A Bistable Circuit Involving SCARECROW-
RETINOBLASTOMA Integrates Cues
to Inform Asymmetric Stem Cell Division
Alfredo Cruz-Ramı ´rez,1Sara Dı ´az-Trivin ˜o,1,10Ikram Blilou,1,10Vero ˆnica A. Grieneisen,3,4,10Rosangela Sozzani,5
Christos Zamioudis,2Pa ´l Miskolczi,6,7Jeroen Nieuwland,8Rene ´ Benjamins,1Pankaj Dhonukshe,1Juan Caballero-Pe ´rez,9
Beatrix Horvath,1Yuchen Long,1Ari Pekka Ma ¨ho ¨nen,1Hongtao Zhang,1Jian Xu,1James A.H. Murray,8Philip N. Benfey,5
Laszlo Bako,6,7Athanasius F.M. Mare ´e,3,* and Ben Scheres1,*
Department of Biology, University of Utrecht, 3584 CH Utrecht, The Netherlands
3Department of Computational and Systems Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
4Department of Cell and Developmental Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
5Department of Biology and Institute for Genome Science and Policy, Center for Systems Biology, Duke University, Durham, NC 27708, USA
6Department of Plant Physiology, Umea ˚ Plant Science Center, Umea ˚ University, S-901 87 Umea ˚, Sweden
7Department of Forest Genetics and Plant Physiology, Umea ˚ Plant Science Center, Swedish University of Agricultural Sciences,
S-901 83 Umea ˚, Sweden
8Cardiff School of Biosciences, Biomedical Sciences Building, Museum Avenue, Cardiff CF10 3AX, UK
9Institute for Systems Biology, 1441 North 107th Street, Seattle, WA 98109, USA
10These authors contributed equally to this work
*Correspondence: email@example.com (A.F.M.M.), firstname.lastname@example.org (B.S.)
In plants, where cells cannot migrate, asymmetric
cell divisions (ACDs) must be confined to the appro-
priate spatial context. We investigate tissue-gener-
ating asymmetric divisions in a stem cell daughter
within the Arabidopsis root. Spatial restriction of
these divisions requires physical binding of the
stem cell regulator SCARECROW (SCR) by the
the stem cell niche, SCR activity is counteracted by
phosphorylation of RBR through a cyclinD6;1-CDK
complex. This cyclin is itself under transcriptional
control of SCR and its partner SHORT ROOT (SHR),
creating a robust bistable circuit with either high or
low SHR-SCR complex activity. Auxin biases this
circuit by promoting CYCD6;1 transcription. Mathe-
matical modeling showsthat ACDs are onlyswitched
tion, determined by SHR and auxin distribution,
respectively. Coupling of cell-cycle progression to
protein degradation resets the circuit, resulting in
a ‘‘flip flop’’ that constrains asymmetric cell division
to the stem cell region.
In Arabidopsis, several factors mediating asymmetric cell divi-
sions (ACDs) have been identified, but little is known on how
control of their activity produces precise spatial patterning,
which is key to the development of the body plan (Abrash and
Bergmann, 2009). Endodermis and cortex tissues are generated
in the Arabidopsis root meristem by two successive ACDs. The
cortex/endodermis initial (CEI) is a stem cell that self-renews
and generates a cortex/endodermis initial daughter (CEID) cell.
The CEID undergoes a single periclinal asymmetric division,
and the progeny generates endodermis and cortex tissues
(Figure 1A). The GRAS family transcription factors SHORT
ROOT (SHR) and SCARECROW (SCR) play a prominent role in
the CEI and CEID ACDs acting as a heterodimer and are requir-
ed for the specification and maintenance of the root stem cell
niche (Cui et al., 2007; Di Laurenzio et al., 1996; Helariutta
et al., 2000; Sabatini et al., 2003). SHR moves from internal
tissues to the endodermis (Helariutta et al., 2000). There, it
gains efficient nuclear localization, and further movement is
restricted by SCR (Heidstra et al., 2004; Cui et al., 2007; Welch
et al., 2007). In addition, ACDs of several root stem cells require
the RETINOBLASTOMA-RELATED (RBR) protein. RBR interacts
genetically with SCR, but the molecular mechanism by which it
restricts ACDs to the stem cell niche has not yet been identified
(Wildwater et al., 2005). The Arabidopsis CYCLIND6;1 gene
(CYCD6;1), which potentially mediates RBR phosphorylation, is
a direct transcriptional target of SCR, suggesting a possible
connection between SCRand RBR activity (Sozzani et al.,2010).
Inanimalsand plants, RBproteins control G1-toS-phase pro-
gression in the cell cycle. In animals, CyclinD/CDK complexes
factor complexes that modulate cell-cycle progression (Temple-
ton et al., 1991; Krek et al., 1994; reviewed in Harbour and Dean,
2000). In plants, RBR is phosphorylated in a cell-cycle-specific
1002 Cell 150, 1002–1015, August 31, 2012 ª2012 Elsevier Inc.
manner by several Cyclin/CDK complexes (Boniotti and
Gutierrez, 2001; Nakagami et al., 2002; Takahashi et al., 2010).
In animals, RBs have also been implicated in cellular differentia-
tion through modulation of the activity of tissue-specific tran-
scription factors (Chen et al., 1996; Chen et al., 2007; Berman
et al., 2008; Nalam et al., 2009; Calo et al., 2010). The plant
RBR protein shares conserved residues with other plant and
animal RBs, primarily in the motifs that define interactions with
E2F transcription factors and those indispensable for binding
diverse proteins containing the conserved Leu-x-Cys-x-Glu
(LxCxE motif; Lee et al., 1998; Lendvai et al., 2007; reviewed in
Local reduction of RBR in the root meristem expands the stem
cell pool without altering cell-cycle rates, suggesting that RBR
regulates stem cell transitions by promoting differentiation of
stem cell daughters (Wildwater et al., 2005). In addition, RBR is
required for the maintenance of stem cells in the shoot and for
differentiation of precursor cells for stomata (Borghi et al., 2010).
its LxCxE motif. We demonstrate that this interaction, together
Figure 1. A Conserved LxCxE Motif in SCR Mediates Direct Binding to RBR
(A) Root stem cell niche organization and cell transitions and divisions defining ground tissue lineages.
(B)Yeast two-hybrid analyses showing and quantifying SCR-RBR interaction. RBR-E2FA and SCR-SHR combinations are positive controls, and RBR-SHRis the
(C) SCR-RBR binding by BiFC in Arabidopsis mesophyll protoplasts. RBR-E2FA and SCR-SHR are positive controls.
