Auxin minimum defines a developmental window for lateral root

Transcription

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Auxin minimum defines a developmental window for
lateral root initiation
Joseph G. Dubrovsky1, Selene Napsucialy-Mendivil1, Jér^o: me Duclercq2,3, Yan Cheng4, Svetlana Shishkova1,
Maria G. Ivanchenko5, Jiřı́ Friml2,3, Angus S. Murphy4 and Eva Benková2,3
1
Instituto de Biotecnologı́a, Universidad Nacional Autónoma de México, Apartado Postal 510-3, 62250 Cuernavaca, Morelos, Mexico; 2Department of
Plant Systems Biology, VIB, 9052 Gent, Belgium; 3Department of Plant Biotechnology and Genetics, Gent University, 9052 Gent, Belgium; 4Department
of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA; 5Department of Botany and Plant Pathology, 2082
Cordley Hall, Oregon State University, Corvallis, OR 97331, USA
Summary
Authors for correspondence:
Eva Benková
Tel: +32 9 3313871
Email: [email protected]
J. G. Dubrovsky
Tel: +52 7773291664
Email: [email protected]
Received: 1 February 2011
Accepted: 3 April 2011
New Phytologist (2011) 191: 970–983
doi: 10.1111/j.1469-8137.2011.03757.x
Key words: Arabidospsis thaliana, auxin
gradients, cell competence, developmental
window, lateral root initiation, Solanum
lycopersicum.
• Root system architecture depends on lateral root (LR) initiation that takes place in a
relatively narrow developmental window (DW). Here, we analyzed the role of auxin
gradients established along the parent root in defining this DW for LR initiation.
• Correlations between auxin distribution and response, and spatiotemporal control of LR initiation were analyzed in Arabidopsis thaliana and tomato (Solanum
lycopersicum).
• In both Arabidopsis and tomato roots, a well defined zone, where auxin content
and response are minimal, demarcates the position of a DW for founder cell specification and LR initiation. We show that in the zone of auxin minimum pericycle
cells have highest probability to become founder cells and that auxin perception
via the TIR1 ⁄ AFB pathway, and polar auxin transport, are essential for the establishment of this zone.
• Altogether, this study reveals that the same morphogen-like molecule, auxin,
can act simultaneously as a morphogenetic trigger of LR founder cell identity and
as a gradient-dependent signal defining positioning of the founder cell specification. This auxin minimum zone might represent an important control mechanism
ensuring the LR initiation steadiness and the acropetal LR initiation pattern.
Introduction
The root system integrates many signals that provide the
plant with an advantage to select growth directions in a
changeable environment (Malamy, 2005; Laplaze et al.,
2007). Root architecture results from a reiterative process of
lateral root (LR) formation. As LR organogenesis takes
place in an extremely dynamic environment within the
simultaneously growing parent roots, an important question
is how the acropetal pattern of root initiation is established
and maintained and what are the mechanisms controlling
recurrent initiation of new LR primordia (LRP).
Auxin is a key regulator of primary (Sabatini et al., 1999;
Friml et al., 2002), lateral (Celenza et al., 1995; Laskowski
et al., 1995, 2008; Casimiro et al., 2001; Benková et al.,
2003; Dubrovsky et al., 2008), and adventitious (Boerjan
et al., 1995; Ullah et al., 2003; Sorin et al., 2005) root
development. In Arabidopsis thaliana and most other
970 New Phytologist (2011) 191: 970–983
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eudicots, LR formation starts from pericycle cells adjacent
to a xylem pole (Laskowski et al., 1995; Dubrovsky et al.,
2000; Beeckman et al., 2001). Consistent with the important role of auxin in root development, several mutants in
various components of the auxin signaling machinery either
lack or have a reduced LR initiation. In the presence of
auxin, the transcriptional repressors AUX ⁄ IAA interact
directly with the auxin receptor, TRANSPORT INHIBITOR
RESPONSE1 (TIR1), and are targeted for degradation,
thus permitting AUXIN-RESPONSE FACTOR (ARF)
proteins to regulate transcription of auxin-responsive genes.
Gain-of-function mutations in IAA14 ⁄ SLR (Fukaki et al.,
2002, 2005; Vanneste et al., 2005) and IAA28 (Rogg et al.,
2001; Dubrovsky et al., 2009) are deficient or strongly
affected in LR initiation, respectively. The double arf7
arf19 mutant does not form any LRs (Okushima et al.,
2005; Wilmoth et al., 2005) and is incapable of LR initiation (Okushima et al., 2007; Dubrovsky et al., 2009).
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Besides transduction, the polar auxin transport represents
an important degree of control of the auxin activity. Auxin
influx (Bennett et al., 1996; Yang et al., 2006) and efflux
(Luschnig et al., 1998; Noh et al., 2001; Petrášek et al.,
2006) carriers are main components of the polar auxin
transport machinery regulating auxin distribution in tissues
and organs, whereas chemical or genetic interference with
the polar auxin transport affects LR initiation (Casimiro
et al., 2001; Benková et al., 2003). Although the role of
auxin in founder cell specification and LRP initiation is
clearly established, the mechanisms regulating the spatiotemporal periodicity of auxin-dependent LRP initiation
events are still not elucidated.
Lateral root formation is a complex process that comprises several growth control points. Protoxylem-adjacent
pericycle cells in the young differentiation zone of
Arabidopsis are capable of proliferating without intervening
mitotic quiescence after they leave the root apical meristem
(Dubrovsky et al., 2000). This and previous studies
(Gladish & Rost, 1993) have suggested that the predetermination of pericycle cells and their subsequent participation
in LRP formation are part of a continuous process that
starts in the root apical meristem, or soon after cells exit the
meristem. Indeed, recent research has suggested that the
first growth control point in LR development consists of
‘priming’, that is, determination of pericycle cells for future
LRP formation. It takes place close to the root apical
meristem, namely in the elongation zone: regular fluctuations in auxin responsiveness in this root zone were found
to correlate with subsequent positioning of LRs (De Smet
et al., 2007; De Rybel et al., 2010; Moreno-Risueno et al.,
2010). Apparently, developmental changes may occur in
pericycle cells at this time; nevertheless, it is still unknown
how activation of auxin response in the protoxylem cell
layer (De Smet et al., 2007) causes the priming in the pericycle cell layer. Interestingly, the auxin responsiveness
simultaneously oscillates with activity of two different sets
of genes, one in phase and the other in antiphase (MorenoRisueno et al., 2010).
