DOI: 10.1126/science.1139531
, 421 (2007);316Science
et al.Hongchang Cui,
Endodermis in Plants
SHR Movement Defines a Single Layer of
An Evolutionarily Conserved Mechanism Delimiting
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37. We thank K. Pollard for valuable suggestions on statistics;
Y. Guo, I. Vasenkova, and R. De Breuil for help in
dsRNA synthesis at Open Biosystems Inc.; I. Cheeseman
for advice on homology searches; A. Straight for sharing
data in advance of publication; and S. Henikoff,
M. Bettencourt-Dias, D. Glover, T. Kaufman, D. Agard,
R. Tsien, and E. Griffis for reagents. G.G. received support
from the Human Frontier Science Program and R.W. from
a University of California GREAT Training Grant. We thank
the Sandler Foundation (UCSF) for support.
Supporting Online Material
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Materials and Methods
Figs. S1 to S7
Tables S1 and S2
References
Movies S1 to S5
Web site S1
14 February 2007; accepted 23 March 2007
Published online 5 April 2007;
10.1126/science.1141314
Include this information when citing this paper.
An Evolutionarily Conserved Mechanism
Delimiting SHR Movement Defines a
Single Layer of Endodermis in Plants
Hongchang Cui,1
Mitchell P. Levesque,1
*† Teva Vernoux,1
*‡ Jee W. Jung,1
Alice J. Paquette,1
Kimberly L. Gallagher,1
§ Jean Y. Wang,1
Ikram Blilou,2
Ben Scheres,2
Philip N. Benfey1
∥
Intercellular protein movement plays a critical role in animal and plant development. SHORTROOT
(SHR) is a moving transcription factor essential for endodermis specification in the Arabidopsis
root. Unlike diffusible animal morphogens, which form a gradient across multiple cell layers, SHR
movement is limited to essentially one cell layer. However, the molecular mechanism is unknown.
We show that SCARECROW (SCR) blocks SHR movement by sequestering it into the nucleus through
protein-protein interaction and a safeguard mechanism that relies on a SHR/SCR-dependent
positive feedback loop for SCR transcription. Our studies with SHR and SCR homologs from rice
suggest that this mechanism is evolutionarily conserved, providing a plausible explanation why
nearly all plants have a single layer of endodermis.
S
tem cell renewal and patterned differentiation
of their progeny are fundamental processes
in the development of multicellular
organisms. The root of Arabidopsis thaliana is
particularly suitable to study these processes,
because it has a simple and stereotyped cellular
organization (fig. S1) (1). SHR and SCR are key
regulators of root radial patterning (2, 3) and
stem cell maintenance (4). In shr and scr mutants,
the cortex/endodermis initial (CEI) cell, which
normally gives rise to two files of ground-tissue
cells (an inner layer of endodermis and an outer
layer of cortex), produces only a single cell layer
(fig. S1) (2, 3, 5). SHR is a transcription factor (6)
expressed in the stele that moves into the adjacent
cell layer where it controls SCR transcription and
endodermis specification (6). By contrast, the
SCR protein is absent from the stele, is predominantly
expressed in the endodermis, the CEI cell,
Fig. 4. Regulation of spindle length and chromosome alignment. (A) Spindle length was altered
after RNAi depletion of the novel protein Ssp4. Scale bar, 5 mm. (B) MT severing (yellow arrow)
frequently occurred after Ssp4 RNAi. Severed MTs often showed treadmilling behavior (red and
green arrows) and then disappeared. Scale bars, 10 mm (left), 2 mm (right). See also movie S4. (C)
Previously unknown Cal1 protein localizes to the centromere (marked by mCherry-Mis12).
(Localization data for other proteins are in fig. S7). Scale bar, 2 mm. (D) Model for kinetochore
assembly in S2 cells based on protein localization and RNAi. (Data are in fig. S7, D to F).
1
Department of Biology and Institute for Genome Sciences
and Policy, Duke University, Durham, NC 27708, USA.
2
Department of Molecular Genetics, Utrecht University,
Padualaan 8, 3584CH Utrecht, Netherlands.
*These authors contributed equally to this work.
†Present address: Max Planck Institute for Developmental
Biology, Department of Genetics and Genomics, Spemannstrasse
35/III, D-72076 Tübingen, Germany.