(D) Coimmunoprecipitation of RBR with a-GFP antibody in WT and 35S::SCR:GFP root extracts. Black arrow marks endogenous RBR intop panel and SCR-GFP
in lower panel.
(E) Protein sequence alignment of SCR orthologs in seed plants and P. patens moss showing conservation of the LxCxE motif.
(F) In vivo interaction strengths from split Renilla luciferase assay in mesophyll protoplasts. RLUs were normalized to H2A-H2B interaction strength. Arrow bar
(G–J) Confocal laser scanning microscope (CLSM) of longitudinal root sections of 5 dpg. scr4-1 plants complemented with WT SCR (G and I) and SCRAxCxA
(H and J). Ep, epidermis; Co, cortex; E, endodermis;*, extra ground tissue layer. See also Figure S1.
Cell 150, 1002–1015, August 31, 2012 ª2012 Elsevier Inc. 1003
with the activity of the RBR regulator CYCD6;1, defines the
precise position of ACDs in the stem cell area through two
bistable switch that is attenuated by a stem-cell-associated
auxin maximum and by mitotic progression, thereby linking the
auxin gradient along the longitudinal axis with the SHR distribu-
tion pattern in the radial axis.
RBR Physically Interacts with SCR through the
Conserved LxCxE Motif
We first investigated the molecular basis for previously observed
genetic interactions between SCR and RBR. Yeast two hybrid
assays indicated that RBR and SCR directly interact in vitro,
although less strongly than the SCR-SHR and RBR-E2FA
combinations used as positive controls (Figure 1B). Direct inter-
action between RBR and SCR was also observed in Arabidopsis
mesophyll protoplasts by using bimolecular fluorescence com-
plementation (BiFC) assays (Figure 1C). By coimmunoprecipita-
tion assays from root extracts, we also observed that SCR and
RBR form a complex in planta (Figure 1D).
An LxCxE motif N-terminal to the GRAS domain in the SCR
protein is highly conserved in SCR orthologs from seed plants
to the moss Physcomitrella patens (Figure 1E). To determine
the relevance of the LxCxE motif for the SCR-RBR interaction,
we generated a variant of the SCR complementary DNA
(cDNA) in which the LxCxE motif was converted into AxCxA
(SCRAxCxA) and tested its capacity to bind RBR. The SCRAxCxA
variant interacted with SHR with the same efficiency as wild-
type (WT) SCR, but the interaction with RBR was disrupted
(Figure 1B). Split-luciferase experiments (Fujikawa and Kato,
2007; Chen et al., 2008) produced SCR-SHR and RBR-E2Fa
interactions equally strong as an H2A-H2B interaction (Fig-
ure 1F). RBR-SCR interaction was weaker, and RBR-SCRAxCxA
was reduced to 11% of the RBR-SCR interaction (Figure 1F).
We concluded that SCR and RBR interact directly and that this
interaction depends on the LxCxE motif in SCR.
Disruption of the SCR-RBR Interaction Promotes CEID-
To test the relevance of the SCR-RBR interaction in planta, we
generated protein fusions to yellow fluorescent protein (YFP)
by using either the SCR wild-type cDNA or the SCRAxCxAcDNA
under the transcriptional control of the SCR promoter. Both
constructs, pSCR::SCR:YFP and pSCR::SCRAxCxA:YFP, were
transformed independently into the scr4-1 mutant background.
Seedlings of multiple stable transformants, homozygous for
the pSCR::SCR:YFP and pSCR::SCRAxCxA:YFP transgenes,
complemented the macroscopic defects in the scr4-1 mutant
and restored cotyledon and primary root size, indicating that
SCR and SCRAxCxAare both functional (data not shown).
scr4-1;pSCR::SCR:YFP roots fully complemented the scr4-1
phenotype and displayed the characteristic SCR expression
pattern in the quiescent center (QC), ground tissue initials and
mature endodermal layer (Figure 1G; compare with Figures
YFP (hereafter referred to as scr4-1;SCRAxCxA) seedlings
similarly complemented the scr4-1 phenotype. However,
meristematic endodermis cells expressing the SCRAxCxAfusion
performed extra periclinal divisions capable of generating a
complete extra ground tissue layer (Figures 1G–1J, asterisks)
from late embryo stage onward (Figures S1E and S1F, asterisk).
We investigated the identity of the extra layer observed in
scr4-1;SCRAxCxAroots by using markers to distinguish cortex
from endodermis tissue. Three markers demonstrated that
disruption of the interaction between SCR and RBR led to an
separates cortex and endodermis identity (Figures S1C, S1D,
S1G, and S1H). The extra ACD in the scr4-1;SCRAxCxAline indi-
cates that RBR interaction with WT SCR counteracts recurrent
In the WT, SCR promotes ACD in the CEI and CEID cells
together with its heterodimeric interaction partner SHR. When
we transformed the scr4-1;shr double mutant with pSCR::
SCR:YFP and pSCR::SCRAxCxA:YFP constructs, both lines dis-
played the shr phenotype lacking ACDs (‘‘M’’ in Figures S1I and
S1J). These results indicate that the extra ACD observed in the
scr4-1;SCRAxCxAroots, like the ACD in WT, is SHR dependent.
RBR Negatively Regulates SCR-SHR Transcriptional
Well-characterized direct targets of SCR and SHR are the
MAGPIE (MGP) and NUTCRAKER (NUC) genes (Cui et al.,
2007). Agenetic link between RBR and SCR-SHR transcriptional
activity was suggested by Wildwater et al. (2005), who showed
that the expression domain of NUC (At5g44160) expands as a
consequence of RBR silencing. After RBR transactivation
induced by dexamethasone (Dex, as previously described in
Wildwater et al., 2005), pMGP::MGP:GFP expression gradually
decreased in roots harboring the RBR-transactivation construct
(Figures 2A–2C). This reduction in expression was evident well
before the full differentiation of the meristem invoked by pro-
longed RBR overexpression.
SHR and SCR directly activate the D-type cyclin CYCD6;1,
whose expression is enriched in the CEI/CEID cells and whose
forced expression in the ground tissue produced extra divisions,
similar to those in scr4-1;SCRAxCxAroots (Sozzani et al., 2010).
CYCD6;1 is a potential regulator of RBR phosphorylation. If
RBR phosphorylation by CYCD6;1 inhibited RBR action on
SCR, this would lead to a feedforward loop (model 1, Figure 3G).
