messenger RNA accumu-
lates in the root stem cell niche with the strongest signal in the
columella stem cell layer (Fig. 1b), in contrast to the predominant
quiescent-centre-localization of PLT1 and PLT2 transcript
3
. At the
heart-stage of embryo development, BBM is expressed in provascular
cells and in the lens-shaped quiescent centre progenitor cell (Fig. 1c).
Post-embryonically, BBM transcript accumulates in the quiescent
centre and columella stem cells—in a similar manner to the PLT
mRNAs—and in provascular cells of the proximal meristem (Fig. 1d).
The plt3-1 mutant allele carries a T-DNA insertion interrupting
the first AP2 domain (Supplementary Fig. 2). No transcript was
detected by PCR with reverse transcription (RT–PCR) or by in situ
hybridization on plt3-1 seedlings (data not shown), suggesting that
plt3-1 is a null allele. Homozygous plt3 single mutants have slightly
shorter roots and meristems compared to wild type, but plt1
2/2
plt2
2/2
plt3
2/2
triple homozygotes are rootless (Fig. 1e, upper
inset). Progeny from plt1
2/2
plt2
2/2
plt3
1/2
, plt1
2/2
plt2
1/2
plt3
2/2
and plt1
1/2
plt2
2/2
plt3
2/2
plants segregate ,25% rootless triple
mutants (Supplementary Table 2), demonstrating linkage between
the rootless phenotype and the three PLT genes. The embryonic root
pole of triple homozygous seedlings is fully differentiated at 3 days
post germination (d.p.g.) and adventitious root primordia arrest at
6 d.p.g. (Supplementary Fig. 3). Mature plt1
2/2
plt2
2/2
embryos
have only subtle defects in the cellular organization of the distal-most
region
3
(Supplementary Fig. 4), but plt1
1/2
plt2
2/2
plt3
2/2
parents
yield ,25% embryos with aberrant root poles that lack a lateral root
cap cell layer (Supplementary Fig. 4).
We previously showed that plt1
2/2
plt2
2/2
mutants have strongly
reduced transcription of the PIN4 gene, which encodes an auxin
efflux facilitator
11
. In triple mutant embryos from plt1
1/2
plt2
2/2
plt3
2/2
parents, PIN1 and PIN3 mRNAs are strongly reduced
(Fig. 1g–j and Supplementary Table 1). Post-embryonic PIN2
mRNA is strongly reduced in triple mutant roots before differenti-
ation (Fig. 1k, l). Therefore, PLT1, PLT2 and PLT3 redundantly
control expression of multiple PIN genes in the embryonic and post-
embryonic root.
bbm-1 and bbm-2 mutant alleles carry T-DNA insertions before
and in the beginning of the first AP2 domain, respectively (Supple-
mentary Fig. 2). Truncated transcripts are detected by RT–PCR and
may be translated, but genetic interactions (described below) suggest
that the insertions cause loss-of-function effects. plt3
2/2
bbm
2/2
double mutants have a shorter root and root meristem than either
single mutant (Fig. 1f, and Supplementary Fig. 3).
Intriguingly, the progeny of plants segregating different plt and
bbm allele combinations lack root and hypocotyl (Fig. 1e, lower inset)
at significant frequencies (Supplementary Table 2), reaching ,10%
of the progeny of selfed plt1
2/2
plt2
1/2
plt3
2/2
bbm-2
2/2
. These
defects initiate in the early basal embryo (Supplementary Fig. 5)
and resemble those in mutants of the auxin response factor
MONOPTEROS
12
and the auxin perception machinery
13,14
. PLT
genes do not seem to strongly perturb early global auxin-dependent
patterning processes, as suggested by essentially normal cotyledon
vasculature in the triple mutant (Supplementary Fig. 4). Segregation
*These authors contributed equally to this work.
1
Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. {Present address: Department of Plant Sciences, University of Oxford,
South Parks Road, Oxford OX1 3RB, UK.
Vol 449
|
25 October 2007
|
doi:10.1038/nature06206
1053
Nature
©2007
Publishing
Group