‡Present address: Reproduction et Développement des
Plantes Laboratory, Unité Mixte de Recherche 5667, Ecole
Normale Supérieure de Lyon, 46, Alleé d’Italie, 69364 Lyon
Cedex 07, France.
§Present address: Department of Biology, University of
Pennsylvania, Philadelphia, PA 19104, USA.
||To whom correspondence should be addressed. E-mail:
philip.benfey@duke.edu
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and the quiescent center (QC), and is required for
the asymmetric cell division that gives rise to the
cortex and endodermis (3, 7). SHR protein does not
move beyond a single layer comprising the endodermis,
CEI cell, and QC. This is in sharp contrast
to moving signal proteins in animals (8). Endodermis
and cortex in the root are derived from the same
initial cells through asymmetric cell divisions.
Notably, although the number of cortex cell layers
varies considerably, nearly all plant species
examined so far have only one layer of endodermis,
suggesting an evolutionarily conserved mechanism
to form this single cell layer. SCR has been found to
play a role in restricting SHRmovement (9, 10), but
the underlying mechanism has remained unclear.
Positive feedback control of SCR transcription.
SHR and SCR belong to the GRAS
family of transcription factors (11). In both
animals and plants, transcriptional regulation
is known to play a key role in development
(12, 13). To elucidate the mechanism by which
SHR and SCR control root radial patterning, we
therefore first dissected their transcriptional circuits.
Previously, it has been shown that SHR
directly controls SCR transcription (14). However,
there was also indication for SCR autoregulation
(9). Using a chromatin immunoprecipitation–
polymerase chain reaction (ChIP-PCR) assay (15),
we found that SCR binds to its own promoter
(Fig. 1A and fig. S2) but not to the promoters of
SHR and a SIN3-like gene (At5g15020), which
does not appear to be regulated by SHR (14). By
reverse transcription PCR (RT-PCR), we confirmed
the previous finding that SCR expression
is reduced in both the shr and scr backgrounds
(9) (fig. S2). Our results thus demonstrated that
SCR is controlled by a SHR/SCR-dependent
positive feedback loop.
SHR and SCR are functionally interdependent.
Recently, we identified a number of additional
putative direct targets of SHR (14). To determine
whether these are also direct targets of
SCR, we assayed binding by ChIP-PCR. Our
initial results showed that SCR binds to the
promoters of MAGPIE (MGP) and SCR-LIKE 3
(SCL3) (Fig. 1A and fig. S3), which are also
bound by SHR (Fig. 1B and fig. S3). We then
checked SHR and SCR binding to other putative
direct SHR targets by promoter scanning
(15) (Fig. 1 and fig. S4). We found that SHR
and SCR both clearly bind to the promoters of
NUTCRACKER (NUC), a receptor-like kinase
(RLK), and MGP (Fig. 1, D to F). Their binding
Fig. 1. SCR binds its own promoter and other SHR targets. (A and B)
ChIP-PCR assay with the use of an antibody to GFP, showing binding by
GFP-SCR and SHR-GFP to the promoters of SCR, MGP, and SCL3. Fold
enrichment values in all panels, from (A) to (F), as determined by
quantitative real-time PCR (QPCR), are means ± SE from technical
replicates. pSHR, SHR promoter; SCR, SCR coding sequence; IN, input
DNA; M, Mock ChIP. (C) SHR-GFP binding to some of its targets is
abolished in scr mutant background. (D to F) SHR-GFP and GFP-SCR bind
to the promoters of NUC (D), MGP (E), and RLK (F), as revealed by promoter
scanning. The asterisks mark the positions of putative binding sites.
Fig. 2. SCR and SHR
directly interact. (A) SHRGFP
is detected in SCR immunoprecipitates
(SCR-IP).
(B) SCR is coimmunoprecipitated
with SHR-GFP.
(C) SCR does not coim-
munoprecipitatewithGFPSCR
(GFP-IP). The SCR-IP
assay shows that SCR is expressed
in the transgenic
plants expressing GFPSCR.
(D) Yeast two-hybrid
assay showing direct interaction
between SHR and
SCR. AD-SHRD, which
lacks the N-terminal 120
amino acids of SHR, still
interacts with SCR. b-Gal,
b-galactosidase; Gal4
UAS, Ga14 binding sites;
BD, Ga14 DNA binding
domain; AD, Ga14 activation
domain; BD-53 and AD-T as a pair are used as a positive control, whereas the BD-lamin and AD-T pair is a
negative control. BD-53, fusion between BD and the p53 protein; BD-lamin; fusion between BD and lamin; AD-T,
fusion between AD and the T protein.