We predicted that the scr4-1;SCRAxCxA
prevents RBR inhibition of SCR activity, leads to CYCD6;1
expression outside of the CEI and CEID. Indeed, whereas the
pCYCD6;1::GFP expression domain in the scr4-1;SCR back-
ground, as in the WT context, was predominantly confined to
the ground tissue initials (Figure 2D), it expanded in the endo-
dermal layer of scr4-1;SCRAxCxAseedlings in perfect correlation
with the extra ACDs (Figures 2E and 2F).
SHR and SCR bind to the NUC promoter (Cui et al., 2007). We
hypothesized that binding of SCR to RBR takes place in the
context of target gene promoters. To test this, we performed
replicated chromatin immunoprecipitation (ChIP) experiments
followed by quantitative PCR (ChIP-qPCR) on the NUC
promoter. We found that RBR consistently associated with
specific NUCpromoter fragments locatedat?4.9Kb (Figure2G,
1004 Cell 150, 1002–1015, August 31, 2012 ª2012 Elsevier Inc.
red asterisk; Student’s t test, p < 0.05) and at ?2.2 Kb upstream
from the ATG (Figure 3A; p = 0.066) but that it was only weakly
enriched in line with its indirect binding to DNA.
We conclude that RBR negatively regulates SHR-SCR tran-
scriptional targets such asNUC and MGP,which in turnpromote
ACD(Welch etal.,2007). Inaddition, RBR constrainsthe expres-
sion of another SHR-SCR target, CYCD6;1, which may itself
regulate RBR activity.
Specific Binding of CYCLIND6;1 to CDKB1 Yields an
Active Complex that Phosphorylates RBR
BiFC assays revealed that CYCD6;1 interacted with RBR in
Arabidopsis protoplasts (Figure 3A) in a manner similar to the
positive control RBR-E2FC (Figure 3B). Previous large-scale
BiFC interaction mapping of Arabidopsis cell-cycle regulators
indicated association between CYCD6;1 and A-, B-, and
D-type CDK subunits (Boruc et al., 2010). We transiently coex-
pressed epitope-tagged CYCD6;1 and CDKs in Arabidopsis
protoplasts and observed specific interaction between CDKB1
and CYCD6;1 proteins (Figure 3C). Immunoprecipitation-protein
kinase assay against histone H1 and a glutathione S-transferase
(GST) fusion to the C-terminal polypeptide encompassing
the last 235 amino acids (GST-RBR-Ct) of Arabidopsis RBR
revealed that a CDKA;1-CYCD3;1 complex phosphorylated
Figure 2. RBR-SCR Interaction Affects the
Expression of SCR-SHR Direct Targets
(A–C) pMGP::MGP:GFP protein fusion expression
in WT (A) and inducible RBR overexpressor line
before (B) and after transfer for 24 hr (C) to Dex-
(D–F) pCYCD6;1::GFP fusion expression in WT (D)
and in scr4-1;SCRAxCxA(E); note the correlation
between expansion of pCYCD6;1::GFP expres-
sion domain and appearance of extra ACDs,
marked with asterisks; asymmetric segregation of
pSCR::SCRAxCxA:YFP in the same plant (F).
specific regions of NUC, PCNA, and IR1 pro-
moters. Black asterisks, Student’s t test (p < 0.05);
fragment ?2.2 showed variable enrichment in
tected no significant increase in histone
levels upon CYCD6;1 binding to CDKB1
(Figure 3D). On the other hand, immuno-
precipitation-kinase assays using a GST
fusion to the 608-amino-acid-long com-
plete pocket domain (see Experimental
Procedures for a detailed description
of RBR fragments used) revealed that
CYCD6;1binding increased basal activity
of CDKB1 (Figure 3E). Our data indicate
that an active CDKB1-CYCD6;1 kinase
complex targets one or more phos-
phorylation sites within the RBR pocket
The CDKB1 kinases to which CYCD6;1 specifically binds
are encoded by two genes, CDKB1;1 and CDKB1;2. Although
CDKB1;1 is expressed in a range of tissues (Segers et al.,
1996), a transcriptional fusion to the b-glucuronidase gene
(pCDKB1;1:GUS) and a translational fusion to GFP (pCDKB1;
1::CDKB1;1:GFP) revealed high levels of CDKB1;1 expression
and protein accumulation in CEI cells as they undergo ACD
(Figures S2C and S2D). To detect the protein fusion, reduction
of proteasome activity with MG132 treatment was necessary,
suggesting additional posttranslational regulation. Double-
mutant cdkb1;1 cdkb1;2 seedlings revealed a higher frequency
of undivided CEIDs as compared to WT (Figures 3F and S2B),
indicating that ACDs are delayed in cdkb1 double mutants.
Consistent with the specific CDKB1-CYCD6;1 interaction, this
phenotype is similar to that reported for the cycd6;1 mutant
(Sozzani et al., 2010). Collectively, our results suggest that
spatiotemporal fine tuning of RBR phosphorylation in the ground
tissue occurs through tight regulation of the CYCD6;1-CDKB1;1
The SHR/SCR/RBR/CYCD6;1 Network Generates
a Bistable Radial Patterning Switch
We addressed whether the nested feedback circuit among
SHR, SCR, RBR, and CYCD6;1 could result in the precise
Cell 150, 1002–1015, August 31, 2012 ª2012 Elsevier Inc. 1005
Figure 3. Specific Interaction between CDKB1 and CYCD6;1 Forms a Protein Complex that Phosphorylates RBR
(A and B) BiFC assays for in vivo interaction between CYCD6 and RBR (A) with E2FC/RBR as positive control (B).
(C) Myc-CDK and HA-CYCD6;1 proteins expressed in Arabidopsis protoplasts. Protein gel blots of complexes immunoprecipitated with anti-c-Myc antibodies
using anti-HA and anti-c-Myc antibodies to detect CYCD6;1 binding and CDK expression, respectively.
(D) Myc-CDK expressed in Arabidopsis protoplasts either alone or together with HA-CYCD6;1 or HA-CYCD3;1 and precipitated with anti-c-Myc antibody. Kinase
(E) Kinase activity of CDKB1 and CDKB1-CYCD6;1 expressed in protoplasts in the presence of [g-32P]ATP using GST-RBR pocket domain (GST-RBR-Pd) as
substrate, detected by autoradiography. Asterisk marks IgG heavy chain.
(F) Frequency of undivided CEI and CEIDs in roots of WT and ckdb1;1 cdkb1;2 seedlings 4 dpg. Differences are significant (t test < 0.05); error bars indicate SEM.