of plt2 in a homozygous bbm background and vice versa yields ,25%
early arrested embryos, and homozygous double mutants could not
be recovered, indicating a redundant function in early embryogenesis
(data not shown).
Ectopic root structures are initiated by constitutive embryonic
expression of PLT genes
3
and after induction of BBM expression
15
.
To test whether PLT induction induces a developmental switch to
root development, we expressed a PLT2–GR fusion protein that
complements plt1
2/2
plt2
2/2
after dexamethasone (dex) induction,
when driven by its own promoter (Supplementary Fig. 6). When
35S-PLT2–GR is activated by application of dex, roots are produced
from the shoot apex (Fig. 1m, n). Our gain- and loss-of-function
experiments indicate that PLT genes are master switches for root
development.
plt1
2/2
plt2
1/2
plt3
2/2
mutants have intermediate root and mer-
istem size between plt1
2/2
plt3
2/2
and plt1
2/2
plt2
2/2
plt3
2/2
(Fig.
1e, f, and Supplementary Fig. 3) and 50% of plt1
2/2
plt2
1/2
plt3
2/2
bbm
2/2
seedlings have shorter roots than plt1
2/2
plt3
2/2
bbm
2/2
, whereas 50% have no primary root (Supplementary Table
2). plt3 alleles are also semi-dominant, because growth and meristem
maintenance defects in plt1
2/2
plt2
2/2
plt3
1/2
seedlings are inter-
mediate between plt1
2/2
plt2
2/2
and plt1
2/2
plt2
2/2
plt3
2/2
(Fig. 1f,
and Supplementary Fig. 3). The semi-dominance of plt2 and plt3
loss-of-function alleles indicates dose-dependent activity.
To test whether PLT genes equally contribute to PLT ‘dosage’, we
transformed plt1
2/2
plt2
2/2
double mutants with PLT1, PLT2, PLT3
and BBM genes fused to the yellow fluorescent protein gene YFP and
driven by the full PLT2 promoter. In independent lines with similar
overall YFP levels, PLT1 and PLT2 fully complemented and PLT3 and
BBM partially complemented root growth in the double mutant. All
PLT proteins rescued columella stem cell activity (Supplementary
Figs 6 and 7). Thus, total PLT levels and to some extent intrinsic
differences in PLT protein activity contribute to root growth and
stem cell maintenance.
Transgenic lines carrying complete promoters of the PLT genes
fused to the cyan fluorescent protein gene CFP reveal highest pro-
moter activity in the stem cell niche, consistent with mRNA levels,
but they also show graded activity in the proximal meristem (Fig.
2a–d). Gradients can be observed in epidermal surface views, exclud-
ing quenching effects, and they are specific to PLT promoters (Fig.
2e). To analyse whether this promoter activity drives a PLT protein
gradient, we combined the PLT–YFP fusions with their correspond-
ing full promoters. PLT1 and PLT2 gene fusions complemented
plt1
2/2
plt2
2/2
mutants (Supplementary Figs 6 and 7, and data not
shown).
j
a db c e
f g h i
Figure 2
|
PLT promoter activity and PLT protein fusions display gradients.
ad, CFP reporter driven by full-size promoters of PLT1 (a) PLT2
(
b) PLT3 (c) and BBM (d). e, Epidermal gradient of PLT2 (left) but not
RCH2 (right) promoter.
fi, YFP reporter fused in-frame to genomic
fragments of PLT1 (
f), PLT2 (g), PLT3 (h) and BBM (i). j, Co-localization in
one plant of PLT2 transcriptional (CFP, left magnification) and translational
(YFP, right magnification) fusion viewed in different regions using separate
channels.
PIN1
PIN2
PIN3
300
60
120
360
180
240
Meristem length (mm)
f
PLT3
BBM
col
pv
a
b
c
d
e
g
h
i
j
k
l
m
n
WS
plt1
plt2
plt3
plt1;plt3;plt2/+
plt2;plt3
plt2;plt3;plt1/+
plt1;plt2
plt1;plt3
plt1;plt2;plt3/+
plt1;plt2;plt3
bbm
plt1;bbm
plt3;bbm
Col0
plt1;plt3;bbm
Figure 1
|
Four PLT genes promote root formation. ad, In situ
hybridization with PLT3- (
a, b) and BBM-(c, d) specific probes in wild-type
embryos at heart-stage (
a, c), and in roots of 3 d.p.g. wild-type plants
(
b, d). Asterisk, quiescent centre; pv, provasculature; col, columella.
e, Seedlings 10 d.p.g., from left to right: wild type, plt3
2/2
, bbm-1
2/2
,
plt1
2/2
plt2
2/2
, plt1
2/2
plt2
2/2
plt3
2/2
and a plt1
2/2
plt2
1/2
plt3
2/2
bbm-
1
2/2
segregant. Insets show magnification of plt1
2/2
plt2
2/2
plt3
2/2
mutant
(upper) and plt1
2/2
plt2
1/2
bbm-1
2/2
segregant (lower). f, Meristem size in
wild type (Col0 and WS) and plt mutants at the indicated d.p.g. For each data
point, n 5 10 to 50; error bars, s.e.m.
gl, In situ hybridization using PIN
probes on wild-type (
g, i) and plt1
2/2
plt2
2/2
plt3
2/2
(h, j) torpedo-stage
embryos and wild-type (
k) and plt1
2/2
plt2
2/2
plt3
2/2
mutant (l) 2 d.p.g.
seedlings.
m, Shoot of 9 d.p.g. 35S-PLT2–GR plant 6 days after
dexamethasone application.
n, Magnification reveals cellular organization of
ectopic root including columella starch granules.
LETTERS NATURE
|
Vol 449
|
25 October 2007
1054
Nature
©2007
Publishing
Group