A second growth control point in the process of root
formation consists of specification of LR founder cells and
their subsequent divisions, forming of an early-stage
primordium. These events occur in the root differentiation
zone soon after completion of the cell elongation. Timelapse analysis in Arabidopsis has shown that LR initiation
only happens within a narrow time window (Dubrovsky
et al., 2006a). This developmental window (DW) was
defined as a period during which pericycle cells in the young
differentiation zone remain in a state that allows LR founder cell specification (Dubrovsky et al., 2006a). This
specification has been associated with increase in auxin
response in small groups of protoxylem-adjacent pericycle
cells (Benková et al., 2003). Live imaging has confirmed
that these auxin-responsive pericycle cells are LR founder
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cells, because soon after they express the auxin-response reporter DR5rev:GFP, these cells divide and form a primordium
(Dubrovsky et al., 2008). Recently, the auxin-responsive
GATA23 transcription factor has been demonstrated to
become active in founder cells c. 10 h after priming of protoxylem cells in the elongation zone; expression of GATA23
depends on the IAA28 function (De Rybel et al., 2010).
When a stage I LRP is formed, the third growth control point
starts to operate, which regulates LRP morphogenesis and
patterning. These processes depend on the AP2 ⁄ EREBP gene
PUCHI (Hirota et al., 2007) and on BDL ⁄ IAA12 and
MP ⁄ ARF5 genes (De Smet et al., 2010).
Previously, we have shown that in the Arabidopsis primary
root, LRP initiation occurs in a regular acropetal pattern at
the beginning of the differentiation zone and that each
new initiation event always takes place distally to a previous
one in the direction of the root apex (Dubrovsky et al.,
2006a). Here, we wondered how the DW is established and
maintained. To achieve a typical acropetal root branching
pattern, founder cell specification must be controlled in
both time and space. Pericycle cell proliferation is rarely
found between developed LRs (Dubrovsky et al., 2000),
indicating that pericycle cells located outside the DW
seldom participate in the initiation of new LRP. External
auxin treatment can induce LRs along the entire parent root,
overcoming the acropetal pattern observed during root
growth under standard growth conditions (Goldacre, 1959;
Blakely et al., 1982; Laskowski et al., 1995). Therefore, we
hypothesized that the maintenance of this acropetal pattern
must require a still unknown spatiotemporal auxin distribution along the root, spatiotemporal restrictions of the auxin
activity, or differential auxin sensitivity along the parent
root. To test this hypothesis, we analyzed the endogenous
auxin response and auxin content gradients along the parent
root, and tested how these gradients correlate with the site
of LRP formation. We found that an auxin minimum zone
in the primary root corresponded to a zone in which pericycle cells could acquire with the highest probability a
founder cell identity. When the gradients in the auxin distribution or perception along the root were altered genetically
or pharmacologically, regular acropetal pattern of LRP
formation could no longer be maintained.
Materials and Methods
Transgenic lines, growth conditions and treatments
Transgenic Arabidopsis thaliana (L.) Heynh. lines in
Columbia-0 (Col-0) background were CYCB1;1DB::GUS
(Colón-Carmona et al., 1999), DR5::GUS (Ulmasov et al.,
1997), DR5rev::GFP (Friml et al., 2003), DR5rev::GFP in
tir1 afb2 afb3 triple mutant background (Dharmasiri et al.,
2005b), and pIAA14::mIAA14-GR (Fukaki et al., 2005).
Tomato (Solanum lycopersicum L.) plants cv Ailsa Craig
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were used. To generate a tomato pIAA2::GUS line, seedlings were transformed with the p1G4 construct carrying
the Arabidopsis pIAA2::GUS reporter (a kind gift from J.
Normanly, University of Massachusetts, MA, USA). The
DR5::GUS tomato line had been reported previously
(Dubrovsky et al., 2008).
Unless otherwise indicated, plants were grown in vertically oriented Petri dishes on 0.2 · MS (Murashige &
Skoog, 1962) medium, pH 5.7, supplemented with 1%
(w ⁄ v) sucrose (Dubrovsky et al., 2006a). To analyze LR initiation following auxin transport inhibition, Arabidopsis
DR5::GUS seedlings were grown on 10 lM 1-naphthylphthalamic acid (NPA) for 10 d, the 1.5 mm apical and basal
root segments were removed, and the remaining root
divided into three equal portions, which were transferred
onto MS medium with or without auxins for three additional days. Tomato DR5::GUS seedlings were grown in
liquid MS medium with 10 lM NPA for 6–7 d, shoots
and root meristems were removed, and roots transferred on
to solid control medium for 3 d. Roots were cleared as
described (Dubrovsky et al., 2006a, 2009).
Dexamethasone (DEX)-inducible transient inhibition of
LR initiation was tested in segmented-agar dishes with
50 lM DEX added only to the lower half of the vertically
oriented dish (Supporting Information, Fig. S3a). Six-dayold pIAA14::mIAA14-GR seedlings were transferred on the
segmented medium so that 1 mm of the root tip was on the
(+) DEX sector. Root portions grown on a DEX sector for
3 d were analyzed either immediately or after transfer on to
MS medium with or without naphthalene-acetic acid
(NAA) for two additional days.
To estimate the DW duration for LR initiation, 24 h
root growth increments between days 7 and 8 were
measured and the average rate of root growth calculated as
V (mm h)1). Roots were cleared and the distance (d) was
measured from the quiescent center (QC) to the most
distal LRP. The DW duration (h) was estimated as
T = (dmax ) dmin)V)1, where dmax and dmin (mm) are the
maximal and minimal distances, respectively.
Quantification of DR5rev::GFP expression along the
Arabidopsis root and microscopy
DR5rev::GFP expression was analyzed by confocal laser
scanning microscopy (CLSM). Live roots were analyzed
with or without staining with 4 lM neutral red at pH 5.7
(Dubrovsky et al., 2006b); some roots were fixed for
6–12 h at 4C in 3.7% para-formaldehyde in phosphatebuffered saline (pH 7.4), supplemented with 1 lg ml)1
propidium iodide. Confocal images spanning the live root
from the tip to the emerged LRs were acquired with a 10·
objective under the same settings. Confocal sections of
60 lm were taken, corresponding to the diameter of the
root central cylinder. Images were assembled in Adobe
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Photoshop (Adobe Systems, San Jose, CA, USA). Green
fluorescent protein (GFP) intensity was measured with
ImageJ (http://rsb.info.nih.gov/) as pixel density within the
tissues of the central cylinder (except pericycle) starting from
the QC and the background level was subtracted. Pixel
densities within the QC cells were considered at 0 lm. For
the most distal 400 lm of the root, intensities were measured in portions of 0–100, 100–200, and 200–400 lm and
averaged over three measurements per portion. Intensities of
three consecutive 167 lm portions were measured for each
500 lm interval. Values were expressed as percentage of
mean pixel density relative to the GFP maximum.