(G) Schematic of model 1.
(H) Bifurcation diagram with equilibrium levels of free SCR as a function of SHR influx into the cell. Dashed line indicates unstable equilibria. Both a high and a low
stable SCR level exist over a wide range of influx.
1006 Cell 150, 1002–1015, August 31, 2012 ª2012 Elsevier Inc.
spatial confinement of ACDs. We generated coupled ordinary
differential equations (ODEs) to describe the wiring of the
network (Figure 3G; Supplemental Information for details) and
focused on analyzing equilibrium levels of nuclear SHR and
free SCR (unbound to RBR), as ACDs occur when both levels
are high and SCR-RBR levels are low. Analysis and numerical
simulations of the model showed that the network ensures
highly robust bistability in which nuclear SHR and free SCR
levels can flip between a low and a high state. Figure 3H depicts
the equilibria in terms of free SCR levels. Bistability can be
found over a very wide range of SHR influx levels. For low values
of SHR influx, there is only one stable state, illustrating that
sufficient SHR influx is needed to gain the potential of
the ACD cell state, whereas very high SHR influx causes the low
equilibrium to disappear, leading to the flip to the ACD cell state.
Conversely, once the ACD cell state is triggered, only very low
SHR levels can bring the cell back into the normal state.
This hysteresis effect is due to the fact that SCR seques-
tering by RBR prevents the positive feedback loops from taking
effect, which is necessary to increase SCR concentrations.
This dynamic behavior is observed over a broad parameter
range, indicating intrinsic robustness within the network wiring.
To address to which extent the bistability is due to the SCR
feedback on its own transcription or to the CYCD6-mediated
feedback loop that promotes free SCR, we mathematically
analyzed the cases in which one or both of the feedback loops
are removed (Figures 3I–3L and S2E–S2H). The qualitative
behavior of bistability was preserved when any one of the
two feedbacks was removed (Figures 3J, 3K, S2F, and S2G).
However, if both are turned off, the system failed to present
bistability for any possible parameter setting (Figures 3L and
S2H). We therefore conclude that it is the combined (and
redundant) action of two feedback loops that determines the
huge potential for bistability underlying the behavior of ACD
To explore how these interactions are influenced by the
cellular environment in the root, we inserted the network in every
cell of an in silico root (Figure 3M). Because SHR production is
limited to the vasculature while degraded everywhere, a radial
gradient in SHR levels was formed. Interestingly, within the
spatial simulations, the SHR influx-dependent bistability leads
to two possible scenarios. One possibility is that the highest
SHR levels, found close to the vasculature, are not sufficient
to trigger the ACD state (Figures 3N, 3P, and 3R). In that case,
SHR keeps its graded radial distribution (Figure 3N), which is
never observed in vivo. Alternatively, the positive feedback
loops are sufficiently strong that all cells close to the vasculature
are flipped to the ACD cell state (Figures 3O, 3Q, and 3S). In the
latter case, due to the efficient sequestering of SHR in the endo-
dermis, CEI/CEID, and QC, cells farther from the stele do not
receive enough SHR to switch to the high equilibrium. This result
was consistent for different parameter settings and spatial im-
plementations (data not shown). The modeling result that the
complete endodermal cell file triggers the ACD state is not
observed in vivo. Hence, the network explains the radial position
of ACDs but does not contain a mechanism to bias this to the
Auxin Promotes SHR/SCR-Dependent CYCD6;1
Transcription to Trigger ACDs in the Longitudinal Axis
The plant growth factor auxin is a key factor in positioning the
niche, and its polar transport is required for longitudinal posi-
tioning of CEI/CEID markers (Sabatini et al., 1999). Therefore,
auxin might mediate restriction of CYCD6;1 expression and the
associated ACD in the longitudinal axis. When we increased
auxin concentration with IAA (the major active auxin) and NPA
(a polar auxin transport inhibitor), expression of pCYCD6;1::GFP
expanded, coinciding with extra periclinal divisions thatoften led
to an extra ground tissue layer in primary roots (Figure 4A). After
the same treatment, Col0;pSHR::SHR:GFP roots revealed the
SHR protein fusion in the inner cell layer, indicating that auxin
induced ACDs (Figure 4B). In scr4-1;SCRAxCxA;pCYCD6;1::GFP
roots, where RBR cannot repress SCR activity, IAA/NPA treat-
ment induced successive ectopic ACDs in the transit amplifying
zone (Figure 4C), correlating with strong shootward-extended
expression of pCYCD6;1::GFP (Figure 4D).
shr;pSHR::SHR:GR mutant background only after DEX-induced
nuclear localization of SHR (Figures 4E and 4E0). When
shr;pSHR::SHR:GR seedlings were grown continuously in both
DEX and IAA/NPA, pCYCD6;1::GFP expression expanded, and
extra ACDs occurred (Figure 4F). We concluded that auxin-
mediated induction of the CYCD6;1 promoter requires SHR
and SCR activity.
To increase auxin levels only in the endodermis by bacterial
biosynthetic enzyme IAAH, we introgressed the pSCR::IAAH
transgene in the Col0;pCYCD6;1::GFP background. Roots of
seedlings grown in IAM-containing medium, but not control
roots (Figure 4G), expanded pCYCD6;1::GFP expression, which
is correlated with extra ACDs (Figure 4H). Because it remained
possible that auxin synthesized in the endodermis fluxed around
to activate an ACD stimulating factor elsewhere, we drove IAAH
from the epidermal/lateral root cap WEREWOLF promoter
(pWER::IAAH). When grown on IAM-containing medium, extra
ACDs occurred in the endodermis, and pDR5::GFP and pPIN1::
PIN1:GFP signals increased in the ground tissue and the
vascular tissue, indicating that auxin synthesized in the outer
layers flows to the inner tissues and triggers extra ACDs (Figures
4I, 4J, and 4J0). However, on IAM- and NPA-containing medium,
reflux of auxin from the WER domain was blocked, as revealed
(I–L) Phase plane analysis of model 1 using quasi-steady-state assumptions (see Figure S2). (I) Model 1 presents two stable equilibria (solid black circles, one of
high SCR and nuclear SHR levels and the other of low SCR and nuclear SHR levels) separated by an unstable equilibrium (open circle). (J and K) When the
activation mediated by CYCD6;1 or the feedback of SCR and nuclear SHR on SCR transcription is turned off, the model continues presenting bistability.
