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First published online 10 January 2007
doi: 10.1242/dev.02753
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1 Department of Plant Systems Biology, Flanders Interuniversity Institute for
Biotechnology (VIB), Ghent University, Technologiepark 927, B-9052 Gent,
Belgium.
2 Plant Sciences Division, School of Biosciences, University of Nottingham,
Sutton Bonington Campus, Loughborough LE12 5RD, UK.
3 Unité Mixte de Recherche 1098, Institut de Recherche pour le
Développement, 911 Avenue Agropolis, F-34394 Montpellier cedex 5,
France.
4 Departamento de Ciencias Morfológicas y Biología Celular y
Animal, Universidad de Extremadura, Avenida de Elvas s/n, E-06071 Badajoz,
Spain.
Author for correspondence (e-mail:
tom.beeckman{at}psb.ugent.be)
Accepted 21 November 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Arabidopsis, Auxin, Basal meristem, Lateral root, Root branching
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Charlton, 1983;
Barlow and Adam, 1988). New
lateral roots are continuously initiated at a predictable distance above the
growing root tip (reviewed by Charlton,
1996). Lateral root primordia and the youngest lateral roots can
be found nearest to the root tip, whereas more mature lateral roots are
encountered higher in the root (Fahn,
1974).
Prior to emergence in the mature zone, lateral root primordia go through an
extensive series of cell divisions (Malamy
and Benfey, 1997). Due to the acropetal development of lateral
roots, early stages can be traced back at more distal positions. Furthermore,
a G2-to-M-specific promoter-reporter construct, CYCB1;1::GUS, marks
the very first divisions in the pericycle during lateral root initiation. In
Arabidopsis thaliana, the first lateral root that is initiated after
embryogenesis is observed in the differentiation zone, at a fixed distance
above the root tip (Casimiro et al.,
2001). This position corresponds with the region where pericycle
cells progress via S phase to G2 (Beeckman
et al., 2001). Initiation of Arabidopsis lateral roots
occurs in a strict acropetal pattern and only in a relatively short zone
distal to the youngest lateral root primordium
(Dubrovsky et al., 2006).
Here, we provide evidence that the events which determine lateral root
positioning take place in a region at the transition between the meristem and
the elongation zone, referred to as the basal meristem
(Beemster et al., 2003), where
several other physiological and growth responses occur, including responses to
gravity, touch and moisture (Ishikawa and
Evans, 1995). The data presented suggest that auxin signaling in
the central cylinder of the basal meristem correlates with regular lateral
root spacing. Furthermore, we show that lateral root initiation is regulated
by periodic fluctuations in DR5 activity. We hypothesize such fluctuations
might be representative for fluctuations in auxin distribution mediating
regular longitudinal spacing of lateral roots. Our data provide a new model
for root branching that highlights the involvement of the root apex.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and aux1-22 (both null alleles),
axr3-1; promoter fusions CYCB1;1::GUS
(Colón-Carmona et al.,
1999), DR5::GUS (Ulmasov et
al., 1997), IAA2::GUS (Swarup
et al., 2001), IAA14::GUS
(Fukaki et al., 2002);
GAL4-GFP enhancer trap lines J0121, J0951, J1701, M0013 and Q1220
(http://www.plantsci.cam.ac.uk/Haseloff/geneControl/catalogues/Jlines);
the promoter trap QC184 (Sabatini et al.,
2003); UAS:AUX1,aux1-22, J0951>>AUX1,aux1-22,
UAS:axr3-1, J0121>>UAS:axr3-1
(Swarup et al., 2005), and
pIAA14::mIAA14-GR lines (Fukaki
et al., 2005).
Growth conditions and drug treatments
Seeds were germinated on standard Murashige and Skoog (MS)-derived medium
on vertically or at 45° oriented square plates (Greiner Labortechnik,
Kremmünster, Austria) under growth conditions described by Vanneste et
al. (Vanneste et al., 2005).
Supplements were 10 µM N-1-naphthylphthalamic acid (NPA; Duchefa,
Haarlem, The Netherlands), 10 µM 
-naphthaleneacetic acid (NAA;
Sigma-Aldrich, St Louis, MO), or 1 µM dexamethasone (Dex;
Sigma-Aldrich).