Microscopy and image acquisition were done as
described (Dubrovsky et al., 2006b). Distances from the
QC to the most distal primordium were measured on
cleared roots prepared as previously described (Dubrovsky
et al., 2009) with an ocular micrometer. Founder cells and
most distal primordia were detected by accumulation of the
DR5rev::GFP activity (Dubrovsky et al., 2008) under a
CLSM with a 40· or a 63· objective. Distances from the
QC to the founder cells and the most distal primordia
detected were measured under CLSM with a 10· objective
and LSM5 Image Examiner program (Zeiss) to the precision of 1 lm.
Analysis of indole-3-acetic acid content and transport
Endogenous free IAA was measured after methylation and
GC-MS analysis as previously described (Kim et al., 2007)
in three independent experiments of 10 seedlings each. The
auxin transport assay was done as previously described
(Geisler et al., 2005; Peer & Murphy, 2007), except that the
Col-0 seedlings were initially grown for 5 d under
120 lmol m)2 s)1 light in Petri dishes with 0.25 · MS,
supplemented with 0.5% sucrose at pH 5.5. Before the assay,
seedlings were transferred to 2-mm-wide, vertically discontinuous filter paper strips saturated with liquid 0.25 · MS
medium and were allowed to equilibrate for 2 h under yellow light. With a micromanipulator, a 10 nl microdroplet
containing 1 lM unlabeled IAA and 1 lM [3H]IAA (specific activity 20 Ci mmol)1, American Radiochemical, St
Louis, MO, USA) in dimethylsulfoxide was applied on the
shoot apex. Seedlings were incubated in yellow light for 4,
4.5, 5, 6 and 7 h, after which the hypocotyls and cotyledons
were removed, and 2 mm root sections sequentially harvested and assayed by scintillation counting (Tri-Carb 3180;
Perkin Elmer, Waltham, MA, USA). A 5 h time point was
chosen for the measurements of transported auxin because,
under the conditions used, auxin could be detected first
at the root apex (> 2 · background) at 4.5 h. Samples
were collected separately, extracted with 100% metanol,
and analyzed according to Östin et al. (1998) for IAA,
2-oxoindole-3-acetic acid (IAAox), and 2-oxoindole-3acetyl-b-D-O-glucopyranose (IAAox-Gluc).
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pIAA2::GUS expression in tomato root
Roots of 6-d-old pIAA2::GUS seedlings were fixed in
50 mM phosphate buffer, pH 7.2, containing 0.3% formaldehyde and 0.3 M mannitol for 10 min, washed three
times with 50 mM phosphate buffer, and incubated overnight in 100 mM phosphate buffer (pH 7.2), 0.5 mM
K3[Fe(CN6)], 0.5 mM K4[Fe(CN6)], 10 mM EDTA,
20% methanol, 0.01% Triton X100, and 2 mg ml)1 Xgluc (Gold BioTechnologies, St Louis, MO, USA). After
clearing, roots were mounted in 25% glycerol and analyzed.
For fluorometric GUS expression assay, 1 mm fragments
of 10 roots were cut at positions as indicated in Fig. 1(e)
with a dissecting microscope, collected on ice, and ground
in 100 ll extraction buffer containing 40 mM phosphate
buffer, pH 7.2, 1 mM EDTA, 0.01% Triton X100, and
0.01% sodium lauroyl sarcosine. Samples were centrifuged
at 10 000 g for 10 min, and 10 ll from each sample was
mixed in duplicates with 40 ll reaction buffer containing
1 mM dithiothreitol and 3 mg ml)1 4-methylumbelliferyl-
b-D-glucuronide (Marker Gene Technologies, Eugene, OR,
USA). Reactions were incubated at 37C for 20 min,
cooled to room temperature, and stopped by 160 ll 0.2 M
Na2CO3. Fluorescence was measured with a Synergy 2
Multi-Detection Microplate Reader (Winooski, VT, USA)
and excitation and emission at 360 and 465 nm, respectively. Protein concentration was measured with a Micro
BCA Protein Assay Kit (Thermo Scientific, Rockford, IL,
USA) according to the manufacturer’s instructions. GUS
expression was calculated as relative fluorescence units mg–1
protein. Values were averaged from two independent
experiments.
Results
LR founder cell specification is restricted to a narrow
developmental zone in the parent root
Previously, we have shown that, in Col-0 plants, LR initiation
occurs within a narrow root portion behind the root tip and
(a)
(b)
(d)
(c)
(e)
Fig. 1 Gradients in auxin response along Arabidopsis and tomato roots. (a) Gradient of DR5rev::GFP activity in 7-d-old Arabidopsis root;
numbers indicate distance from the quiescent center (QC) (mm). (b, c) DR5 activity measured in the central cylinder as relative mean pixel
density of the green fluorescent protein (GFP) signal and expressed as a percentage of maximum activity (measured in the QC (for b)) and in
the proximal part of the root (for c) of each individual root in 5-d-old (n = 7) (b) and 14-d-old (c) Arabidopsis plants (n = 5). Insets show the
mean pixel density within the first 400 lm from the QC. (d, e) Gradient in the pIAA2::GUS auxin reporter activity along the primary root of
tomato. (d) pIAA2::GUS reporter expression in a 6-d-old seedling; numbers indicate distance from the QC (mm). (e) Quantification of GUS
expression with a fluorometric assay in 1 mm root segments collected at the indicated distances. Values are averages from two independent
experiments with 10 roots each. Mean ± SEs are shown (b, c, e).
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have calculated that each subsequent LRP is initiated within a
time-frame interval of c. 10 h, referred to as the ‘developmental window for LR initiation’ (Dubrovsky et al., 2006a).