(L) Without either of these feedbacks, no bistability occurs for any parameter setting.
(M) Root layout for all spatial simulations.
(N–S) Cytosolic SHR (N and O), nuclear SHR (P and Q), and free SCR (R and S) for weak (N, P, and R) and strong (O, Q, and S) activation mediated by CYCD6;1
(see ‘‘Modeling Procedures,’’ Figure S2, and Tables S1 and S2). Molecular weight standards are indicated in kilodaltons.
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by higher levels of pDR5::GFP in the LRC and low expression of
both markers in the vascular and ground tissues; under these
conditions, no extra ACDs were observed (Figures 4K and 4K0).
Laser ablation experiments, which alter auxin flow in predictable
manners (van den Berg et al., 1997; Xu et al., 2006), indepen-
dently supported that a local increase of auxin levels in the
endodermis is critical to trigger new ACDs but needs the pres-
ence of SHR and SCR (Figures S4A–S4H).
An Auxin-Attenuated Switch Promotes ACD at the Stem
To explore the spatial-dynamical consequences of auxin feed-
back on CYCD6 transcription, we added this interaction to our
ODE network, now representing model 2 (Figure 4L, green
arrow). For an isolated cell, in a nonspatial setting, we found
a switch-like response in free SCR levels linked to the auxin
concentration (Figure 4M). Next, we generated a spatial model
by concomitantly describing auxin dynamics based on experi-
mentally observed PIN distributions (Grieneisen et al., 2007;
nization of the stem cell niche and root cap (Figure S3, details in
Supplemental Information). The modeled auxin dynamics in this
context predicted that, through the auxin-attenuated network
within the root context, CYCD6;1, nuclear SHR, and SCR
become localized to the QC and CEI (Figures 4N–4R).
Although, compared to model 1, model 2 clearly presents an
improvement in capturing the regulation of the ACDs, it fails to
explain the confinement of SHR to the endodermis. This is
because, in model 2, only the cells that enter the ACD state
can block the SHR throughput from the vasculature to the
external cell files. Thus, SHR moves from endodermal cells
that are not in the ACD state into the next cell layer (Figure 4Q),
as in scr mutants (Heidstra et al., 2004; Sena et al., 2004).
The lack of SHR confinement contradicted experimental data,
stream targets by binding the SHR-SCR complex. To test
whether a ternary complex can indeed be formed, we coex-
pressed SHR-GFP fusion protein with epitope-tagged SCR
and RBR in Arabidopsis protoplasts and purified complexes
on a GFP affinity matrix. Immunoblot analysis of affinity-purified
complexes confirmed the previously observed SCR-SHR
heterodimer and indicated that SCR-SHR interaction is not
compromised by the RBR protein (Figure 5B). Western analysis
also revealed copurification of the RBR protein, suggesting
thatthe threeproteinscan formacomplex (Figure5B).Wethere-
fore extended model 2 to explicitly take into account not only the
SCR-RBR complex but also the SHR-SCR and the SHR-SCR-
RBR ternary complex (Figure 5A). The third model presents, in
a parsimonious way, our current understanding of the regulatory
circuit and the distribution of key factors underlying ACDs
(Figures 5C–5G and S4I–S4M).
Model 3 revealed that, whereas RBR complexes are absent in
the ACD region, as basically all RBR is in its phosphorylated
form, high levels of the ternary complex in the endodermis
prevent both ACDs and SHR transport into the cortex (Figures
5G and S4M). Implementing the SCRAxCxAmodification, which
decreases the binding of RBR to SCR, resulted in high levels of
the SHR-SCR complex not only the CEI but also in the first endo-
dermal cell and CEID, which is in agreement with the observed
extended CEI activity (Figures 5H–5J). After simulating auxin
immersion (Grieneisen et al., 2007), high levels of the SHR-
together with a further reduction of the inhibitory complex (Fig-
ure 5L). This confirms the quantitative impact of auxin levels on
the process of ACD specification through the network and
extra ACDs. However, although model 3 explains how auxin and
SHR influx trigger the ‘‘on’’ state of the bistable SHR-SCR-RBR
circuit, as well as the radial confinement of SHR, it does not
reveal how the system is reset. Because the network functions
through a double feedback loop (one of which is auxin depen-
dent), once the system is flipped to the ‘‘on’’ state to undergo
ACDs, even a dramatic decrease in auxin levels will not be
able to turn it back ‘‘off’’ (Figures S4N and S4O).
Protein Degradation Can Reset the Bistable ACD Switch
The solution to this apparent failure was suggested by the ACD
itself. The triggering of an ACD is a cell-cycle event, and regula-
tory proteins are often degraded during specific cell-cycle
phases. Therefore, we analyzed the levels of functional RBR,
SHR, and SCR protein fusions expressed from their own
promoters during the cell cycle. Prior to mitosis, endodermis-
expressed RBR:YFP, SHR:YFP, and SCR:YFP protein fusions
wereprimarily localized to thenucleus.However,elongated cells
prior to cell plate formation displayed a weaker and cytoplasmic
signal (Figures S5A–S5C0), and postdivision nuclear levels were
Figure 4. Network Model within Tissue Context Presents Bistable Switch Where Auxin-Dependent CYCD6;1 Activation by SHR-SCR Limits
ACD to the CEI
(A–K) IAA+NPA treatments in Col-0;pCYCD6;1::GFP (A), Col-0;pSHR::SHR:GFP (B), and scr4-1;ACA; pCYCD6;1::GFP (C) and (D). shr;pSHR::SHR:GR;
treated with both IAA+NPA and Dex for 24 hr (F). Effect of local increase of auxin levels in endodermis revealed in the pSCR::IAAH; pCYCD6;1::GFP line in
absence (G) and presence (H) of the substrate IAM. Phenotype and expression patterns of Col-0;pWER::IAAH;pPIN1::PIN1:GFP;pDR5::GFP roots of 5 dpg
seedlings grown in MS media and transferred for 2 days to media in absence (I) and presence of the substrate IAM (J and J0) and in the presence of IAM and NPA
(K and K0). Arrowheads in (J) and (K) point to increased pDR5::GFP expression in the LRC, indicating auxin synthesis. Ep, epidermis; Co, cortex; E, endodermis;
Eco, extra cortex layer; M, mixed identity ground tissue layer; *, extra ground tissue layer.
(L) Schematic of model 2. The green arrow indicates the extension of the model, which takes the effect of auxin into account.