For all time-course experiments, the highest synchronization level was obtained by incubating the agar plates, after sowing, for 2 days at 4°C in the dark and then under continuous light at 22°C. Under these conditions, germination started at the earliest 20 hours after transfer to the growth chamber and was nearly 100% at 48 hours. After transfer to the growth chamber, the plates were screened for germinated seeds with a dissecting microscope, to indicate the early (at 24 hours) and late (at 34 hours) germinating population. The positions of germinating seeds (i.e. seeds with a radicle protruding the seed coat) were marked on the plate using a felt-tip pen. Only the marked seedlings were used for further analyses. In each time course, samples were taken at intervals of 7.5 hours (see Fig. S1 in the supplementary material for corresponding seedling stages). By considering the appearance of the radicle as time 0 hours, we obtained highly uniform seedling stages as supported by the homogenous seedling size at each time point determined by time-lapse recordings (see Fig. S1 in the supplementary material).
Histochemical and histological analysis
The ß-glucuronidase (GUS) assays were performed as described by
Beeckman and Engler (Beeckman and Engler,
1994) or according to the protocol of Malamy and Benfey
(Malamy and Benfey, 1997). For
anatomical sections, GUS-stained samples were treated as described previously
(Beeckman and Viane, 2000;
De Smet et al., 2004).
Microscopic analyses
For whole-mount microscopic analysis, samples were cleared by mounting in
lactic acid (Acros Organics, Geel, Belgium) or according to Malamy and Benfey
(Malamy and Benfey, 1997). All
samples were analyzed by differential interference contrast microscopy (DMLB;
Leica Microsystems). For fluorescence microscopy, whole seedlings were stained
with 10 µg/mL propidium iodide (Sigma-Aldrich) and mounted in water under
glass coverslips for green fluorescent protein (GFP) signal analysis with a
confocal microscope 100M with software package LSM 510 version 3.2 (Zeiss,
Jena, Germany). Images were collected with a 488-nm emission filter.
Imaging and root length measurements
Photographs were taken with a CAMEDIA C-3040 zoom digital camera (Olympus,
Tokyo, Japan) and processed with Photoshop 7.0 (Adobe Systems, San
José, CA). Whole plates were scanned on a color copier CLC-iR C3200
(Canon, Tokyo, Japan). For measuring the interlateral root distances, the
positions of lateral roots and emerged primordia were indicated under a
dissecting microscope (Stemi SV11 Apo, Zeiss) on the back of the plates using
a felt-tip pen prior to scanning. Plate scans were measured with ImageJ
(http://rsb.info.nih.gov/ij/).
Toner labeling
Distances from root tips (including root cap) to the start of DR5::GUS
expression at 10, 25, 40 and 55 hours after germination (HAG) were first
determined using a stereomicroscope (Stemi SV11 Apo, Zeiss) with an eyepiece
and measurement unit. To label that part of the root tip where DR5::GUS
expression is anticipated, black toner particles from a copier were positioned
with an eyelash on the root. Likewise, roots at off-peak time-points (17.5,
32.5 and 47.5 HAG) were labeled at a position that was extrapolated from the
measurements at the high-level time-points. In this way, the epidermal cells
of this region become permanently marked
(Beemster and Baskin,
1998).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
resulting in an enhanced waving of the root when seedlings are grown at a
45° angle (Okada and Shimura,
1990). On agar plates, vertically grown Arabidopsis roots
also display a wavy pattern (Fig.
1A), which is accompanied by lateral root development at outer
sides of bends (Fig. 1B). To
investigate the correlation between these two processes, Arabidopsis
seedlings were grown on 1.5% hard agar at an inclination of 45°
(Fig. 1C). At 12 days after
germination (DAG), seedlings showed on average 20 measurable curves per root
representing 16% of the total root length
(Fig. 1D;
Table 1). Of the total number
of lateral roots, approximately 51% were positioned precisely in this 16%
region of the root (Fig. 1D,
red mark). A 
2 test (with one degree of freedom) revealed that
this peculiar lateral root distribution does not occur by chance in wild type
(P<0.001) and suggested a correlation between lateral root
formation and root waving.