All PLT protein fusions revealed conspicuous gradients that
extend into the transit-amplifying cells and, for the PLT2 and
PLT3 fusions, into the elongation zone (Fig. 2f–i). The promoter
and protein gradients fully match when combined in one plant
(Fig. 2j). We previously reported accumulation of PLT transcripts
in the stem cell area
3
, but, after extended staining, PLT1 in situ hybri-
dizations also reveal a broader expression domain (Supplementary
Fig. 8). We concluded that PLT promoter activity leads to protein
gradients with maximum expression in the stem cell niche. PLT1 and
PLT2 expression maxima broadly encompass the niche, whereas
PLT3 and BBM are more restricted.
We asked whether differences in PLT expression domains affect
the ability of PLT proteins to compensate for redundant partners.
Indeed, PLT1 and PLT2 only partially complement a plt1
2/2
plt2
2/2
mutant when driven by the BBM promoter (Supplementary Figs 6
and 7).
Our experiments suggested that the PLT protein concentration
gradient instructs different outputs in different regions, even though
each gene slightly differs in activity and expression profile. We there-
fore tested whether altering the level or shape of the PLT2 gradient
affects the position of developmental boundaries. We expressed the
PLT2–YFP fusion in plt1
2/2
plt2
2/2
mutants under the RCH2 pro-
moter, which has low activity in the stem cell area but is active in
meristematic and elongating cells at a level comparable to that of
the PLT2 promoter (Fig. 3d–g). RCH2-PLT2 YFP prolongs transit-
amplifying cell divisions but fails to maintain stem cells at 7 d.p.g.
(Fig. 3b, d). The transit-amplifying cell pool is lost at 12 d.p.g.
(Fig. 3c). We concluded that intermediate PLT levels in the meristem
promote transient cell cycling.
To validate that meristem size is controlled by a PLT gradient, we
analysed plt1
2/2
plt2
2/2
mutants complemented with the PLT2–YFP
construct driven by a truncated 1.3-kb PLT2 promoter fragment
(pPLT2s). This truncated promoter drives significant expression in
the stem cell area but the gradient declines more rapidly (Fig. 3e, f).
Accordingly, stem cells are rescued but root and meristem sizes are
,50% smaller (Supplementary Fig. 7). The amount of YFP signal per
mid-nuclear section in the stem cell zone, halfway the meristem, and
in the first expanding cells, provides three clearly separated intensity
ranges that match with zonation in the full- and truncated-promoter
driven gradients (Fig. 3g), suggesting that the PLT2 gradient defines
meristem zonation.
A dose-dependent gradient model predicts that PLT over-
expression shifts the meristem boundary. Indeed, dex induction of
35S-PLT2–GR plants promotes continuous growth of the transit-
amplifying cell pool and meristem size increases (Fig. 4a–c). Ink
toner marks marking the elongation zone boundaries at the time of
induction reveal that PLT overexpression sustains cell division only
in cells that are still cycling and inhibits cell expansion in the elonga-
tion zone. These data reinforce the idea that distinct PLT levels dic-
tate cell proliferation and mitotic exit.
The auxin response marker DR5-GUS
8
and PIN3 transcription do
not change in 35S-PLT2–GR plants just before the onset of meristem
size expansion, but only at later stages, indicating that PLT-induced
expansion of the division zone is not caused by rapid changes in PIN
expression (Supplementary Fig. 9).
Notably, the stem cell area in PLT2–GR plants is not altered after
induction (Fig. 4c). The RETINOBLASTOMA (RBR) pathway was
recently identified as an independent stem cell input
16
, so we rea-
soned that this pathway might still limit stem cell pool size in the
presence of higher PLT levels. Therefore, we combined a root-specific
RNA interference (RNAi)-mediated silencing construct (RCH1-
RBRi)
16
with 35S-PLT2–GR. After induction with dex in the double
transgenic, root meristem size increases as in 35S-PLT2–GR, but
clusters of dividing cells in the root cap area expand beyond that seen
in RCH1-RBRi alone (Fig. 4d, e). Moreover, periclinal divisions
normally associated with stem cells occur throughout the proximal
area (Fig. 4f). These data suggested that the high expression region of
the PLT gradient can be instructive for stem cell fate. Dramatic sup-
port for this notion is provided by duplications of the distal stem cell
area in ,10% of RCH1-RBRi;35S-PLT2–GR root meristem zones
(Fig. 4gi). We concluded that high PLT levels define the stem cell
domain, confirming PLT dosage-dependent stem cell specification.
Average pixel intensity
0
50
100
150
200
250
a
b
c
d
e
f
g
pPLT2–PLT2–YFPa2
pPLT2–PLT2–YFPb1
pPLT2–PLT2–YFPb2
pPLT2s–PLT2–YFPa1
pPLT2s–PLT2–YFPa2
pPLT2–PLT2–YFPa1
pRCH2–PLT2–YFP
Figure 3
|
PLT expression regulates stem cell maintenance and meristem
boundary. a
d, Meristem prolongation but not stem cell rescue in RCH2-
PLT2–YFP plants. Nomarski optics image of root tip of 7 d.p.g. plt1;plt2
(
a), and of plt1;plt2 RCH2-PLT2YFP at 7 d.p.g. (b) and 12 d.p.g. (c). Starch
granule staining (brown) shows no rescue of columella stem cells below the
quiescent centre. Confocal view of 7 d.p.g. plt1;plt2 RCH2-PLT2–YFP root
(
d) shows that the meristem is rescued and reveals no expression of
PLT2–YFP in the stem cell area. Asterisk in
b, quiescent centre. e, f, Promoter
truncation shifts the meristem boundary. CLSM views at identical pinhole
and laser settings for RCH2-PLT2–YFP (
d), pPLT2-PLT2–YFP (e) and
pPLT2s-PLT2–YFP (
f). g, Quantification of fluorescence per nucleus in
pRCH2-PLT2–YFP transient meristem (red circles in
d, and red graph
areas), and in stem cells (yellow in
e, f and graph area), mid-meristem (green
in
e, f and graph area) and first elongating cells (blue in e, f and graph area) of
pPLT2-PLT2–YFP and pPLT2s-PLT2–YFP (a and b indicate independent
transformants, 1 and 2 indicate different roots).
NATURE
|
Vol 449
|
25 October 2007 LETTERS
1055
Nature
©2007
Publishing
Group