Spatially, this time-frame should correspond to a specific zone
a few mm above the QC. In the Col-0 wild-type, the distance
from the QC to the most distal LRP ranged from 2.4 to
6.8 mm, defining a zone with the highest capability for LRP
initiation, corresponding to the DW (Fig. 2). In terms of time
this DW corresponds to 8.5 h. Because the earliest known sign
of LR founder cell identity is the increased DR5 activity in
pericycle cells (Benková et al., 2003; Dubrovsky et al., 2008),
we used DR5rev::GFP to detect founder cells by CLSM and
established their position relative to the most distal primordium in the same roots (Table 1). The distance between the
site of founder cell specification and that of primordium initiation was short, on average 0.13–1.1 mm, indicating that once
the founder cell specification occurs, primordium initiation
follows relatively soon after. These data show that founder
cell specification and primordium initiation in Arabidopsis
(Col-0) occur, on average, at 4.21 ± 1.01 mm (mean ± SD,
n = 77, 8-d-old plants) from the QC, i.e. in the young differentiation zone. Thus, both of these processes are separated in
time and space from the pericycle cell priming that takes place
within the elongation zone (De Smet et al., 2007; MorenoRisueno et al., 2010), that is, at c. 0.2–1.2 mm from the QC
in Arabidopsis (Tapia-López et al., 2008; J. G. Dubrovsky,
pers. obs.). Thus, founder cell specification and LRP initiation
Fig. 2 Developmental window operating during lateral root
initiation shown as the distribution of distances from the quiescent
center (QC) to a most distal primordium in 8-d-old Arabidopsis
thaliana Col-0 plants (n = 77).
represent a growth control point in the LR formation that
operates in the differentiation zone.
Auxin reporters reveal an auxin response minimum
along the primary root
Because exogenous auxin can activate pericycle cells and
promote new LR formation along the entire parent root,
whereas in the intact root initiation can only take place in a
narrow zone, we wondered whether endogenous auxin distribution and response can be related to the establishment
of the DW for founder cell specification. In the Arabidopsis
root, two auxin maxima have been reported, one in the QC
(Sabatini et al., 1999; Friml et al., 2002; Ljung et al., 2005;
Petersson et al., 2009) and another at the root base
(Bhalerao et al., 2002; Marchant et al., 2002; Ljung et al.,
2005), forming distal and proximal auxin gradients, respectively. As not much is known about the auxin response
distribution in the root zones where LRP initiate and
develop, the expression of the auxin-sensitive DR5rev::GFP
reporter (Friml et al., 2003) was analyzed along the entire
root of Arabidopsis with CLSM (Fig. 1a). We observed the
DR5 maximum in the QC cells as previously reported
(Sabatini et al., 1999). Within the root meristem, the DR5
activity was maintained in a one-cell-thick layer of immature xylem-precursor cells, occurred at a low level in
elongating and differentiating protoxylem cells, and was not
detected in pericycle cells (Fig. S1). Starting at c. 1.2 mm
from the root tip, no DR5 activity was detected in the
young differentiation zone (Fig. S1). Quantification of
DR5rev::GFP expression confirmed that its level became
very low at 0.2 mm from the QC (insets in Fig. 1b,c) and
that, at 5–6 mm from the root tip, the DR5 activity started
to steadily increase in a proximal direction within the tissues
of the root central cylinder, excluding pericycle cell layer
(Fig. 1a–c). This gradient seems to be independent of
seedling age, because the DR5 activity had a similar pattern
in roots of 5-d-old (Fig. 1b), 7-d-old (data not shown), and
14-d-old (Fig. 1c) seedlings. A similar pattern of auxin
response was also observed based on fluorimetric GUS measurements in roots of tomato expressing the pIAA2::GUS
auxin reporter (Fig. 1d,e). Thus, there is a well defined
Table 1 Location of founder cells and distal lateral root primordium detected along the root of Arabidopsis DR5rev::GFP line
Distance to founder cells (mm)
Distance to distal lateral root primordium (mm)
Plant age
Minimal
Maximal
Mean
Minimal
Maximal
Mean
7d
14 d
2.724
2.920
4.861
5.112
3.348 ± 0.198 (12)
4.170 ± 0.198 (14)
3.336
3.010
5.402
5.270
4.404 ± 0.260 (9)
4.299 ± 0.204 (14)
Data are means ± SE, n is indicated in the parentheses. The distance was measured from the quiescent center (QC). In 14-d-old plants, founder cells were slightly more distant from the QC than in 7-d-old plants (P = 0.007, Student’s t-test); the distance to the distal lateral root
primordium was the same in 7 and 14-d-old plants (P = 0.753, Student’s t-test).
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auxin response minimum zone that corresponds spatially to
the DW for LR initiation that is found at the start of the
differentiation zone in both Arabidopsis and tomato.
Monitoring auxin content and distribution along the
root
Free IAA concentrations were assayed in primary roots of 5d-old Col-0 seedlings. Steady-state free IAA concentrations
decreased progressively in the primary root from the shootto-root transition zone downward to the root apex and then
increased again at the root apex (Fig. 3). The lowest free
IAA content was detected in root segments taken 2–4 mm
from the apex, which was 40% less than that in the 12–
14 mm root segments (P = 0.002, Student’s t-test), and
was accompanied with a concurrent increase in the oxidative breakdown products IAAox and IAAox-Gluc (data not
shown). This pattern of free auxin distribution closely
corresponded to the pattern we detected in DR5 and IAA2
promoter activities along the root of Arabidopsis and
tomato, respectively (Fig. 1), and is in agreement with
previous reports on overall auxin distribution in the root
(Ljung et al., 2005; Petersson et al., 2009).
To correlate the steady-state free auxin concentrations
with the auxin transport activity, we performed nanoscale
polar auxin transport assays from the shoot to root apex. In
these assays, 10 nl [3H]IAA (1 lM, 20 Ci mmol)1) was
applied to the apex of 10 seedlings that were incubated
Fig. 3 Transport of shoot-derived auxin along the root and free
auxin content. The line plot represents radioactively detected signal
in pooled 2 mm segments from 10 seedlings (n = 6) 5 h after
10 fmol 3[H]IAA was deposited at the shoot apex of 5.5-d-old
seedlings. The asterisks indicate differences in each subsequent root
segment when compared with the previous segment at P < 0.005
(Student’s t-test). The histogram represents free IAA content of
seedlings sampled in the same manner after treatment with a
solvent control (n = 3). The asterisk on the histogram indicates a
difference between the root segments of 0–2 and 2–4 mm
(P = 0.001, Student’s t-test). Mean ± SEs are shown.