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), the
time to bridge this distance could be calculated to approximately 15
hours.
The wavy growth pattern is the consequence of an alternation between
right-turn and left-turn root bending
(Rutherford and Masson, 1996).
As lateral roots are formed on top of the bends, the wavy growth will result
in a left-right alternation of lateral roots and in an equal distribution of
laterals over both sides of the root. In vertically grown 10-DAG-old
Arabidopsis seedlings (n=11), lateral roots (including
primordia) were indeed distributed equally at both sides (49.6% left and 50.4%
right) (Fig. 1B,E), with 66% of
the roots in a strict left-right alternating sequence. This result is in
agreement with previous analyses in tomato, another species with lateral roots
positioned on two longitudinal rows
(Newson et al., 1993).
The agravitropic aux1 mutant
(Bennett et al., 1996) lacks
the normal wavy growth pattern. Instead of the left-right bending found in
wild-type roots, aux1 roots mainly bent constitutively to the right,
with a right-handed root coiling as a consequence
(Fig. 1F). In 10-DAG-old
aux1 roots (n=18), lateral roots predominantly appeared on
the outer (left) side of the coiling root (69.7% left and 30.3% right;
Fig. 1E). This uneven
positioning of lateral roots resulted in a clear deviation from left-right
alternation in 66% of the successive lateral roots investigated, representing
a significantly higher percentage than was found in wild-type roots as
determined by a Student's t-test (P<0.001).
Both lateral root initiation and gravitropic response depend on
AUX1-facilitated auxin transport (Casimiro
et al., 2001; Swarup et al.,
2005), so we asked whether lateral root initiation might be
controlled by local activity of AUX1. Targeted expression of AUX1 to
the lateral root cap and epidermal tissues of aux1 roots fully
restores the aux1 agravitropic defect
(Swarup et al., 2005). Hence,
we analyzed whether the same targeted expression of AUX1, using a
GAL4 driver line (J0951; Fig.
2A) could also restore the lateral root initiation defect of
aux1 (Marchant et al.,
2002). Seedlings expressing UAS:AUX1 under the control of
the GAL4 driver line J0951 in an aux1-22 mutant background
(Swarup et al., 2005) were
grown for 10 DAG on 1.5% agar at 45° inclination. The number of lateral
roots per cm in the aux1-22 mutant was significantly reduced compared
with that of the Col-0 control (Fig.
2B; Table 2). In
contrast, targeted expression of AUX1 to the lateral root cap and
epidermis of aux1 restored the lateral root number to that of the
wild type (Fig. 2B;
Table 2). Furthermore, the
left-right alternation in lateral root formation could be rescued in
vertically grown J0951>>AUX1,aux1-22 plants to levels similar to
those of the Col-0 control (Fig.
2C; Table 2). Other
GFP driver lines restoring AUX1 functioning only in the lateral root cap
(M0013) or in stele and columella tissues (J1701)
(Swarup et al., 2005) did not
complement the lateral root initiation defect in the mutant background, but
complementation could be obtained with another lateral root cap and
epidermis-specific driver line (Q1220) (see Fig. S2 in the supplementary
material). In the absence of an epidermis-specific driver line, we conclude
that AUX1 action in lateral root cap and/or epidermal cells influences lateral
root initiation and positioning.
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), and the basipetal transport towards this region also
involves AUX1 (Swarup et al.,
2001; Swarup et al.,
2005). Therefore we investigated whether the basal meristem
displayed an increased susceptibility for auxin-induced lateral root
initiation. Wild-type seedlings (5 DAG) that had already formed a few lateral
roots in a left-right alternating fashion were transferred to a high
concentration of NAA (10 µM) and incubated for 1 week. Although lateral
roots were induced along the entire length of the root, proliferation was
especially excessive in the basal meristem
(Fig. 3A). The same experiment
was repeated with a quiescent center-expressed promoter trap (QC184)
(Sabatini et al., 2003). Even
though no clear discrete quiescent centers could be visualized in the
proliferating cell population in the basal meristem, the marker line had an
intensely stained band at the periphery of the structures
(Fig. 3B), reflecting some
level of apical differentiation and suggesting intensive primordia formation
in this part of the root with fused structures as a consequence. We analyzed
whether this increased sensitivity was also reflected in the expression of the
synthetic auxin-responsive marker DR5::GUS
(Ulmasov et al., 1997). At 40
HAG, transgenic seedlings were transferred from MS medium to medium containing
10 µM NAA. On MS medium, after 20 minutes of GUS staining, the auxin
reporter DR5::GUS was detected in two short strands, just above the
meristem (Fig. 3C). After 6
hours of NAA treatment, DR5::GUS expression had increased all over
the root of the seedlings, but more intensely in the basal meristem
(Fig. 3D).