This effect is normally limited by RBR. Low RBR levels in the RCH1-
RBRi transgenic display limited expansion of the stem cell domain
16
because the PLT levels dictated by the gradient are limiting.
Our data indicate that PLT protein gradients define three outputs
in the growing root primordium: stem cell programming, mitotic
activity and exit to differentiation. Analysis of PLT target genes will
be required to assess how much of the response to graded activity is
due to additive concentration effects on the same targets and to
differences in target specificity.
Although the molecular link between auxin action and PLT gene
activation may not be direct
3
, auxin distribution and response
systems are essential for correct PLT gene transcription. This raises
the possibility that PLT proteins promote stem cells and transit-
amplifying cells as a graded read-out of auxin distribution. In an
accompanying paper, we provide evidence that PIN-mediated polar
auxin transport establishes a dynamic gradient spanning the root
meristem
17
. Hence it is tempting to speculate that an auxin gradient
underlies the observed PLT gradients. Classical morphogen systems
were conceptualized as independent from the response system.
However, several gradients in animal development involve compli-
cated dynamics (for example, ref. 18) and the static concept of posi-
tional information is being challenged
19
. We show that PIN polar
auxin transport facilitator expression that is essential for correct
auxin distribution is regulated by PLT activity, which is a clear
example of entanglement between positional information and its
response system.
METHODS SUMMARY
Plant work. plt1-4 and plt2-2 alleles were described in ref. 3, plt3-1, and bbm-1
and bbm-2 are salk T-DNA insertion lines 127417, 097021 and 067917, respect-
ively, provided by the Signal Insertion Mutant Library (http://signal.salk.edu/).
The T-DNA insertion in PLT3 was confirmed by genotyping. The plt1;plt2;plt3
triple mutant was generated by crossing plt3-1 to plt1-4;plt2-2. bbm-1 and bbm-2
were crossed to plt1-4;plt2-2 and plt3-1 and allelic combinations were selected
from F
2
populations. The T-DNA insertion site on bbm-1 and bbm-2 lines was
verified by genotyping. Primers for genotyping are indicated in Supplementary
Table 3. Promoter and genomic sequences were amplified from Col-0 genomic
DNA using the primer combinations listed in Supplementary Table 3.. Promoter
fragments were fused to the endoplasmic reticulum targeted CFP coding
sequence in a pGreenII vector
20
. For translational fusions, PLT genomic
sequences were fused at the 39 end to either the YFP coding sequence or the
carboxy-terminal-encoding region of the rat glucocorticoid (GR) receptor
21
and
placed under the control of particular promoters (amplified regions are
described in Supplementary Table 3). Promoter swaps were performed by fusing
5.8 kb of PLT2 and 4.2 kb of BBM promoter fragments to the YFP-fused PLT
genomic sequences. Transgenic plants were generated by transforming Col-0
wild-type or plt1-4;plt2-2 plants, as described
22
.
Phenotype analysis and microscopy. Light microscopy
23
, confocal microscopy
and aniline blue staining
24
of mature embryos was performed as described. Root
length was measured, as before
3
. Meristem cell length was measured using
ImageJ (v.1.36) and mature cortical cell length as well as fluorescence levels were
determined using Zeiss LSM Pascal (3.2SP2) software.
In situ hybridization. Whole-mount RNA in situ hybridization was performed
as described
11
. The PLT3 and BBM riboprobes, specific for non-conserved
sequences downstream of the AP2 repeats, were prepared from templates
amplified from complementary DNA (for primers, see Supplementary Table
3). The PLT1 probe is as in ref. 3; the PIN1, PIN2 and PIN3 probes are as in
ref. 25.
Received 5 July; accepted 30 August 2007.
1. Tabata, T. & Takei, Y. Morphogens, their identification and regulation.
Development 131, 703
712 (2004).
2. Gurdon, J. B. & Bourillot, P. Y. Morphogen gradient interpretation. Nature 413,
797
803 (2001).