(M)Bifurcationdiagramshowingequilibrium levelsoffreeSCRasafunctionofauxinlevels. Aswitch-like behavioroccurswhenauxin levelsareincreased,butthe
system does not ‘‘turn off’’ when the levels are subsequently decreased.
(N–R) The spatial simulations show the triggering of the ACD cell state in the QC and CEI/CEID cells. (N) auxin; (O) CYCD6;1; (P) free SCR; (Q) cytosolic SHR; and
(R) nuclear SHR levels. (See also ‘‘Modeling Procedures,’’ Figure S3, and Tables S2 and S3.)
Cell 150, 1002–1015, August 31, 2012 ª2012 Elsevier Inc. 1009
To record multiple cells in specific cell-cycle phase, we stan-
dardized conditions for in planta hydroxyurea (HU)-induced
cell-cycle synchronization (Figures S5D–S5K0; Cools et al.,
2010). pSHR::SHR:YFP decreased in abundance in individual
endodermal cells followed between 19 and 22 hr HU, when the
35S::H2B:RFP protein fusion resolved into a mitotic figure, and
such decreasewas also observed
pRBR::RBR:CFP fusions (Figures S5L–S5S).
To investigate further whether specific protein degradation
machinery mediated the decrease in SHR, SCR, and RBR
concentration, we utilized the 26S proteasome inhibitor
MG132. In the presence of MG132, the fluorescence of
a CYCB1;1:GFP control line increased (Figures 6A and 6B;
n R 7). Roots expressing RBR and SCR protein fusions also
revealed increased signal after MG132 treatment (Figures
6C–6F). No increase of pSHR::SHR:YFP signal was evident after
ure 6G). These data suggest that SCR and RBR are subject to
26S-proteasome-mediated degradation, whereas the decrease
by other factors.
Some plant D Cyclins are targets for degradation by the 26S
proteasome (Genschik et al., 1998; Lechner et al., 2002; Plan-
chais et al., 2004; Sanz et al., 2011). Indeed, when CYCD6;1:
YFP was expressed in cortex and endodermal layers along
the root meristem, fluorescence was detected in the nucleus
of some endodermis cells and in the cytoplasm of others. In
contrast, after MG132 application, strong nuclear-localized
YFP signal appeared throughout both cortex and endodermis
layers (Figures 6I and 6J). We concluded that proteasome
activity destabilizes the CYCD6;1:YFP protein. We validated
the dynamics of protein degradation during divisions in vivo
by recording the behavior of SCR and SHR proteins in endo-
dermis cells by using the double marked lines p35S::
H2B:RFP;pSCR::SCR:YFP and p35S::H2B:RFP;pSHR::SHR:GFP
and observed rapid protein degradation (Figure 6K and Movies
S3 and S4).
We analyzed whether the observed cell-cycle-mediated
degradation was sufficient to reset the ACD switch in model 3.
Figures 6L and S5X show bifurcation diagrams for the equilib-
rium level of SHR-SCR as a function of the increased level of
were three equilibria, two of which were stable. This implies that
the first cell within the endodermal cell file could indeed be either
in the ‘‘on’’ state or in the ‘‘off’’ state, but, having inherited its
state fromthe CEID, it would beexpected to be in the ‘‘on’’ state.
If, however, cell division was accompanied by increased protein
degradation, it would be sufficient to flop cells from the ‘‘on’’
state back down to a lower state, switching the system off (see
Figures 6L and 6M).
The observed rapid protein degradation corresponded to the
balance between the level of accelerated protein degradation
Figure 5. Auxin Concentration Influences CYCD6;1 Expression and Modulates Ground Tissue ACDs
(A) Schematic of model 3.
(B) Complex formation between SHR-GFP, HA-SCR, and Myc-RBR proteins expressed in Arabidopsis protoplasts and purified by binding to GFP affinity beads.
(C–G)Spatial simulationsshowthetriggeringoftheACDcellstateintheQCand CEI/CEID cellsand theconfinement ofSHRintheendodermis.Profileswithin the
root tip of (C) CYCD6;1; (D) free SCR; (E) SHR; (F) SHR-SCR; and (G) SHR-SCR-RBR levels. (See also ‘‘Modeling Procedures,’’ Figure S4, and Movie S1.)
(H–J) A 75% reduction of the binding of SCR to RBR, as to mimic the SCR (see also Movie S2).
(K and L) Treatment of the SCR with auxin (see also Movie S2). Color bar represents relative concentration levels. See Supplemental Information, Figure S4, and
Table S4 for full details.
1010 Cell 150, 1002–1015, August 31, 2012 ª2012 Elsevier Inc.
place is further illustrated in Figure 6M, in which shorter time
‘‘off’’ without relapse to the ‘‘on’’ state. This behavior of our
model predicted that protease inhibitor treatment could itself
trigger ACDs. MG132 application indeed consistently triggered
ectopic periclinal cell divisions associated with CycD6 expres-
sion that were asymmetric based on the persistence of SCR
expression only in inner cells (Figures S5T–S5V).
We describe a nested feedback circuit in Arabidopsis that
ensures the accurate spatiotemporal control of asymmetric cell
divisions. Modeling studies reveal that this circuit integrates
information of two developmental axes, auxin-mediated tissue
polarity, and the radial SHR expression domain and translates
this into a precisely located asymmetric cell division state, which
is schematized by the simplified model in Figures 7A–7C.
Our results suggest a specific role for the CYCD6-CDKB1
complex in which it phosphorylates RBR to influence SCR
transcriptional activity. Based on our genetic and biochemical
data, RBR phosphorylation is carried out by a complex of
CYCD6;1 with either CDKB1;1 or CDKB1;2. The cell-type-
specific transcriptional regulation of some of the Arabidopsis
CYCDs and studies reporting 26S-proteasome-dependent
CYCD3;1 and CYCD2;1 degradation (Planchais et al., 2004;
Sanz et al., 2011) suggest that tight spatiotemporal regulation
of CYCDs at both transcriptional and posttranslational levels
Figure 6. Protein Degradation Turns ‘‘Off’’ the Switch
(A–K) Expression patterns prior to and after MG132 treatment for pCYCB1;1:: CYCB1;1:GFP (A and B), pRBR::RBR:CFP/pRBR::RBR:YFP (C and D),
pSCR::SCR:YFP/p35S::H2B:RFP (E and F), pSHR::SHR:YFP/p35S::H2B:RFP (G and H), and J0571/pUAS::CYCD6;1:YFP (I and J). Quantification of total
fluorescence intensity before and after mitosis for pSHR::SHR:YFP/p35S::H2B:RFP, pSCR::SCR:YFP/p35S::H2B:RFP, and pRBR::RBR:CFP/p35S::H2B:RFP
plants (K); Student’s t test was used to assess the statistical significance of the distributions (*p < 0.05). Arrow bar represents SEM.