20 APRIL 2007 VOL 316 SCIENCE www.sciencemag.org422
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sites on the NUC and MGP promoters were
located in regions relatively far upstream of the
translation start sites (TSS) (Fig. 2, D and E),
which explains why we were unable to confirm
NUC as a direct SHR target by ChIP-PCR using
PCR primers that amplify more proximal sequences
(14). Notably, most binding sites for SHR
and SCR at these promoters appear to coincide.
The observation that SHR and SCR bind to
a common set of genes suggests functional interdependence
between these two transcriptional
regulators. We therefore examined SHR binding
to some of its targets in an scr background. In
the absence of SCR, SHR binding to these
targets is abolished (Fig. 1C). Expression levels
of all these genes are reduced in the shr and scr
mutants (fig. S2), indicating that SCR is required
for SHR to regulate these genes. To determine
the extent of overlap between SHR and
SCR targets, we performed genome-wide expression
analysis in shr and scr mutants. Nearly
all putative direct SHR targets that we previously
identified (14) show significant reduction in their
expression in both mutant backgrounds (Table 1).
Moreover, a large portion of SHR indirect targets
also showed reduced expression in the scr background
(table S1).
SHR and SCR proteins directly interact.
Functional interdependence between SHR and
SCR could be achieved through their cooperative
binding to the same promoter or through
direct interaction. To determine whether SHR
and SCR form a complex, we performed coimmunoprecipitation.
Reciprocal pull-down experiments
showed that SCR and SHR are in a
complex (Fig. 2, A and B). In yeast cells, SHR
and SCR interact directly (Fig. 2D), and the
central domain spanning the two leucine heptad
repeats and the VHIID (Val-His-Ile-Ile-Asp)
motif is responsible for this interaction (fig.
S5). However, SCR does not appear to interact
with itself (Fig. 2C). The finding that SCR
physically interacts with SHR provides a
molecular basis for their functional interdependence.
However, clearly not all aspects
of SHR activity rely on interaction with SCR,
because the mutant ground-tissue layer in scr
still expresses endodermal markers that are not
detected in the shr mutant background. One
hypothesis is that the SHR/SCR complex controls
some aspects of SHR function, such as
asymmetric cell division, QC specification, and
stem cell maintenance, whereas complexes
formed between SHR and other proteins fulfill
other aspects of SHR function, particularly
endodermis specification.
SCR affects SHR subcellular localization
and movement. As the SHR protein with a
strong nuclear localization signal is no longer
capable of moving (7), the finding that SHR and
SCR directly interact suggests that one role for
SCR might be to sequester SHR into the nucleus,
thus preventing its movement. Indeed, the
fusion protein between green fluorescent protein
(GFP) and SHR (SHR-GFP) becomes largely
cytoplasmic in the mutant cell layer of scr (scr-1
in Fig. 3C) (10), in contrast to its exclusive
nuclear localization in the endodermis of wildtype
(WT) roots (Fig. 3C). However, because of
the low amount of SHR-GFP in the mutant cell
layer, it is unclear whether SHR moves out of
the mutant layer into the epidermis.
A large pool of SCR would be required to
completely block SHR movement. The positive
feedback loop for SCR transcription could provide
such a mechanism (16). To test this hypothesis,
we examined the effect of reduction in
SCR expression on SHR movement using an
RNA interference (RNAi) construct. We reasoned
that, if SCR levels were reduced below a
threshold level, some SHR protein might be able
to move into the presumptive cortex where it
would activate SCR transcription and endodermis
specification. Asymmetric cell division would
also occur, giving rise to an additional layer of
ground tissue. This process could be repeated
until free-moving SHR was exhausted. In support
of our hypothesis, plants from the RNAi transgenic
lines that we generated produced multiple
layers of cells. Two lines that have different
levels of SCR transcript were further examined
(Fig. 3A). As shown in Fig. 3C, the extra cell
layers in both lines express the endodermal
marker pSCR::GFP, and SHR-GFP expressed
in the stele is also present in these supernumerary
cell layers. Notably, SHR-GFP is detected in both
daughter cells of the CEI cell, whereas its levels
appear to decrease in the outer cell layer after each
additional cell division (Fig. 3C, insets). Moreover,
the number of supernumerary cell layers is
inversely correlated with the level of SCR
transcript in the two independent transgenic lines.