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under dim yellow light. Starting at 2 h, 2 mm sections were
excised serially from the root apex upward to the root-toshoot transition zone. The [3H]IAA could first be detected
(by scintillation counting of the pooled sections) at the primary root apex (> 2 · background concentrations) at 4.5 h
and signals increased progressively in the pooled sections
closer to the root-to-shoot transition zone. However, at 5 h
and later, [3H]IAA signals at the root apex increased steadily
and decreased progressively in the rest of the root (data not
shown) in a manner consistent with the observed steady-state
free IAA concentrations (Fig. 3). Altogether, the auxinreporter analysis and the direct measurements of auxin
content and transport reveal that auxin concentrations and
responses along the root obey a certain regularity that is
probably maintained through polar auxin transport. An
auxin minimum is defined between a distal auxin gradient at
the root tip, and a proximal gradient along the central cylinder in Arabidopsis and tomato roots.
The zone of auxin minimum in the root overlaps with
the developmental window for founder cell specification and primordium initiation
As already described, the distance from the most distal primordium to the QC was between 2.4 and 6.8 mm in Arabidopsis
(Fig. 2) in a zone overlapping that of minimal auxin response
and content (between 1.5 and 5–6 mm from the QC), from
which the proximal auxin gradient starts (Table 1; Figs 1, 3).
Similarly, in tomato roots, the distance from the QC to the
most distal LRP ranged from 4.9 to 10.7 mm (mean ± SE,
7.9 ± 0.30 mm, n = 23), corresponding to a zone with low
auxin response (Fig. 1d,e). When we plotted the distance from
the QC to the most distal LRP against the distance to the start
of the proximal auxin gradient for each root, a clear correlation
was found for both Arabidopsis DR5rev::GFP plants
(R = 0.80; Fig. 4a) and tomato pIAA2::GUS plants (R = 0.83;
Fig. 4b). Thus, founder cell specification and initiation of an
early-stage primordium correlates statistically with the start of
the proximal auxin gradient.
Lack of PIN3 and PIN7 auxin efflux carriers has been
demonstrated to interfere with LR initiation (Benková
et al., 2003). As both PIN3 and PIN7 participate in the regulation of the polar auxin transport (Friml et al., 2002,
2003), we examined whether the zone of auxin minimum
in pin3 and pin7 mutants was altered compared with that
of the wild-type and, if so, how this correlated with the
positioning of the LR initiation site. In both pin3 and pin7,
the DR5rev::GFP signal behind the root elongation zone
started at a shorter distance from the QC, namely 2–3 and 3–
4 mm from the QC, respectively, while it was between 4 and
7 mm from QC in the wild-type (Fig. 4a). In pin3 and pin7,
there was low (R = 0.44) or no correlation (R = 0.04),
respectively, between relative positions of the most distal
LR initiation events and the start of the proximal auxin-
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(a)
Fig. 4 Correlation between the lateral root (LR) initiation site and
the start of the proximal auxin gradient in Arabidopsis and tomato
root. (a) Correlation between the start of the proximal auxin
gradient (expressed as 5% of maximum auxin response in the
quiescent center (QC)) and the position of the most distal LR
primordia (LRP) in 7-d-old DR5rev::GFP Arabidopsis plants and pin3
and pin7 mutants crossed with DR5rev::GFP. (b) Correlation
between the start of the detectable pIAA2::GUS expression and the
position of the most distal LRP in 6-d-old tomato plants. Regression
line and correlation coefficient are shown (n = 7–8 (a) and n = 23
(b)). For Arabidopsis and tomato wild-type, P < 0.05 (Pearson’s
correlation test); for pin3 and pin7, P = 0.273 and 0.908,
respectively (Pearson’s correlation test).
response gradient (Fig. 4a). In both of these cases, the correlation was not statistically significant (P > 0.05, Pearson
correlation test) in contrast to the wild-type Arabidopsis and
tomato seedlings (P < 0.05, Pearson correlation test), indicating that in the wild-type, maintenance of the auxin
minimum zone in the root central cylinder depends on the
polar auxin transport and that the auxin minimum zone
defines the DW for the LR initiation. When the zone
becomes altered, the correlation between the sites of
primordium initiation and location of the auxin minimum
zone is lost.
Pericycle cells have the highest probability to become
founder cells in the auxin minimum zone
If the zone of the auxin minimum defined the DW, we
hypothesized that pericycle cells transiently present in this
(b)
(c)
Fig. 5 Irreversible nature of the transient inhibition of the lateral root
(LR) initiation in Arabidopsis. (a) Col-0 seeds germinated and
seedlings grown on medium supplemented with 10 lM
1-naphthylphthalamic acid (NPA) for 6 d; seedlings were transferred
to NPA-free Murashige–Skoog (MS) medium for an additional 3 d;
LRs and primordia were formed exclusively in the root portion
formed after transfer. The numbers of both primordia and lateral
roots present within the root portions formed before and after
treatment are shown in one of three independent experiments
(n = 22). (b) Six-day-old pIAA14::mIAA14-GR were transferred on
to plates either without ()) or with 50 lM dexamethasone (DEX)
(+) sectors for 3 d. Seedlings had decreased density of pericycle
activation events (LRs and primordia) in the root portion formed on
DEX-supplemented (+) sectors. (c) After DEX treatment, seedlings
were transferred on to DEX-free medium without ()) or with 1 lM
(+) naphthalene-acetic acid (NAA) for an additional 2 d (see
Supporting Information, Fig. S3, for experimental setup). On NAAfree medium, the density of pericycle activation events did not
increase in the previously DEX-treated root portion, but it increased
upon transfer to NAA-containing medium. Mean ± SE (n = 19–23)
of two independent experiments. (+) or ()) DEX treatments differed
at P < 0.001 (Student’s t-test).
