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First published online September 7, 2007
doi: 10.1242/10.1242/dev.010298
1 Division of Biology, California Institute of Technology, Pasadena, CA,
USA.
2 Department of Botany and Plant Sciences, University of California, Riverside,
CA, USA.
3 Laboratorie RDP, Ecole Normale Superieur de Lyon, Lyon, France.
* Author for correspondence (e-mail: meyerow{at}caltech.edu)
Accepted 21 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Auxin, Callus, Cytokinin, Regeneration, Self-organization, Shoot meristem, Arabidopsis thaliana
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The small Cnidarian, Hydra, for example, can self-assemble a new
correctly patterned body from re-aggregated cells derived from dissociated
somatic cells of adult tissue (Gierer et
al., 1972). Recently, the observation that several genes critical
for proper embryonic development in higher animals are expressed during de
novo Hydra head regeneration has lead to important insights into the
molecular basis of animal self-organization
(Hobmayer et al., 2000).
However, animal model systems for studying de novo patterning, such as
Hydra, are not well developed for molecular analysis or genetics
compared to classical model organisms with established collections of mutants,
transgenic lines and protocols (Lowenheim,
2003; Wittlieb et al.,
2006).
Assembly of a complete organism from fragments of adult somatic tissue is
rare among animals, but many plants are capable of this type of regeneration.
A half century ago Skoog and Miller demonstrated an in vitro system for
regenerating flowering plants from fragments of adult somatic tissue
(Skoog, 1950;
Skoog and Miller, 1957).
Remarkably, the identity of induced tissues in this in vitro system was shown
to be driven by the ratio of two plant hormones: auxin and cytokinin. It was
shown that transfer of tissue explants to medium with higher levels of auxin
induced development of root regenerative tissues, whereas transfer of explants
to medium with higher levels of cytokinin induced new shoot regenerative
tissues, and inductive media containing both auxin and cytokinin induced a
proliferation of cells termed callus.
During post-embryonic development in flowering plants such as
Arabidopsis thaliana, all above ground organs of the plant originate
from stem cells within the apical tip of the shoot meristem. The origin of the
primary shoot meristem during embryogenesis can be traced back to a small
group of apical precursors (West and
Harada, 1993). Throughout embryogenesis the apical lineage is
marked by precisely regulated expression of many genes, which are required for
proper patterning of the shoot meristem
(Aida et al., 1997;
Barton and Poethig, 1993;
Laux et al., 1996;
Long et al., 1996). For
example, early patterning during embryogenesis is recognizable by expression
of the auxin transporter, PIN-FORMED 1 (PIN1), required for
the initiation and maintenance of auxin gradients within various tissues of
the plant (Friml et al., 2003;
Heisler et al., 2005). In the
two-cell pro-embryo, PIN1 expression coincides with an initial
differential activation of auxin response in the apical cell. Expression of
the homeodomain transcription factor WUSCHEL (WUS) begins in
the 16-cell stage embryo in two inner apical cells and maintains a tightly
restricted pattern throughout embryogenesis
(Mayer et al., 1998). The
dynamic expression of the redundant transcription factors CUP-SHAPED
COTYLEDON 1 and 2 (CUC1 and CUC2) and the
homeodomain transcription factor, SHOOT MERISTEMLESS (STM),
marks a small number of apical cells in the mid-globular stage embryo that are
required for meristem initiation (Aida et
al., 1997; Aida et al.,
1999; Long and Barton,
1998).
Although much is known about patterning of the shoot meristem during
embryogenesis, there is little understanding of patterning that must occur
during de novo induction of plant tissues in culture
(Cary et al., 2002;
Long and Barton, 1998). The
cell proliferation observed during callus formation ensures that the ordered
morphology of normal tissue is severely disrupted
(Cary et al., 2002;
White, 1939). Furthermore, new
shoot meristems can be induced from root-derived explants, which differ in
cell lineage, gene expression, and tissue structure from the shoot meristem
(West and Harada, 1993), thus
raising the question of how root cells react to changes in environment and
initiate patterned shoot tissues.
Live imaging of the Arabidopsis meristem has been recently applied
to the analysis of cell lineage and cell fate during active growth of the
shoot meristem, to understand genetic control of meristem size, and to cell
type specification leading to flower primordium initiation and patterning
(Heisler et al., 2005;
Reddy et al., 2004;
Reddy and Meyerowitz, 2005).