|
). Therefore, the DR5::GUS reporter line was
grown under conditions where auxin transport was blocked with 10 µM NPA.
After 72 hours on NPA and subsequent GUS staining, no expression was detected
in the basal meristem (Fig.
3E), arguing for auxin transport dependent expression of
DR5::GUS in the basal meristem.
A detailed anatomical analysis of DR5::GUS stele expression on MS
medium revealed that the GUS reporter was restricted to the two protoxylem
strands and was absent from the adjacent pericycle cells
(Fig. 3F). To confirm this
pattern, the expression of a more sensitive auxin-responsive marker,
IAA2::GUS (Swarup et al.,
2001), was analyzed in detail in the basal meristem of 72-HAG-old
seedlings grown in the presence or absence of the auxin transport inhibitor
NPA. IAA2 had been shown previously to be expressed in the root
meristem and in both protoxylem poles
(Swarup et al., 2001). In our
analysis of the basal meristem, IAA2::GUS was expressed in the
central cylinder at and around the phloem and xylem poles. Strong staining, in
agreement with the radial pattern of DR5::GUS expression could be
observed in the protoxylem cells of IAA2::GUS lines
(Fig. 3G). When seedlings were
grown on NPA, staining was equal, although weaker, in the entire central
cylinder (Fig. 3H) in contrast
to the pattern observed when grown without NPA.
A recurrent auxin signal in the basal meristem controls regular longitudinal lateral root initiation
The above lines of evidence suggested that the basal meristem might
represent a site of auxin accumulation distinct from the distal auxin maximum
in the quiescent center and surrounding cells
(Sabatini et al., 1999). To
elaborate on its potential significance for lateral root initiation, we
monitored spatial and temporal expression patterns of the DR5::GUS
reporter line in the basal meristem. Starting from 10 HAG, seedlings were
harvested every 7.5 hours and subsequently stained for GUS activity. Temporal
changes were observed in the staining pattern of the GUS-positive strands in
the stele of the basal meristem. The recorded temporal variations revealed an
oscillating DR5::GUS expression pattern in the basal meristem with an
interval of approximately 15 hours (Fig.
4A; Table 3). Over
the entire time course we obtained two populations of seedlings, one with a
high and one with a low percentage of strong DR5::GUS staining.
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) we observed a basipetal shift of the DR5::GUS
expression in the later time points compared with the early ones. To be able
to label the correct zone of the root tips where DR5 activity is expected to
occur, the distances between the root tip, including the root cap, to the
start of the DR5::GUS staining were measured at the time points with high
level of expression (Table 3).
Taking these distances into account, toner ink particles were positioned on
the roots of seedlings of the CYCB1;1::GUS reporter line, as
described in Materials and methods. After the seedlings had been labeled, they
were allowed to grow for another 30 hours, sufficient for development of at
least one lateral root initiation site
(Dhooge et al., 1999), and
subsequently stained histochemically for GUS
(Fig. 4B). By assuming that the
auxin signal in this part of the root that corresponds to the basal meristem
triggers lateral root initiation, the label that remained attached to the
epidermis after 30 hours of growth would be expected to colocalize with an
early lateral root initiation site (Fig.