Furthermore, SHR-GFP is primarily nuclearTable
1. Expression levels of SHR direct targets in shr and scr mutants, relative to WT, as measured by
whole-genome Affymetrix ATH1 microarray. FC, fold change (reduction).
shr
(FC)
P value
scr
(FC)
P value
NUC 2.8 6.4 × 10–27
1.6 9.2 × 10–5
MGP 2.5 3.7 × 10–19
1.5 1.7 × 10–3
SCR 2.5 5.0 × 10–8
4.0 4.3 × 10–7
Br6ox2 1.9 7.4 × 10–23
1.8 3.0 × 10–4
RLK 1.4 2.8 × 10–5
1.4 3.8 × 10–1
SCL3 1.3 6.3 × 10–3
1.4 7.6 × 10–2
Tropinone reductase (TRI) 1.2 2.7 × 10–1
1.2 7.0 × 10–1
SNEEZY (SNE) 1.0 6.6 × 10–1
0.8 5.9 × 10–5
Fig. 3. SCR determines
SHR subcellular localization
and its range of
movement. (A) SCR transcript
levels in two independent
SCR RNAi lines
(SCRi-1 and SCRi-2), relative
to that in WT, as
determined by RT-QPCR.
(B) Root lengths of the
SCR RNAi lines and WT 6
days after germination.
Error bars in (A) and (B)
indicate SD. (C) Confocal
images of 6-day-old roots
of WT, scr-1, SCRi-1, and
SCRi-2 seedlings, showing
their structure [propidium
iodide (PI) staining], an
endodermal marker expression
(pSCR::GFP),
and SHR-GFP localization
(pSHR::SHR-GFP). The insets
in the bottom panels
are enlarged images of
the framed areas. C, cortex;
E, endodermis; M,
mutant cell layer; S, supernumerary
cell layers.
Scale bars, 10 mm.
www.sciencemag.org SCIENCE VOL 316 20 APRIL 2007 423
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localized in the supernumerary layers of SCRi-1,
the weaker RNAi line, but is largely cytoplasmlocalized
in SCRi-2, the stronger RNAi line (Fig.
3C). The two lines also showed reduced root
length that correlates with the strength of RNAi (P
values are 2.9 × 10–5
and 2.8 × 10–13
, respectively;
Student’s t test, n = 39 roots), although the QC,
CEI, and other initials appear normal (Fig. 3, A
and B). These results demonstrate the critical role
of the positive feedback mechanism for SCR in
restricting SHR movement, root radial patterning,
and root growth.
Our results support a mechanism by which
SCR tightly restricts SHR movement, as described
below. On the one hand, SCR sequesters
SHR into the nucleus through protein complex
formation, making SHR incapable of further
movement. On the other hand, the SHR/SCRdependent
positive feedback loop for SCR
transcription ensures no free-moving SHR can
escape from the endodermis by driving a rapid
buildup of SCR that does not self-interact but
rather preferentially interacts with SHR. This
mechanism would require a basal level of SCR
expression to initiate the feedback loop. Notably,
a substantial level of SCR mRNA is still detectable
in both the shr and scr backgrounds, and
its specific radial expression pattern is largely
unaltered. This SHR/SCR-independent basal
SCR transcription may be one of the key factors
defining the boundary for SHR movement.
The model that we propose for SCR to
restrict SHR movement could also account for
the fact that different cell fates are rapidly acquired
by the progeny of the daughter cells of
the CEI cell (9). After this asymmetric cell division,
the concentration of the SHR/SCR
complex will remain high in the inner cell of
the endodermal lineage driven by a sustained
supply of SHR from the stele, which activates
the SCR feedback loop. This high concentration
of the SHR/SCR complex would maintain the
expression of SCR as well as other downstream
patterning genes. By contrast, the SHR/SCR concentration
in the other cell of the cortex lineage
would drop rapidly, resulting from the inability of
SHR to move beyond the endodermis coupled
with protein turnover and the dilution accompanying
cell division. Indeed, although SCR is
detected in both cells immediately after the
asymmetric cell division, SCR and other endodermal
markers are only expressed in the
endodermis soon thereafter (9).