3. Aida, M. et al. The PLETHORA genes mediate patterning of the Arabidopsis root
stem cell niche. Cell 119, 109
120 (2004).
4. Skoog, F. & Miller, C. O. Chemical regulation of growth and organ
formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol. 54, 118
130 (1957).
5. Weigel, D. & Jurgens, G. Stem cells that make stems. Nature 415, 751
754
(2002).
6. Spradling, A., Drummond-Barbosa, D. & Kai, T. Stem cells find their niche. Nature
414, 98
104 (2001).
7. Xu, J. et al. A molecular framework for plant regeneration. Science 311, 385
388
(2006).
8. Sabatini, S. et al. An auxin-dependent distal organizer of pattern and polarity in the
Arabidopsis root. Cell 99, 463
472 (1999).
9. Nole-Wilson, S., Tranby, T. L. & Krizek, B. A. AINTEGUMENTA-like (AIL) genes
are expressed in young tissues and may specify meristematic or division-
competent states. Plant Mol. Biol. 57, 613
628 (2005).
10. Birnbaum, K. et al. A gene expression map of the Arabidopsis root. Science 302,
1956
1960 (2003).
11. Blilou, I. et al. The PIN auxin efflux facilitator network controls growth and
patterning in Arabidopsis roots. Nature 433, 39
44 (2005).
12. Hardtke, C. S. & Berleth, T. The Arabidopsis gene MONOPTEROS encodes a
transcription factor mediating embryo axis formation and vascular development.
EMBO J. 17, 1405
1411 (1998).
13. Hellmann, H. et al. Arabidopsis AXR6 encodes CUL1 implicating SCF
E3 ligases in auxin regulation of embryogenesis. EMBO J. 22, 3314
3325
(2003).
14. Dharmasiri, N. et al. Plant development is regulated by a family of auxin receptor F
box proteins. Dev. Cell 9, 109
119 (2005).
15. Srinivasan, C. et al. Heterologous expression of the BABY BOOM AP2/ERF
transcription factor enhances the regeneration capacity of tobacco (Nicotiana
tabacum L.). Planta 225, 341
351 (2007).
16. Wildwater, M. et al. The RETINOBLASTOMA-RELATED gene regulates stem cell
maintenance in Arabidopsis roots. Cell 123, 1337
1349 (2005).
17. Grieneisen, V. A., Xu, J., Mare
´
e, A. F. M., Hogeweg, P. & Scheres, B. Auxin
transport is sufficient to generate a maximum and gradient guiding root growth.
Nature doi:10.1038/nature06215 (this issue).
18. O’Connor, M. B., Umulis, D., Othmer, H. G. & Blair, S. S. Shaping BMP morphogen
gradients in the Drosophila embryo and pupal wing. Development 133, 183
193
(2006).
19. Jaeger, J. & Reinitz, J. On the dynamic nature of positional information. Bioessays
28, 1102
1111 (2006).
20. Hellens, R. P., Edwards, E. A., Leyland, N. R., Bean, S. & Mullineaux, P. M. pGreen: a
versatile and flexible binary Ti vector for Agrobacterium-mediated plant
transformation. Plant Mol. Biol. 42, 819
832 (2000).
21. Aoyama, T. & Chua, N. H. A glucocorticoid-mediated transcriptional induction
system in transgenic plants. Plant J. 11, 605
612 (1997).
a
b
cd
e
f
g
h
i
Figure 4
|
Inducible expansion of meristem and stem cell area with
PLT2
GR fusions. ac, 35S-PLT2–GR 7 d.p.g. without dex (a) and 1 d after
5 mM dex application (
b, c). Overview shows positioning of ink toner
particles that mark the meristem boundary (black arrowhead) and upper
elongation zone boundary at the onset of induction (
b); the elongation zone
boundary is defined as the position where cortical cells rapidly expand.
Induced PLT2–GR roots reveal cell division below the meristem boundary
and incomplete cell elongation (
c). df, 35S-PLT2–GR;pRCH1-RBR RNAi
plants: 10 d.p.g. without dex revealing the two RBRi-induced stem cell layers
below the quiescent centre (blue arrowhead, inset), asterisk indicates the
quiescent centre (
d); with 3 d of dex application, revealing excessive root cap
stem cells (blue arrowhead) and periclinal divisions in the proximal
meristem (
e); magnification with ectopic periclinal divisions (f , white
arrowhead).
gi, Duplication of the stem cell area (red arrowheads) and
distal cell types (brown starch granules) in , 10% of 8 d.p.g. 35S-PLT2–GR,
pRCH1-RBRi plants after dex application. Early (
g), mid- (h) and late
(
i) stages of ectopic stem cell centre; note the prolonged activity of both stem
cell centres (
i, inset).
LETTERS NATURE
|
Vol 449
|
25 October 2007
1056
Nature
©2007
Publishing
Group

22. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-
mediated transformation of Arabidopsis thaliana. Plant J. 16, 735
743 (1998).
23. Willemsen, V., Wolkenfelt, H., deVries, G., Weisbeek, P. & Scheres, B. The HOBBIT
gene is required for formation of the root meristem in the Arabidopsis embryo.
Development 125, 521
531 (1998).
24. Bougourd, S., Marrison, J. & Haseloff, J. Technical advance: an aniline blue staining
procedure for confocal microscopy and 3D imaging of normal and perturbed
cellular phenotypes in mature Arabidopsis embryos. Plant J. 24, 543
550 (2000).
25. Friml, J. et al. AtPIN4 mediates sink-driven auxin gradients and root patterning in
Arabidopsis. Cell 108, 661
673 (2002).
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank the Netherlands Genomics Initiative (M.L.) and the
Portuguese Foundation for Science and Technology (C.G.) for funding,
A. Shimotohno and J. M. Perez-Perez for sharing data and Frits Kindt for
photography.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. Correspondence and requests for materials should be
addressed to B.S. (b.scheres@uu.nl).
NATURE
|
Vol 449
|
25 October 2007 LETTERS
1057
Nature
©2007
Publishing
Group
Figures (4)
Citations
More filters

Journal ArticleDOI
28 Nov 2008-Science
TL;DR: The cell differentiation and division balance necessary for controlling root meristem size and root growth is the result of the interaction between cytokinin and auxin through a simple regulatory circuit converging on the SHY2 gene.
Abstract: Plant growth and development are sustained by meristems. Meristem activity is controlled by auxin and cytokinin, two hormones whose interactions in determining a specific developmental output are still poorly understood. By means of a comprehensive genetic and molecular analysis in Arabidopsis, we show that a primary cytokinin-response transcription factor, ARR1, activates the gene SHY2/IAA3 (SHY2), a repressor of auxin signaling that negatively regulates the PIN auxin transport facilitator genes: thereby, cytokinin causes auxin redistribution, prompting cell differentiation. Conversely, auxin mediates degradation of the SHY2 protein, sustaining PIN activities and cell division. Thus, the cell differentiation and division balance necessary for controlling root meristem size and root growth is the result of the interaction between cytokinin and auxin through a simple regulatory circuit converging on the SHY2 gene.

733 citations


Journal ArticleDOI
25 Oct 2007-Nature
TL;DR: A robust auxin gradient associated with the maximum, in combination with separable roles of auxin in cell division and cell expansion, is able to explain the formation, maintenance and growth of sharply bounded meristematic and elongation zones.
Abstract: The plant growth regulator auxin controls cell identity, cell division and cell expansion. Auxin efflux facilitators (PINs) are associated with auxin maxima in distal regions of both shoots and roots. Here we model diffusion and PIN-facilitated auxin transport in and across cells within a structured root layout. In our model, the stable accumulation of auxin in a distal maximum emerges from the auxin flux pattern. We have experimentally tested model predictions of robustness and self-organization. Our model explains pattern formation and morphogenesis at timescales from seconds to weeks, and can be understood by conceptualizing the root as an 'auxin capacitor'. A robust auxin gradient associated with the maximum, in combination with separable roles of auxin in cell division and cell expansion, is able to explain the formation, maintenance and growth of sharply bounded meristematic and elongation zones. Directional permeability and diffusion can fully account for stable auxin maxima and gradients that can instruct morphogenesis.