In this study we use a live imaging approach to characterize stage-specific
molecular patterning events during de novo organization of the shoot meristem
from callus (Fig. 1).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
strong wus-1 mutant allele and the strong pin1-4 allele have
been described previously (Bennett et al.,
1995; Mayer et al.,
1998).
Construction of GFP reporters
The translational protein fusion constructs including the
pPIN1::PIN1-GFP, pSTM::STM-VENUS, pREV::REV-VENUS, and
pCUC2::CUC2-VENUS constructs have been described previously
(Heisler et al., 2005). The
upstream regulatory sequence reporters including the pDR5rev::3XVENUS-N7,
pCUC2::3XVENUS-N7 and the pFIL::DsRED-N7 markers were described
previously (Heisler et al.,
2005; Sieber et al.,
2007). The transcriptional pCLV3::GFP-ER reporter was
described previously in plants bearing a construct consisting of a 35S
promoter driving 29.1 plasma membrane-localized yellow fluorescent protein
(YFP) (Reddy and Meyerowitz,
2005). The pARR5::GFP reporter in the WS ecotype has been
described previously (Yanai et al.,
2005) and was generously provided by Joseph Kieber (Department of
Biology, University of North Carolina, Chapel Hill, USA).
The previously published pWUS::mGFP5-ER construct
(Jonsson et al., 2005)
contains 3 kb of upstream and 1.5 kb of downstream WUS genomic regulatory
sequences separated by the mGFP-ER coding sequence in the T-DNA
vector pPZP222 conferring gentamycin resistance in plants
(Hajdukiewicz et al., 1994).
The pWUS::DsDed-N7 construct, also in pPZP222, is composed
of 4.4 kb of upstream and 1.5 kb of downstream WUS genomic regulatory
sequences separated by the DsRed coding region fused to the N7 nuclear
localization sequence. The pWUS::DsDed-N7 construct was transformed
into Ler harboring the pCLV3::GFP-ER reporter. The
pWUS::DsDed-N7 reporter line gave a pattern of expression confined to
the rib zone of shoot meristems and floral meristems. A putative additive
signal or strong autofluorescence was detected in the older leaves of the
pWUS::DsDed-N7 transformants, which was not found in
pWUS::mGFP5-ER transformants. Spatial expression of the
pWUS::DsRed-N7 marker was verified by semi-quantitative RT-PCR to
strictly correspond to areas of callus samples with WUS transcript
(see Fig. S1A in the supplementary material), in contrast to random samples of
callus.
The pRIBO::2XCFP-N7 construct in the T-DNA vector pPZP222
was composed of 2.6 kb of upstream regulatory sequence from the 60S ribosomal
protein L2 gene (At2g18020) fused to two tandem copies of eCFP (Clontech)
followed by the N7 nuclear localization sequence
(Cutler et al., 2000).
The pML1::GFP-ER construct in the T-DNA vector pPZP222
was composed of 3.4 kb of upstream regulatory sequence from the ML1
gene containing a fragment demonstrated to drive L1-specific expression, fused
to mGFP-ER (Sessions et al.,
1999).
The pPIN1::PIN1-CFP construct was created by substituting the CFP coding sequence for the GFP coding sequence in the published pPIN1::PIN1-GFP construct. Plants bearing multiple transgenes and the mutant alleles were combined by genetic crossing.
Regeneration conditions
Root explants were harvested from 2-week-old seedlings grown in sterile
culture on Murashige and Skoog basal salt mixture (MS) plates. Explants were
cultured on callus-inducing medium (CIM) consisting of modified Gamborg's B-5
medium (Sigma) containing 20 g/l glucose, 0.5 g/l MES (Sigma) and supplemented
with 1x Gamborg's vitamin solution (Sigma), 500 µg/l of 2,4-D (Sigma)
and 50 µg/l of kinetin (Sigma). Samples were incubated on CIM tissue
culture plates for 2 weeks. Callus samples were cut into 2 cm length sections
which were cultured on shoot-inducing medium (SIM) plates, consisting of MS
medium containing 10 g/l sucrose, 0.5 g/l MES and supplemented with 1x
Gamborg's vitamin solution, 2 µg/ml zeatin (BioWorld, Dublin, OH, USA), 1
µg/ml d-biotin (Sigma), and 0.4 µg/ml indole-3-butyric acid (IBA;
Sigma).