zone would be able to acquire founder cell identity and initiate a LR, and that once they had moved out of the zone as a
result of the primary root growth, this ability would disappear under normal growth conditions. To test these
hypotheses, we transiently inhibited the LRP initiation and
examined the pattern of its restoration after removal of the
restrictive conditions. We first used NPA to block the LRP
initiation (Casimiro et al., 2001; Himanen et al., 2002,
2004) during the first 6 d of root growth. When the seedlings were transferred onto NPA-free medium, initiation was
restored exclusively in the root portion formed after the
inhibitor removal (Fig. 5a), suggesting that a new DW with
pericycle cells competent for LR initiation had been established at the root tip. The IAA polar transport was restored
within 24 h after NPA removal (Fig. S2), thus confirming
the transient character of the NPA inhibition of the polar
auxin transport. After transfer to NPA-free medium, no LRP
were detected in the zone grown in the presence of NPA,
even after prolonged incubation without the inhibitor, demonstrating the irreversible loss of the capability to initiate a
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primordium in the root portion where the polar auxin transport had been inhibited. Next, founder cell activation was
inhibited in a genetically controlled manner with the
pIAA14::mIAA14-GR Arabidopsis line (Fukaki et al., 2005) in
which a stabilized form of the IAA14 ⁄ SLR repressor protein
accumulates in the nucleus upon DEX treatment (Fukaki
et al., 2005). LR formation in the pIAA14::mIAA14-GR seedlings was inhibited transiently upon the DEX treatment (De
Smet et al., 2007), but it is unclear whether LRP initiation
was abolished. In roots grown on agar plates with DEXsupplemented sectors (Fig. S3a), LRP initiation was not
completely eliminated in the presence of DEX (Fig. S3b,c),
but the density of the pericycle activation events (including
LR and LRP) was reduced 1.8-fold in the root portion growing on the sector with 50 lM DEX (Fig. 5b). Importantly,
when seedlings were transferred to DEX-free medium for an
extra 2 d, the density of pericycle activation events did not
increase (P > 0.05, Student’s t-test) in the DEX-treated root
portion, confirming that the loss of ability to initiate LRP is
irreversible once pericycle cells exit the auxin minimum zone.
However, the density of pericycle activation events in the
DEX-treated segment was increased 12.9-fold upon seedling
transfer to NAA-containing medium (Fig. 5c), demonstrating
that the competence for founder cell specification was not lost.
Thus, these data provide consistent experimental evidence
that LR initiation is restricted to a DW. Although cells outside
the DW retain their competence to acquire founder cell identity, they require additional auxin stimulation.
Indeed, a temporal 9 h application of Sephadex beads
loaded with 10 lM NAA at different portions of the root
further confirmed that the highest number of initiation
events occurred in the most distal (1–10 mm) root zone,
comprising the zone of auxin minimum (Fig. S4), and
dramatically decreased with distance from the root tip.
Dynamics of pericycle activation of NPA-pretreated roots
induced by NAA revealed that pericycle cells were activated
first, within 2 h of the treatment, in the root portion corresponding to the DW (within the apical 5 mm of the root).
However, a 6 h auxin treatment was necessary before LR
initiation events appeared in the more proximal parts of the
primary root (Fig. S5). These experiments demonstrate that
pericycle cells in the auxin minimum zone and within the
DW portion have the highest auxin responsiveness compared with other parts of the root. Owing to the Casparian
strip in the endodermis cells, we could not exclude that the
pericycle was less accessible to NAA in mature root portions
than in the root tip. To facilitate auxin access to the pericycle, we also analyzed auxin responsiveness in three physically
separated consecutive root segments lacking LRPs after NPA
treatment. In both untreated and auxin-treated root segments, the density of pericycle activation events was the
greatest in the apical root segment (with the meristem and
elongation zone excised), comprising the auxin minimum
zone, whereas in the middle and basal segments, initiation
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Fig. 6 Pericycle cell competence to form lateral roots (LRs).
DR5::GUS Arabidopsis seedlings were grown on 10 lM 1naphthylphthalamic acid (NPA) for 10 d, and then 1.5 mm segments
at the root base and at the root apex were excised and discarded.
Then the root was divided into three approximately equal root
segments: basal, median, and apical. Every cut is marked with an X
on the scheme. The root segments were transferred for 3 d on to
control (n = 29–33; white bars), 0.1 lM 2,4-Dichlorophenoxyacetic
acid (n = 22–33; gray bars), or 1 lM naphthalene-acetic acid (NAA)
(n = 20–28; black bars) media; the density of pericycle activation
events (LRs and primordia) per mm of the root is shown. The
combined data of three independent experiments are shown.
Means ± SE are shown. Different letters indicate the difference
between apical, median, and basal segments within the same
treatment at P < 0.05 (Kruskal–Wallis one-way ANOVA on ranks).
The average length of the root segments was 4.8 ± 1.2 mm
(mean ± SD; n = 146).
progressively decreased (Fig. 6). A similar gradual decrease
in induced initiation events outside the DW was found in
tomato DR5::GUS seedling roots (Fig. S6). This analysis
reveals that although all pericycle cells along the root of
young plant seedlings can respond to auxin with pericycle
activation, the probability of pericycle cells to acquire a founder cell identity is the greatest in the root portion
corresponding to the zone of auxin minimum. Hence, in the
auxin minimum zone, the developmental state of the pericycle cells makes them more sensitive to external auxin and
the enhanced pericycle activation in this zone depends on
the original auxin status of this tissue. Overall, our data show
that under normal growth conditions the ability to acquire a
founder cell identity is transient in the auxin minimum zone
and lost when the cells exit this zone. When auxin is
provided externally, the pericycle is activated both inside and
outside the DW, but the cells in the zone of auxin minimum
are still more prone to LR formation.
Auxin perception is required for proper spatial pattern
of primordium initiation
TRANSPORT INHIBITOR RESPONSE1 (TIR1), and
the homologous auxin receptors AUXIN-SIGNALING-
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Table 2 Primary root length and lateral root (LR) development in 8d-old Arabidopsis wild-type (DR5rev::GFP) and triple tir1 afb2 afb3
mutant (DR5rev::GFP)
DR5rev::GFP
tir1 afb2 afb3
DR5rev::GFP
Student’s t-test
Distance from
the root tip to
the distal LRP
(mm) (10)
Number of plants
with emerged
LRs (15)
Root length
(mm) (10)
4.87 ± 0.77
7.36 ± 2.96
15 ⁄ 15
3 ⁄ 15
43.0 ± 4.8
22.1 ± 5.2
P < 0.05
P < 0.001
LRP, lateral root primordia.
Mean ± SD, n is indicated in parentheses in the column headers.