714 citations


Journal ArticleDOI
TL;DR: The role of identified molecular components that convert auxin gradients into local differentiation events, which ultimately defines the root architecture is reviewed, in order to understand how this class of hormones participates in the control of root development.
Abstract: A plant's roots system determines both the capacity of a sessile organism to acquire nutrients and water, as well as providing a means to monitor the soil for a range of environmental conditions. Since auxins were first described, there has been a tight connection between this class of hormones and root development. Here we review some of the latest genetic, molecular, and cellular experiments that demonstrate the importance of generating and maintaining auxin gradients during root development. Refinements in the ability to monitor and measure auxin levels in root cells coupled with advances in our understanding of the sources of auxin that contribute to these pools represent important contributions to our understanding of how this class of hormones participates in the control of root development. In addition, we review the role of identified molecular components that convert auxin gradients into local differentiation events, which ultimately defines the root architecture.

586 citations


Cites background from "PLETHORA proteins as dose-dependent..."

  • ...Based on several lines of evidence, including the ability of ectopically expressed PLT2 to direct the formation of root meristems in shoots and the inability of plt1/plt2 plants to specify root stem cells, even in the presence of high exogenous auxin, it appears that high PLT levels promote stem cell identity and maintenance, that intermediate PLT levels promote mitotic activity of stem cell daughters, and that cells with the lowest PLT levels are capable of differentiation (Aida et al. 2004; Galinha et al. 2007)....

    [...]

  • ...…of auxin, in turn contribute to the regulation of gene expression that defines cell fate and pharmacological or genetic disruptions of auxin movement dramatically impacts root Cite this article as Cold Spring Harb Perspect Biol 2010;2:a001537 3 patterning (Aida et al. 2004; Galinha et al. 2007)....

    [...]

  • ...…of high exogenous auxin, it appears that high PLT levels promote stem cell identity and maintenance, that intermediate PLT levels promote mitotic activity of stem cell daughters, and that cells with the lowest PLT levels are capable of differentiation (Aida et al. 2004; Galinha et al. 2007)....

    [...]

  • ...Furthermore, although these genes are differentially expressed in a graded fashion in the root tip, their overlapping expression patterns leads to maximal PLT protein levels in the stem cell zone (Galinha et al. 2007)....

    [...]


Journal ArticleDOI
René Benjamins1, Ben Scheres1Institutions (1)
TL;DR: This review focuses on the feedback loops that form an integrative part of these regulatory mechanisms in the phytohormone auxin and their role in plant growth and development.
Abstract: The phytohormone auxin is a key factor in plant growth and development. Forward and reverse genetic strategies have identified important molecular components in auxin perception, signaling, and transport. These advances resulted in the identification of some of the underlying regulatory mechanisms as well as the emergence of functional frameworks for auxin action. This review focuses on the feedback loops that form an integrative part of these regulatory mechanisms.

517 citations


Cites background from "PLETHORA proteins as dose-dependent..."

  • ...Transcriptional and translational studies with PLT fusion proteins reveal gradients with highest expression in the stem cell area, intermediate levels in the division zone, and low levels in the elongation zone (41)....

    [...]

  • ...Changing the expression pattern and thereby the shape of the gradient together with the detailed analysis of the different mutant combinations suggested that high PLT levels maintain stem cells, intermediate levels control cell division, and low levels are needed for cell elongation (41)....

    [...]

  • ...PLT genes are necessary for the expression of PIN genes in the embryonic root pole and root meristem, which can in part explain the severe mutant phenotype (17, 41)....

    [...]