4B,C). Indeed, toner particles were detected at the position of a
lateral root in a high percentage of the cases for 10, 25, 40 and 55 HAG
(Fig. 4D;
Table 3). At 17.5, 32.5 and
47.5 HAG, in-between two predicted auxin signals and, hence, with low
probability to detect the auxin signal, the percentage of the lateral root
initiation sites that correlated perfectly with the toner particles was
strongly reduced (Fig. 4D;
Table 3). In conclusion,
significant colocalization between toner particles and lateral root primordia
could be obtained by labeling the basal meristem at the time when we recorded
the DR5::GUS expression. The correlation between the recurrent DR5::GUS expression in the basal meristem and the initiation of lateral roots implies an oscillating auxin response driving the process of lateral root initiation. To evaluate whether a similar oscillation was present in the formation of lateral roots, the timing of the initiation of consecutive lateral roots was determined. A time-series experiment with Col-0 seedlings containing the CYCB1;1::GUS marker was performed from 10 HAG until 60 HAG and samples were taken and stained for GUS every 5 hours. Up to 25 HAG, no initiation site could be detected. At 30 HAG, 71% of the seedlings contained only one lateral root initiation event (n=43). Seedlings with two lateral root initiation sites appeared at the earliest at 45 HAG in 20% of the analyzed population (n=31), and three lateral root initiation sites were observed at the earliest at 60 HAG (n=11; Fig. 4E). This order of sequence fits well with a period of 15 hours between the initiations of consecutive lateral roots and is in agreement with the periodicity found with the DR5 activity in the basal meristem. In this respect, the first auxin signal at 10 HAG might correspond to the first primordium at 30 HAG, the second auxin signal at 25 HAG to the second primordium at 45 HAG, and so on (Fig. 4F). Based on these data, a time period of 20 hours between the auxin signal in the basal meristem and the corresponding lateral root initiation event can be deduced.
By assuming that the auxin response and lateral root initiation are correlated, the recorded periodicity would imply a regular longitudinal spacing of lateral roots. In Col-0 seedlings (10 DAG, n=37) the distances between two neighboring lateral roots or emerged primordia, irrespective of their radial position were measured. The distance between consecutive primordia was relatively constant along the primary root, namely 2225±35 µm (n=856).
Having emphasized the AUX1-dependency of lateral root spacing (see above), we analyzed whether the recorded oscillations in the DR5::GUS expression pattern were affected in the aux1 mutant. Indeed, the number of aux1 seedlings with DR5::GUS expression in the basal meristem was severely reduced at all time points investigated (Fig. 4A).
Priming xylem pole pericycle cells for lateral root initiation depends on intact auxin response but is independent of IAA14
In Arabidopsis, pericycle founder cell identity and, hence,
lateral root initiation are limited to the pericycle cells opposite the xylem
pole (Casimiro et al., 2003).
However, the presented data suggested that the auxin response was stronger in
the protoxylem elements than in the adjacent pericycle cells.
To show that auxin response in the xylem pole pericycle cells from the
basal meristem onward is required for lateral root initiation, this response
was selectively disrupted by regional expression of a dominant mutant version
of the IAA17 protein (axr3-1)
(Leyser et al., 1996). An
UAS:axr3-1 construct was expressed under the control of the GAL4
driver line J0121 that is active in the main root xylem pole pericycle from
the basal meristem onward (Fig.
5A) (Laplaze et al.,
2005). Detailed analysis of the lateral root number revealed a
strong reduction in J0121>>UAS:axr3-1 compared with the controls
(Fig. 5B;
Table 4). A microscopic
analysis (n=10) showed that the number of lateral roots was reduced
clearly in all developmental stages [as defined by Malamy and Benfey
(Malamy and Benfey, 1997)] and
that lateral roots rarely developed beyond stage I
(Fig. 5C). Interestingly,
pericycle cells with nuclei displaced toward each other could be observed in
J0121>>UAS:axr3-1 (Fig.
5D). Longitudinal sections of four segments (with 5 mm intervals)
in the region just above the root meristem demonstrated that both J0121 and
J0121>>UAS:axr3-1 revealed the same frequency of lateral root
initiation events per seedling, but the portion of displaced nuclei per
seedling in J0121 was significantly lower than that in
J0121>>UAS:axr3-1 (Fig.