F-BOX1 (AFB1), AFB2 and AFB3 regulate developmental
responses to auxin (Dharmasiri et al., 2005a,b; Kepinski &
Leyser, 2005). To examine their impact on LR initiation,
we studied the tir1 afb2 afb3 mutant in the DR5rev::GFP
background and found, in agreement with published results
(Parry et al., 2009), that in 70% of the seedlings the primary root was shorter than 3 mm (n = 96). Therefore, we
analyzed only those mutant seedlings that had an elongated
primary root, on average 22.1 mm (Table 2). As DR5 activity is not detected in this triple mutant (Dharmasiri et al.,
2005b), we could not use this reporter to detect founder
cells and to monitor their position relative to the auxin gradients along the primary root. However, we observed that
the mutant seedlings exhibited an irregular primordium initiation that occurred at an increased distance from the QC
and varied among the seedlings (Table 2). In other words,
the DW for founder cell specification was apparently less
precisely defined in the mutant than in the wild-type. We
also found various defects in pericycle cell development and
primordium morphology, including pericycle cell division
without LRP formation (Fig. 7d), two-cell layered pericycle
(Fig. 7e), and abnormal LRP formed from two-cell layered
pericycle (Fig. 7f). Exogenous auxin induced cell proliferation in the xylem-adjacent pericycle cells of the tir1 afb2
afb3 mutant with density of activation events, dynamics,
and distribution along the root axis similar to those of
untreated roots (Fig. 7g–i). Overall, these data suggest that
accurate auxin perception is required for correct establishment of the zone of auxin minimum, DW, and normal
LRP initiation process.
Discussion
The developmental importance of the auxin distribution
has been demonstrated in different organogenic events
(Benková et al., 2009). Auxin acts as a morphogenetic trigger of LR initiation and organogenesis (Dubrovsky et al.,
2008). Here, we demonstrate that auxin gradients along the
root control the positioning of the site of founder cell speci-
fication and their posterior activation, resulting in the
acropetal pattern of the LR formation.
The auxin minimum zone defines the DW for LR
initiation
Our detailed analyses of the DR5 activity along the primary
root supported by direct auxin measurements revealed that
in roots the distal and the proximal auxin gradients are separated by a zone of auxin minimum. The distal gradient in
the Arabidopsis root typically ceases at 0.2 mm from the
root tip, with the DR5rev::GFP expression turning out to be
very low (Fig. 1b,c), and at c. 1.2 mm from the QC, no
DR5 activity is detected (Fig. S1). The proximal auxin
gradient starts in wild-type Arabidopsis seedlings 4.5 to
6 mm from the QC. A similar pattern was found in tomato
roots (Fig. 1d,e). In both species, the zone of auxin minimum overlaps with a DW in which specification of founder
cells followed by their division occurs with the highest probability (Table 1; Figs 1, 4).
In NPA-pretreated Arabidopsis roots lacking LRP, new
initiation events occur exclusively in the root portion
formed after removal of NPA and no LRP formation takes
place in the pericycle previously exposed to NPA (Fig. 5a).
Absence of new pericycle activation events in the NPAtreated zone is not a consequence of NPA retention in this
root portion because the IAA polar transport is restored
within 24 h upon elimination of the inhibitor. Accordingly,
NPA becomes hydrolyzed and inactivated within 24 h at
the root tip of treated seedlings of Arabidopsis (Murphy &
Taiz, 1999). Interestingly, in seedlings of Arabidopsis
(Fig. 6) and tomato (Fig. S6) that were able to form LRs
after NPA treatment, the primary root was separated from
the shoot and the meristem was removed. This indicates
that endogenous auxin gradients established in intact roots
are essential for the irreversible arrest of pericycle cell activation for LRP formation. When these endogenous auxin
gradients are broken by excisions, the pericycle acquires the
capability for founder cell specification and LRP formation.
Mutants in the PIN3 and PIN7 genes coding for auxin
efflux carriers of the PIN family (Friml et al., 2003, 2004)
have previously been shown to be defective in LR initiation
(Benková et al., 2003; Dubrovsky et al., 2009). The findings reported here reveal that PIN3 and PIN7 are
important players in the maintenance of the proximal auxin
gradient and in correct positioning and operation of the
DW for LR initiation. In both pin3 and pin7 mutants, the
auxin minimum zone is reduced and correlation between
the start of the proximal auxin gradient and the site of primordium initiation is absent (Fig. 4a), whereas positioning
of the LR initiation is more random.
Temporal accumulation of the mIAA14 repressor
strongly reduces the density of pericycle activation events
and, as with the transient inhibition by NPA, no additional
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(a)
(h)
(b)
(c)
(i)
(d)
(e)
(f)
(g)
Fig. 7 Defects in lateral root (LR) initiation and primordium organogenesis in auxin receptor tir1 afb2 afb3 triple mutant in comparison with
wild-type (DR5rev::GFP) in 8-d-old Arabidopsis seedlings. (a–c) Wild-type without treatment (n = 10). (a) Protoxylem-adjacent pericycle cell.
(b) Stage I primordium. (c) Emerging primordium; asterisks mark flanking pericycle cells. (d–f) Defects in untreated roots of the tir1 afb2 afb3
(DR5rev::GFP) line (n = 10). (d) Unusually short pericycle cells. (e) Two-layered pericycle found in some root portions; p, epicycle. (f)
Abnormal primordium formed from a two-layered pericycle. Asterisks mark the margins of the primordium. (g) Quantitative analysis of
pericycle activation events in wild-type (DR5rev::GFP; circles) and in tir1 afb2 afb3 (DR5rev::GFP; triangles) upon 10 lM naphthalene-acetic
acid (NAA) treatment. Means and 95% confidence intervals are given (n = 10). (h) DR5rev::GFP treated with 10 lM NAA during a 24 h
period (n = 10). (i) Upon a 6 h treatment with 10 lM NAA, pericycle cells in the tir1 afb 2 afb 3 (DR5rev::GFP) triple mutant become shorter
as a result of cell division; at 12 h, a two-layered pericycle or stage-II primordia are formed. At 24 h, division of pericycle cells in the area
between primordia or LRs (24 h-1), formation of an abnormal primordia from activation of a two-layered pericycle (asterisk, 24 h-2), and
formation of a multilayered pericycle in the distal root portion (24 h-3) are observed. The vertical and horizontal arrowheads indicate end walls
of pericycle cells and new cell walls, resulting from periclinal cell division, respectively (n = 10). Bar, 50 lm.
activation takes place in the DEX-treated root zone at a later
time, as would be expected if the acropetal pattern of the
LR initiation were not maintained. Accumulation of the
mIAA14 protein is not sufficient to prevent the LR initiation when seedlings are treated with NAA (Fig. 5c).
Altogether these findings reveal the existence of a mechanism implicated in the regulation of an acropetal pattern of
LRP formation. This mechanism depends on auxin transport and distribution, particularly on a zone of auxin
minimum that defines a DW for founder cell specification
and LR initiation.