Journal ArticleDOI
Alexandra Schlereth1, Barbara Möller2, Weilin Liu2, Marika Kientz1  +6 moreInstitutions (3)
08 Apr 2010-Nature
TL;DR: The microarray-based isolation of MP target genes that mediate signalling from embryo to hypophysis is described, with the small TMO7 transcription factor representing a novel MP-dependent intercellular signal in embryonic root specification.
Abstract: During Arabidopsis embryogenesis, a single cell — called the hypophysis — is specified to become the founder cell of the root meristem in response to signals from adjacent cells. Hypophysis specification requires an auxin responsive transcription factor, MONOPTEROS, which promotes transport of auxin from the embryo to the hypophysis precursor. In this study, Dolf Weijers and colleagues identify MONOPTEROS target genes and show how they mediate root formation. During Arabidopsis embryogenesis, a single cell is specified to become the founder cell of the root meristem — the hypophysis — in response to signals from adjacent cells. Hypophysis specification requires an auxin-responsive transcription factor, MONOPTEROS (MP), which promotes transport of auxin from the embryo to the hypophysis precursor. Here, MP target genes are identified and the means by which they mediate root formation is shown. Acquisition of cell identity in plants relies strongly on positional information1, hence cell–cell communication and inductive signalling are instrumental for developmental patterning. During Arabidopsis embryogenesis, an extra-embryonic cell is specified to become the founder cell of the primary root meristem, hypophysis, in response to signals from adjacent embryonic cells2. The auxin-dependent transcription factor MONOPTEROS (MP) drives hypophysis specification by promoting transport of the hormone auxin from the embryo to the hypophysis precursor. However, auxin accumulation is not sufficient for hypophysis specification, indicating that additional MP-dependent signals are required3. Here we describe the microarray-based isolation of MP target genes that mediate signalling from embryo to hypophysis. Of three direct transcriptional target genes, TARGET OF MP 5 (TMO5) and TMO7 encode basic helix–loop–helix (bHLH) transcription factors that are expressed in the hypophysis-adjacent embryo cells, and are required and partially sufficient for MP-dependent root initiation. Importantly, the small TMO7 transcription factor moves from its site of synthesis in the embryo to the hypophysis precursor, thus representing a novel MP-dependent intercellular signal in embryonic root specification.

489 citations


References
More filters

Journal ArticleDOI
Steven J. Clough1, Andrew F. Bent1Institutions (1)
01 Dec 1998-Plant Journal
TL;DR: The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Abstract: Summary The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA

17,334 citations



Journal ArticleDOI
Ikram Blilou1, Jian Xu1, Marjolein Wildwater1, Viola Willemsen1  +6 moreInstitutions (3)
06 Jan 2005-Nature
TL;DR: This work shows that five PIN genes collectively control auxin distribution to regulate cell division and cell expansion in the primary root and reveals an interaction network of auxin transport facilitators and root fate determinants that control patterning and growth of the root primordium.
Abstract: Local accumulation of the plant growth regulator auxin mediates pattern formation in Arabidopsis roots and influences outgrowth and development of lateral root- and shoot-derived primordia. However, it has remained unclear how auxin can simultaneously regulate patterning and organ outgrowth and how its distribution is stabilized in a primordium-specific manner. Here we show that five PIN genes collectively control auxin distribution to regulate cell division and cell expansion in the primary root. Furthermore, the joint action of these genes has an important role in pattern formation by focusing the auxin maximum and restricting the expression domain of PLETHORA (PLT) genes, major determinants for root stem cell specification. In turn, PLT genes are required for PIN gene transcription to stabilize the auxin maximum at the distal root tip. Our data reveal an interaction network of auxin transport facilitators and root fate determinants that control patterning and growth of the root primordium.

1,633 citations


Journal ArticleDOI
Roger P. Hellens1, E. A. Edwards2, Nicola Leyland2, Samantha Bean2  +1 moreInstitutions (2)
TL;DR: The pGreen plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids.
Abstract: Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids. Its size and copy number in Escherichia coli offers increased efficiencies in routine in vitro recombination procedures. pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain. pSoup provides replication functions in trans for pGreen. The removal of RepA and Mob functions has enabled the size of pGreen to be kept to a minimum. Versions of pGreen have been used to transform several plant species with the same efficiencies as other binary Ti vectors. Information on the pGreen plasmid system is supplemented by an Internet site (http://www.pgreen.ac.uk) through which comprehensive information, protocols, order forms and lists of different pGreen marker gene permutations can be found.

1,438 citations


Journal ArticleDOI
01 Nov 2001-Nature
TL;DR: Technical advances now make it possible to characterize small zones that maintain and control stem cell activity in several organs, including gonads, skin and gut, and promise to advance efforts to use stem cells therapeutically.
Abstract: The concept that stem cells are controlled by particular microenvironments known as 'niches' has been widely invoked. But niches have remained largely a theoretical construct because of the difficulty of identifying and manipulating individual stem cells and their surroundings. Technical advances now make it possible to characterize small zones that maintain and control stem cell activity in several organs, including gonads, skin and gut. These studies are beginning to unify our understanding of stem cell regulation at the cellular and molecular levels, and promise to advance efforts to use stem cells therapeutically.

1,432 citations


Network Information
Performance
Metrics
No. of citations received by the Paper in previous years
YearCitations
20222
202175
202060
201947
201843
201730