5E; Table 4).
|
|
). IAA14
(SOLITARY ROOT, SLR) has been shown to play a prominent role in lateral root
initiation (Fukaki et al.,
2002; Fukaki et al.,
2005; Vanneste et al.,
2005). Although IAA14 is expressed in the pericycle and
during lateral root initiation (Fukaki et
al., 2002) expression analysis of IAA14::GUS revealed
that IAA14 is not expressed in the basal meristem
(Fig. 6A). Therefore, auxin
response in the xylem pole pericycle cells in the basal meristem is most
likely still intact in the slr-1 mutant and initial priming of
pericycle cells might still occur. To demonstrate this possibility we made use
of plants expressing the stabilized mutant mIAA14 under the native
IAA14 promoter controlled by an inducible system with the
glucocorticoid receptor (pIAA14::mIAA14-GR)
(Fukaki et al., 2005). This
allows the evaluation of a temporal effect of the mutated IAA14 protein on the
initiation of lateral roots, as release from medium containing Dex to medium
without Dex restores the wild-type IAA14 functioning. We asked more
specifically whether pericycle cells that passed through the basal meristem in
plants having a stabilized mutant mIAA14, were still competent to
initiate lateral roots when the wild-type IAA14 was restored. As reported
previously, 10-DAG-old seedlings expressing pIAA14::mIAA14-GR in the
presence of Dex did not form lateral roots
(Fukaki et al., 2005).
Subsequently, seedlings (10 DAG) grown in the presence of Dex were transferred
to media with and without Dex. Seedlings that continued to grow on Dex had no
lateral roots (Fig. 6B), but
pIAA14::mIAA14-GR seedlings that had been transferred onto Dex-free
media formed lateral roots (Fig.
6C). Interestingly, also in the distal part of the region
previously subjected to Dex (8.3±0.7 mm, n=10), roots could be
formed whereas this was not the case in the more proximal regions of the roots
where the inhibition appeared to be permanent
(Fig. 6C). |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Swarup et al., 2005). Here, we
demonstrate that the processes for lateral root initiation and gravitropic
response are intertwined and operate in the same zone of the root tip:
gravitropic response mediated waving of the primary root is correlated with
the formation of lateral root primordia. As a consequence, lateral root
development displays a left-right alternating pattern, which is disturbed in
aux1 mutants. Several mutations that simultaneously affect lateral
root initiation and gravitropic response corroborate this connection
(Hobbie and Estelle, 1995;
Muday et al., 1995;
Simmons et al., 1995;
Marchant et al., 2002;
Benková et al., 2003;
Lin and Wang, 2005).
|
;
Vanneste et al., 2005) that
could hitherto not be explained. Our new data support the physiological
relevance of this time lag. The initial auxin signal transduction takes place
in the basal meristem already at 10 HAG, subsequently priming divisions of
founder cells will occur at late time stages higher up in the root. Recently,
the basal meristem has been shown to recycle auxin channeling back from the
root tip through the root cap (Blilou et
al., 2005; Leyser,
2005). Redistribution of this recycled pool might generate an
auxin response in the basal meristem as visualized by the DR5::GUS
reporter, which also revealed the existence of an auxin maximum in columella
initials (Sabatini et al.,
1999). Fundamental changes in cell fate, cell division plane, and
cell polarity have been observed when this root tip auxin maximum is
disturbed. Although we could not demonstrate directly the existence of a
second concentration maximum in the basal meristem, it is tempting to
speculate that auxin accumulation itself primes the initiation of a new
lateral organ. The occurrence of an auxin maximum in the basal meristem fits
with the auxin signaling center defined in the basal part of the elongation
zone based on genome-wide expression profiles in the root
(Birnbaum et al., 2003;
Beeckman, 2004) and is in
agreement with the previously proposed inductive signal (hormonal or
environmental) that affects founder cell formation and/or division of
pericycle cells close to the root tip
(Barlow and Adam, 1988;
Dubrovsky et al., 2001;
Dubrovsky et al., 2006).
At the anatomical level the DR5::GUS staining is restricted in the
basal meristem to the two protoxylem cell files neighboring the pericycle
cells (Fig. 7A). This peculiar
radial staining pattern is easily disturbed in the presence of NPA
(Fig. 7B), a treatment known to
inhibit lateral root initiation (Casimiro
et al., 2001). We hypothesize that a radial gradient with a
maximum in the protoxylem cells might be required for lateral root initiation
to take place. However, this interpretation is still very speculative and only
based on the radial expression pattern of GUS markers. Further studies of the
radial auxin distribution patterns and mechanisms in the basal meristem are
required to support this hypothesis.