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The auxin minimum defines the positioning of the LR
initiation
Oscillation in DR5 activity within the elongation zone (De
Smet et al., 2007; De Rybel et al., 2010; Moreno-Risueno
et al., 2010) and subsequent activation of pericycle cells
taking place in the differentiation zone are two key growth
control points in the LR formation. The aim of this work
was to analyze how the processes of the second growth
control point, the founder cell specification and their first
divisions, are regulated in space and time. We show that a
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zone of auxin minimum and a DW defined by this minimum are essential. When the DW was pharmacologically
or genetically modified, cells could pass through this part of
the root without primordium initiation. Our data demonstrate that at time intervals £ 8.5 h, the next pericycle
activation event takes place within the zone of auxin minimum and that the cells are triggered to become founder
cells. This estimation is close to the time intervals between
consecutive oscillation events in gene expression activity at
pre-branch sites (Moreno-Risueno et al., 2010). This timerelated mechanism seems to be well coordinated with the
rate of root growth, and the start of the proximal auxin
gradient is apparently displaced towards the root tip at time
intervals equal to those between subsequent primordium
initiation events.
Our work reveals that, paradoxically, LRP initiation triggered by auxin (Dubrovsky et al., 2008) occurs with the
highest probability in a zone of auxin minimum at the tip
of the primary root. The possibility that pericycle cells in
the DW exhibiting an auxin minimum are preferentially
activated for LR initiation might be that reduced auxin
concentrations in this zone sensitize pericycle cells to small
fluctuations in auxin concentrations. In support of this
explanation are the experiments with the tir1 afb2 afb3
mutant that suggest a correct auxin perception is essential
for DW establishment. Interestingly, when TIR1 ⁄ AFBdependent signaling is inhibited pharmacologically, the
newly grown root portion no longer shows GATA23
promoter activity typical for founder cells (De Rybel et al.,
2010). Developmental mechanisms involved in founder cell
specification and stage I LRP formation are not completely
understood. We provide a mechanistic explanation for the
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link between auxin gradients and pericycle activation events
and suggest that an auxin minimum zone restricts the
occurrence of founder cell specification and LR initiation in
time and space. The establishment of this zone might represent an important control mechanism to ensure steadiness
and acropetal pattern of primordium initiation.
A developmental window model for LR initiation
defined by auxin gradients established along the
parent root
We propose the following model of auxin-dependent establishment of a DW for founder cell specification and
primordium formation (Fig. 8). In an intact growing root,
two distal and one proximal auxin gradients are established,
defining a morphogenetic zone that corresponds to a DW for
LR initiation and includes founder cell specification and their
first divisions leading to a stage I primordium. Within this
zone, a minimum auxin concentration coincides with the
maximum probability of cells to become founder cells. This
window, between c. 2 and 6 mm from the QC, is dynamic
and displaced in the same direction and at the same rate as the
root growth rate. NPA-sensitive polar auxin transport and
auxin receptors of the TIR1 ⁄ AFB family are important for
this DW operation. When external auxin is applied, pericycle
cells outside the DW can be activated because they maintain
their competence to become founder cells. Under nonstandard growing conditions, such as exogenous auxin or root
damage in the natural environment, the DW is no longer
operational and initiation can occur outside the DW zone.
In conclusion, our data indicate that a DW in which the
LR initiation takes place in an acropetal sequence correlates
Fig. 8 Model of developmental window (DW) operation. In an intact growing root, two distal and one proximal auxin gradients are
established that define a DW for lateral root (LR) initiation. Within this morphogenetic zone, the minimal auxin concentration correlates with
the highest probability of pericycle cells to acquire founder cell (FC) identity. This morphogenetic zone, between 2 and 6 mm from the tip, is
dynamic and is displaced in the same direction and at the same rate as the root growth rate. Auxin transport and receptors of the TIR1 family
are required for operation of this DW. When external auxin is applied, its distribution is rearranged and cells outside the DW can be activated,
because they maintain their competence to form FCs. NPA, 1-naphthylphthalamic acid (NPA).
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spatially and temporally with the establishment and maintenance of the auxin minimum zone, which is defined as a
zone between distal and proximal auxin gradients and is
functionally important to maintain steady LRP formation.
Functionality of this window relies on active auxin transport
and perception and on response mechanisms.
Acknowledgements
We thank J. Murfett for the DR5::GUS line, M. Estelle for
the tir1 afb2 afb3 mutant, H. Fukaki for pIAA14::mIAA14GR, P. Doerner for the CycB1;1DB::GUS line, J. Normanly
for the p1G4 AtIAA2::GUS reporter construct, A. Saralegui,
A.L. Martı́nez-Valle and S. Ainsworth for excellent technical help, N. Doktor for help with artwork and pixel-density
analysis, P. Benfey for critical reading of the manuscript,
and M. De Cock for help in preparing it. This research was
supported by the European Research Council starting independent research grant (to E.B.), National Research
Initiative Competitive Grants Program (grant 2006-03434
to M.G.I.), the Odysseus program of the Research
Foundation-Flanders (J.F.), the Dirección General de
Asuntos del Personal Académico – Programa de Apoyo a
Proyectos de Investigación e Innovación Tecnológica,
Universidad Nacional Autónoma de México (grant IN212509
to S.S. and grants IN225906 and IN212009 to J.G.D.),
Consejo Nacional de Ciencia y Technologı́a, Mexico (grant
79736 to S.S. and grant 49267 to J.G.D.), and Programa de
Apoyos para la Superación del Personal Académico –
Universidad Nacional Autónoma de México (J.G.D.).
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Supporting Information
Additional supporting information may be found in the
online version of this article.
2011 The Authors
New
Phytologist
Research
Fig. S1 Auxin-response gradient in the central cylinder of
the distal root portion of the DR5rev::GFP Arabidopsis line.
Fig. S5 Dynamics of pericycle cell activation after auxin
treatment.
Fig. S2 Restoration of IAA transport after 1-naphthylphthalamic acid (NPA) treatment.
Fig. S6 Gradient in probability of pericycle cells to initiate
lateral roots in tomato plants.
Fig. S3 Experimental setup to evidence the irreversible nature of transient inhibition of lateral root initiation in
Arabidopsis using pIAA14::mIAA14-GR line.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting information
supplied by the authors. Any queries (other than missing
material) should be directed to the New Phytologist Central
Office.
Fig. S4 Gradient in probability of pericycle cells to initiate
lateral roots upon local auxin treatment in Arabidopsis
CYCB1;1DB::GUS line.
2011 The Authors
983

Auxin minimum defines a developmental window for lateral root

Transcription

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