Interaction and expression of SHR and SCR
homologs in rice. The observations that nearly
all plants examined so far have only a single
layer of endodermis (even though the number of
cortex layers can be highly variable) and that
SCR orthologs are exclusively expressed in the
endodermis (17–19) suggest that the mechanism
described above is likely to be evolutionarily
conserved. However, the only SHR homolog
cloned so far, which is claimed to be the closest
SHR homolog from rice, shows an expression
pattern that is distinct from SHR in Arabidopsis
(19), thus casting doubt on this hypothesis.
Database searches revealed that there are, in
fact, two close rice homologs for both SHR
(Os03g31750 and Os07g39820) and SCR
(Os11g03110 and Os12g02870). We named the
more similar SHR and SCR homologs OsSHR1
(Os07g39820) and OsSCR1 (Os11g03110) and the
more dissimilar ones OsSHR2 (Os03g31750) and
OsSCR2 (Os12g02870), respectively (table S2).
The rice genes that were previously reported
as homologs of SHR and SCR are OsSHR2 and
OsSCR1 (19). We therefore cloned OsSHR1 and
analyzed its expression in rice roots by in situ
hybridization. As shown in Fig. 4A, OsSHR1
and OsSCR1 are both expressed in tissues analogous
to those of their counterparts in Arabidopsis.
OsSCR1 and OsSHR1 interact in yeast
as strongly as Arabidopsis SHR and SCR do
(Fig. 4B). They also interact equally well with
SHR and SCR, but no interaction was observed
between OsSHR2 and OsSCR1 (Fig. 4B). These
results strongly suggest that OsSHR1 and OsSCR1
are functional homologs of SHR and SCR in rice.
They further suggest that the functional relationship
between SHR and SCR, as well as their role
in radial patterning in higher plants, is evolutionarily
conserved.
Proteins that move as signaling molecules
play a critical role in both animal and plant development
(8, 20, 21). Although the list of
transcription factors that are able to move is
growing (22–28), little is known about the
mechanisms regulating intercellular movement.
Decapentaplegic (Dpp), for example, a wellcharacterized
example from animals, moves
passively by diffusion and forms a gradient
across multiple layers of cells as a result of
unregulated binding to and internalization by its
receptors located on the surface of the cells that
it passes through (8, 29). By contrast, both SHR
movement and its range of action are actively
regulated, and the mechanism that we have
uncovered in this study is quite distinct from
those previously described. Although some
aspects of this mechanism have been reported
for other proteins, this is the first example where
both protein-protein interaction and transcriptional
control are involved to achieve tight
control of protein movement. This difference
may extend to other moving plant proteins and
indicate a fundamental difference between plant
and animal signaling during development.
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30. We thank X. Dong (Duke University) for the p35S::GFP
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members of the Benfey Laboratory for critical reading of
the manuscript and helpful comments. We acknowledge
fellowship support from the Human Frontiers Science
Program and the European Molecular Biology
Organization (for T.V.), NIH (for A.J.P.), and NSF (for
M.P.L.). This work was supported by a grant to P.N.B.
from NIH (RO1-GM043778).
Supporting Online Material
www.sciencemag.org/cgi/content/full/316/5823/421/DC1
Materials and Methods
Figs. S1 to S5
Tables S1 to S3
References
4 January 2007; accepted 6 March 2007
10.1126/science.1139531
REPORTS
Nonequilibrium Phase Transitions in
Cuprates Observed by Ultrafast
Electron Crystallography
Nuh Gedik,1
Ding-Shyue Yang,1
Gennady Logvenov,2
Ivan Bozovic,2
Ahmed H. Zewail1
*
Nonequilibrium phase transitions, which are defined by the formation of macroscopic transient
domains, are optically dark and cannot be observed through conventional temperature- or
pressure-change studies. We have directly determined the structural dynamics of such a
nonequilibrium phase transition in a cuprate superconductor. Ultrafast electron crystallography
with the use of a tilted optical geometry technique afforded the necessary atomic-scale spatial and
temporal resolutions. The observed transient behavior displays a notable “structural isosbestic”
point and a threshold effect for the dependence of c-axis expansion (Dc) on fluence (F), with
Dc/F = 0.02 angstrom/(millijoule per square centimeter). This threshold for photon doping
occurs at ~0.12 photons per copper site, which is unexpectedly close to the density
(per site) of chemically doped carriers needed to induce superconductivity.