(L) Bifurcation diagram showing equilibrium SHR-SCR levels as a function of the level of enhanced protein degradation, d.
(M) Time dynamics for intermediate (40 min, thin lines) and long (2 hr, thick lines) time span of enhanced protein degradation. Red, d = 10; orange, d = 4. See also
Cell 150, 1002–1015, August 31, 2012 ª2012 Elsevier Inc. 1011
may be common among the highly diversified plant D cyclins.
The influence of CYCD6;1-CDKB1-mediated RBR phosphoryla-
tion on the SCR-SHR activity needs to be resolved at the mech-
anistic level but points to a role for RBR and cyclin-CDK
complexes in modulating the transcriptional capacity of factors
controlling cell transitions in a specific cell lineage. Our findings
are in line with recent in vivo studies in animals in which RBs are
shown to promote cellular differentiation by interaction with
tissue-specific transcription factors (Calo et al., 2010) but
provide, in addition, a mechanism by which this role is orches-
trated in space and time.
Our data demonstrate that the auxin maximum spatially
correlates with and positively influences CYCD6;1 transcription
in the CEI/CEID cells, in which high levels of CYCD6;1 (along
with CDKB1) phosphorylate RBR. An auxin-responsive element
(ARE) in the promoter region of the CYCD6;1 gene suggests
that auxin-responsive factors (ARFs) could read out auxin
concentrations on the CYCD6;1 promoter. This would serve
to potentiate SHR-SCR complex activity on the CYCD6;1
promoter because the auxin input is unable to act in the absence
of SHR. This scenario is consistent with the additive stimulation
of ACDs observed after auxin increase and simultaneous
uncoupling of SCR action from RBR inhibition by the SCRAxCxA
We show that the mechanism of correct ACD localization
depends on a radial SHR gradient and a longitudinal auxin
gradient (Figure 7A). The feedback of the network on SHR trans-
port guarantees that there is only a single cell file with ACD
potential, and auxin triggers the correct cells within that file. A
strong bistability due to a nested positive feedback loop,
combined with resetting of the high state through protein degra-
dation, underlies the ‘‘readout’’ of the axial information. Other
scenarios could program this readout as well but do not lead
to such a robust cell fate specification.
Figure 7. ‘‘Flip-Flop’’ Mechanism Exploiting Cell Cycle
(A–C) Schematic of root with SHR and auxin gradient. Red asterisk indicates CEI/CEID cell in ACD ‘‘on’’ state, receiving higher auxin levels (position A); blue
asterisk indicates endodermal/cortex cell in CEI ‘‘off’’ state, receiving lower auxin levels (position B).
(D) Due to natural auxin variations at position A and B, a sigmoidal response would lead to an imprecise outcome.
(E) (left) With a bistable switch, a small window of bistability would be required to change the ACD cell state (right). Such a small window of bistability, combined
with highly distinct cell differentiation states, lacks robustness.
(F) A robust wide window of bistability leads to sustained ACDs in the endodermal cells.
in sustenance of the ACDcell state (left). Afterward, arapid recovery of theACDcell state (flipon) can take placeinCEI/CEID cells due tohigh auxin levels, but not
in endodermal and cortex cells, which turn off their ACD cell state (right).
1012 Cell 150, 1002–1015, August 31, 2012 ª2012 Elsevier Inc.
A first alternative scenario could rely on a steep sigmoidal
response curve. However, this would not allow for the sudden
switch in the cellular response that is critical for daughter cells
to present very different behaviors after cell division. More-
over, a sigmoidal response curve would make root develop-
ment very sensitive for auxin levels within the CEI/CEID
region, which naturally present many fluctuations during devel-
opment (due to gravitropism, variable shoot-derived auxin
influx, etc.). As indicated in Figure 7D, in the sigmoidal
scenario, natural fluctuations in auxin levels in the CEI/CEID
and in endodermal cells could present them with comparable
A second alternative scenario could be that of a quantitatively
different bistable switch. In this scenario, bistability is possible
only within a small window of auxin levels, such that high auxin
levels trigger the ACD state, whereas slightly lower levels cause
the dynamics to fall back to the low SHR-SCR state, thereby
removing the requirement of increased protein turnover rates
(Figure 7E). This scenario, however, imposes strict requirements
on the network dynamics. First, both bifurcations have to be very
carefully tuned because the auxin concentration within the cell
undergoing the ACD should be high enough to promote the
‘‘on’’ state coming from the ‘‘off’’ state. Concomitantly, the
concentration within one of the daughter cells should be low
enough to promote the ‘‘off’’ state coming from the ‘‘on’’ state.
These requirements impose strong constraints on the kinetics
of the network. For a dynamical system to present two states
that differ substantially, it tends to be accompanied by a broad
window for bistability as well (Figure 7E, inset). A network pre-
senting both very different characteristic states and a narrow
window of bistability would require high cooperativity, which
makes it very sensitive to parameter changes.
In short, robustness can most readily be achieved through
a broad window for bistability (Figures 7F and 7G), which,
however, comes with a price—an extra requirement for the
resetting of the cells.
Cell-cycle factors also produce a bistable behavior, which is
fundamental to establish the reentry in cell cycle after serum
deprivation (Yao et al., 2008). In our case, the circuit not only
provides bistability but also the spatial and temporal constraints
that attenuate it to make it specific to the stem cell area. Given
the advantages of an attenuated bistable switch over other
mechanisms, it will be interesting to find out whether this regula-
tory cassette forms a recurring basic regulatory circuit among
others (Alon, 2007). We propose the term ‘‘flip-flop circuit’’ for
this and similar genetic networks.
There remain at least two issues to be addressed to further
match our model’s predictions and experimental observations.
First, our model predicts that high levels of SHR-SCR activity
should also form within the QC. As this is not the case, additional
network components within the QC may suppress the high
activity levels of SHR-SCR complex at that location. The QC
contains a modified transcriptional program (Sarkar et al.,
2007), and it remains to be elucidated how this program
represses the ACD-promoting activity of the SCR complex.
Second, targets of SHR and SCR such as NUC and MGP are
themselves required for SHR movement (Welch et al., 2007).
Thisindicates thatmoreintricate feedbackloopsmaybepresent
standing of its function.