A recurrent auxin signal in the basal meristem controls longitudinal lateral root distribution
We demonstrated that an auxin response reporter in the basal meristem shows
rhythmic expression with the same periodicity as lateral root initiation. This
phasing of approximately 15 hours is in agreement with the temporal window
between the initiation of two successive lateral roots as was recently
calculated for Arabidopsis by Dubrovsky et al.
(Dubrovsky et al., 2006). The
recurrence of the auxin signal may be caused (at least in part) from periodic
gravitropism-induced fluctuations in auxin redistribution within the root
apex. The existence of an auxin signal in the basal meristem stresses the
importance of the root tip for the regulation of root branching and supports
the idea that the auxin pool in the root tip drives the initial stages of
lateral root primordia formation (Bhalerao
et al., 2002). As lateral roots are almost never found in opposite
positions, the appearance of the auxin signal simultaneously at both
protoxylem poles (Fig. 7A)
necessitates an attenuation determining the left-right positioning of lateral
roots. How this attenuation is brought about is not known.
Auxin-dependent signaling in the basal meristem presumably represents the
very first checkpoint toward lateral root initiation
(Fig. 7C,D). It cannot be
neglected that other auxin sources, such as shoot-derived auxin, play a role
in later steps of lateral root formation
(Reed et al., 1998;
Bhalerao et al., 2002), for
instance in triggering the asymmetric division and further primordium
development.
Auxin response of xylem pole pericycle cells in the basal meristem required for determination of founder cell identity is independent of IAA14/SLR
Lateral roots were nearly totally absent when auxin response in the xylem
pole pericycle cells was abolished by specific expression of a stabilized form
of IAA17 (AXR3). Microscopic inspection of such roots revealed a pre-mitotic
stage of Arabidopsis lateral root initiation that has, until now,
only occasionally been reported (Casero et
al., 1993; Barlow et al.,
2004). Just prior to the asymmetric cell division, the nuclei of
two neighboring pericycle cells migrate to the common anticlinal cell wall
(Fig. 7C). In wild-type roots,
this process is probably rapidly followed by the division event, explaining
the lack of reports on this stage in the literature.
|
;
Fukaki et al., 2005;
Vanneste et al., 2005). Xylem
pole pericycle-specific expression of a stabilized form of IAA14, which is
closely related to IAA17, can repress lateral root formation
(Fukaki et al., 2005). Our
data suggest that IAA14 control of lateral root initiation acts downstream of
auxin signaling in the basal meristem and is not required for the priming of
the founder cells. First, IAA14 is not expressed in the basal
meristem. Second, releasing pericycle cells from the repression of a
stabilized form of IAA14 (mIAA14) results in the formation of lateral
roots even in a region of the root that was earlier subjected to
mIAA14. We conclude that expressing the stabilized form of IAA14 from
its native promoter does not interfere with the very early phases of lateral
root initiation and that priming of the pericycle cells could take place in
the absence of normal IAA14 functioning. In Fig. 7D a model is proposed that illustrates the possible spatiotemporal events that occur in the root tip prior to lateral root initiation. Our results suggest that via AUX1 action in lateral root cap and/or epidermal cells, the pericycle cells in the basal meristem might be primed through an IAA14-independent pathway (Fig. 7D). Next, in more proximal regions of the root, an IAA14-dependent auxin response is required for initiation of cell division of pericylce cells as can be visualized by the expression of CYCB1;1. It is likely that IAA14 is not the only Aux/IAA protein involved in this process but further functional characterization of other Aux/IAA proteins such as IAA17 during lateral root initiation is required.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/4/681/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Present address: Department of Plant and Animal Sciences, Faculty of
Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
Present address: Institut des Sciences du Végétal, Centre de
la Recherche Scientifique, Groupe Perception et Transport de l'Auxine, Avenue
de la Terrasse, Bâtiment 23, F-91198 Gif sur Yvette Cedex, France
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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