T
he physical and chemical properties of
materials can be altered as a result of
the generation of metastable structures
(1), electronic and/or structural modifications
(2, 3), and phase transitions (4). For the latter,
much of the work has been done on solids at
equilibrium, namely when temperature or
pressure becomes the variable of change. In
contrast, transient structures of nonequilibrium
phases, which are formed by collective
interactions, are elusive and less studied
because they are inaccessible to conventional
studies of the equilibrium state. Initiated by
photons, the structural changes underlying
such transitions involve charge redistribution
and lattice relaxation, culminating in a process
termed a photoinduced phase transition (5–7).
In order to understand the nature of these optically
dark phases, it is important to observe
the structural changes with the use of timeresolved
methods, especially those that use
ultrafast electron microscopy (8–10), electron
diffraction (10–12), and x-ray absorption and
diffraction (13–17). Here, the direct observation
of the nonequilibrium structural phase
transition in superconducting cuprates is
reported.
We have previously established ultrafast
electron crystallography (UEC) (10) as a
method for studying surfaces and nanometerscale
materials with atomic-scale resolutions.
Our apparatus integrates a femtosecond laser
system into an ultrahigh vacuum (UHV) assembly
of three chambers (Fig. 1A). In this
technique, the output of a Ti:sapphire femtosecond
laser (with a pulse width of 120 fs) is
split into two beams: an 800-nm pulse used
to excite the sample and a 266-nm pulse
(generated by frequency tripling) used to
produce an electron packet via the photoelectric
effect. The electrons are then accelerated
at 30 kV, resulting in a de Broglie
wavelength of l = 0.07 Å. The diffraction
patterns of these electrons from the sample
are recorded on a charge-coupled device
(CCD) camera with single-electron sensitivity.
The time delay between the initiating
laser pulse and electron probe packet is controlled
by changing the optical path length
between the two pulses. Diffraction patterns
at different delay times (diffraction frames)
provide a movie of structural change, with
atomic-scale spatial and ultrashort temporal
resolutions (10, 18).
The material that we chose to study is
oxygen-doped La2CuO4+d; although the undoped
material is an antiferromagnetic Mott
insulator, doping confers superconductivity
below the critical temperature (Tc) and metallic
properties at room temperature. Thin films
were grown on a LaSrAlO4 substrate by
means of an atomic-layer molecular beam
epitaxy system (19). The films under study
were characterized during growth by reflection
high-energy electron diffraction and ex
situ by atomic force microscopy (AFM), x-ray
diffraction (XRD), and measurements of resistivity
and magnetic susceptibility as a function
of temperature (20).
In order to observe lower-order Bragg diffractions
from the material, the incident angle
of electrons (q/2) is set typically between ~1°
and 2°. Because the speed of electrons is
about one-third that of light, a large groupvelocity
mismatch occurs between the laser
pulse and the electron packet. Moreover, the
electron beam has, at this angle of incidence, a
large footprint on the surface of the material:
in this case, the (001) planes with the c axis
defining the surface normal direction. We
have implemented a wavefront tilting scheme,
1
Physical Biology Center for Ultrafast Science and Technology,
California Institute of Technology (Caltech),
Pasadena, CA 91125, USA. 2
Brookhaven National Laboratory
(BNL), Upton, NY 11973–5000, USA.
*To whom correspondence should be addressed. E-mail:
zewail@caltech.edu
www.sciencemag.org SCIENCE VOL 316 20 APRIL 2007 425
onAugust7,2009www.sciencemag.orgDownloadedfrom
ERRATUM
www.sciencemag.org SCIENCE ERRATUM POST DATE 21 DECEMBER 2007 1
CORRECTIONS&CLARIFICATIONS
Research Articles: “An evolutionarily conserved mechanism delimiting SHR movement
defines a single layer of endodermis in plants” by H. Cui et al. (20 April 2007, p. 421). In
two instances in the fifth paragraph on page 424, one of the rice homologs for SHR,
Os03g31880, was mistyped as Os03g31750.
Post date 21 December 2007
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