Finally, it has recently been shown how plant transcription
factors can trigger cytoskeletal rearrangements for division
plane reorientation (Dhonukshe et al., 2012). It will be interesting
to explore whether the network described here connects to
similar mechanistic factors that trigger division plane rotation
during asymmetric cell division.
Arabidopsis thaliana plants were grown as described in Sabatini et al. (2003)
(see Extended Experimental Procedures). IAA/NPA treatments were done for
24 to 48 hr in liquid media similarly to Pe ´rez-Torres et al. (2008). For CEI
frequency measurements, plants were grown and roots were prepared for
confocal microscopy as described (Nieuwland et al., 2009). To test 26S-
proteasome-dependent protein degradation, 4 days postgermination (dpg)
seedlings were transferred to liquid MS media supplemented with 50 mM
MG132 (Sigma-Aldrich). Similar conditions were used to enable visualization
of CDKB1;1::GFP protein fusion. For confocal microscopy, roots were
mounted in 10 mM propidium iodide. Root tissue sections were performed
as in Willemsen et al. (1998) and were visualized by using Nomarski optics.
Casparian strip staining with berberine hemisulphate (Sigma) is described in
Lux et al. (2005).
Yeast Two-Hybrid Assay
Yeast two-hybrid (Y2H) interactions were studied by using the ProQuest
Two-Hybrid System (Invitrogen Life Technologies). Coding sequences of
RBR, SCRAxCxA, and E2FA were amplified and fused to both pDEST32 BD
and pDEST22 AD vectors. Compatible Y2H constructs for SCR and SHR
were previously generated, and Y2H analyses were performed as described
(Welch et al., 2007). To quantify interaction strengths, three experimental
and technical replicates of b-galactosidase assays, with CPRG as substrate,
Bimolecular Fluorescence Complementation Assays
For BiFC analysis, we subcloned RBR and E2Fa cDNAs in vectors pARC233,
pARC234, pARC235, and pARC236 by Gateway LR reactions to generate
C- and N-terminal fusions to the two YFP fragments. SHR and SCR were
generated as previously described (Welch et al., 2007). YFP fluorescence
was recorded by using a Leica SP2 CLSM. Col-0 mesophyll protoplasts
were transfected according to Yoo et al. (2007). Results are from three biolog-
ical replicates with three technical replicates each.
For split Renilla luciferase analysis, cDNAs were cloned by Gateway LR
recombination into pDuExB and pDuExP vectors (Fujikawa and Kato, 2007).
A minimum of two biological replicates of 50,000 mesophyll protoplasts
were transfected in triplicate with 2 mg of each DNA and 1 mg of transfection
control. Proteins were extracted and analyzed for firefly and renilla luciferase
96 microplate luminometer (Promega). Relative luciferase activity was calcu-
of average H2A-H2B interaction.
ChIP was carried out on root material of 5-day-old Col-0 seedlings. IP was
performed in the absence (negative control) and presence of antibody specific
for AtRBR1 protein as described in Horva ´th et al. (2006). NUC promoter qPCR
primers were designed to amplify fragments between 100 and 200 bp span-
ning the 6 kb promoter region of the gene and were designed to be in
ascending order upstream away from the NUC ATG (see Table S5). Enrich-
ment was calculated by comparison of samples after IP with and without
antibody. We used as negative control a random intergenic region (IR)
(between At3g03660–70) and as positive control, the promoter region of
PCNA1 (At1g07370) (Kosugi and Ohashi, 2002). Each primer pair was tested
Cell 150, 1002–1015, August 31, 2012 ª2012 Elsevier Inc. 1013
with a minimum of two biological replicates, with three technical replicates
each. Student’s t tests were performed to analyze statistical significance.
For protein kinase assays, CDK-cyclin complexes were immunoprecipitated
with anti-c-Myc antibody from lysate of transfected Arabidopsis protoplasts.
Immunocomplexes were washed as described in Extended Experimental
Procedures and were washed once with kinase buffer containing 25 mM
Tris-HCl (pH 7.8), 15 mM MgCl2, and 1 mM DTT. Phosphorylation assays
were performed in 20mlkinase buffer supplementedwith 0.25mg ml?1histone
H1 or 0.06 mg ml?1GST-RBR-C-terminal protein and 5 mCi of [g-32P]ATP
(PerkinElmer) for40minatroomtemperatureandthenterminatedby theaddi-
tion of Laemmli sample buffer. Reaction mixtures were separated by SDS-
PAGE and stained with CBB, and substrate phosphorylation was revealed
by autoradiography. For details in other methods, see Extended Experimental
Regulatory Network Modeling
Regulatory networks were formulated as coupled ordinary differential equa-
tions or were embedded within a spatial context of the root (see Grieneisen
et al., 2007), where simulations of auxin dynamics were performed by con-
currently solving for diffusion, permeability, and decay of auxin by using an
Alternating Direction Implicit (ADI) method (Peaceman and Rachford, 1955).
For details on formal descriptions of regulatory networks, see Extended
figures, five tables, and four movies and can be found with this article online at
We thank Dominique Bergmann for critically reviewing this manuscript; Fred
Sack, Lieven de Veylder, Naohiro Kato, Dorus Gadella, Joachim Goedhart,
Tom Beeckman, and Tom Bennett for materials; and Anahı ´ Pe ´rez-Torres,
Luis Herrera-Estrella, and Juan Carlos del Pozo for technical advice. B.S.
was supported by an ERC Advanced Investigator Fellowship and by
ALW-ERAPG grant 855.50.017. A.C.-R. was supported by EMBO-ALTF
1114-2006 and CONACYT 000000000092916 grants. S.D.-T. was funded by
Ministerio de Educacion y Ciencia, Spain, and by Marie Curie IEF (IEF-2008-
237643). V.A.G. was supported by the Dorothy Hodgkin Fellowship. V.A.G.
and A.F.M.M. were supported by the UK Biological and Biotechnology
Research Council (BBSRC) via grant BB/J004553/1 to the John Innes Centre.
I.B. was sponsored by an NWO VIDI grant. P.N.B. was funded by NIH grant
R01-GM043778. L.B. was supported by Swedish Research Council and by
Carl Tryggers Stiftelse grants. Work in James A.H. Murray laboratory was
supported by the BBSRC grant BB/G00482X and by the ERASysBio+ initiative
under the EU FP7 ERA-NET Plus scheme.
Received: July 5, 2011
Revised: May 24, 2012
Accepted: July 11, 2012
Published online: August 23, 2012
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