For quantifying shoot meristem induction, samples were cultured in tall
tissue culture plates (USA Scientific) for a further 2 weeks, at which point
the number of shoots per 2 cm callus explant was recorded. Shoots were defined
as described previously (Daimon et al.,
2003). Each experiment contained independent wild-type controls
using the same batch of medium and growth conditions.
Exogenous application of IAA
Indole-3-acetic acid (IAA) lanolin paste from Carolina Biological Supply
Company at a concentration of 500 ppm labeled with 1 µg/ml of propidium
iodide was applied directly to callus in the vicinity of developing shoot
meristems.
Imaging conditions
Callus and regenerating shoots were imaged directly on respective media.
For each marker line, at least 25 samples were imaged to confirm that observed
patterns were representative of respective markers. Propidium iodide for
staining root cell outlines of root tissues was applied to samples at a
concentration of 10 µg/ml 10 minutes prior to imaging. The
lipophilic dye FM4-64 (Molecular Probes) was used at a concentration of 10
µg/ml to demarcate cell membranes and specifically labeled
regenerating shoot tissues initiating from root-derived callus.
All imaging was done using a Zeiss 510 Meta laser scanning confocal
microscope with either a 10x air objective, 20x air objective, or
a 40x 0.8 NA water dipping lens using the multi-tracking mode. Specific
sets of filters used for the respective markers were similar to those already
described (Heisler et al.,
2005; Reddy and Meyerowitz,
2005). Projections of confocal data were exported using Zeiss LSM
software. Alternatively, volume renderings were made using Amira (Mercury
Computer Systems).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ulmasov et al.,
1997) driving expression of tandem VENUS yellow fluorescent
protein localized to the cell nucleus, pDR5rev::3XVENUS-N7. In
non-induced root explants, the DR5 reporter (green) marked root
pericycle cells, a subset of lateral root progenitors, and the distal tip of
lateral roots including columellar root cap cells
(Fig. 2A), as previously
reported (Benkova et al.,
2003). However, after 5 days incubation on CIM, proliferative
growth was marked by the DR5 reporter and was initiated in the
vicinity of lateral roots, root meristems and to a lesser extent, the root
pericycle (Fig. 2B).
DR5 response diminished over time and was not observed within large
callus outgrowths after 1 week of culture
(Fig. 2C). In addition, after
2-3 days induction on CIM, a reporter for the auxin efflux carrier
PIN-FORMED 1 (PIN1), was induced in callus outgrowths (green
in Fig. S1B in the supplementary material), but was later downregulated and
was not detected after 10 days of induction.
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). We used transgenic plants bearing ARR5 regulatory
sequences driving GFP expression, pARR5::GFP, to dynamically monitor
cytokinin response. In the non-induced root, pARR5::GFP activity was
observed in the root stele, root meristems and lateral root progenitor cells
(Fig. 2D, green). After 8 days
of induction on CIM, signal from the ARR5 reporter was detected in
the root explant vasculature and strongly marked proliferating callus cells
(Fig. 2E), and after 2 weeks of
induction had expanded throughout callus
(Fig. 2F).
A recent study using an enhancer trap for CUC1 demonstrated that
CUC1 upregulation is associated with callus formation on CIM
(Cary et al., 2002). We
determined if transcription of the partially redundant gene CUC2 is
also upregulated on CIM. Prior to induction, a reporter consisting of
CUC2 regulatory sequences driving tandem VENUS expression localized
to the cell nucleus, pCUC2::3XVENUS-N7, was active in a subset of
cells of the root vascular cylinder and lateral root primordia founder cells
(Fig. 2G, green). After 8 days
of induction on CIM, the CUC2 reporter was upregulated in small
proliferating callus cells (Fig.
2H) and was later observed throughout the callus
(Fig. 2I). By contrast,
WUS, STM and CLV3 were not expressed in callus, consistent
with previous RT-PCR data (Cary et al.,
2002), and no FILAMENTOUS FLOWER (FIL) and
REVOLUTA (REV) reporter activity was observed.
CIM contains tenfold higher levels of the synthetic auxin 2,4-D, than of the cytokinin, kinetin. We next investigated which of these hormones was responsible for callus induction and upregulation of the CUC2 reporter. Modified CIM, containing 2,4-D as the sole hormone, induced callus and CUC2 reporter expression (Fig. 2J, green). On the same medium, the cytokinin responsive pARR5::GFP reporter was upregulated at sites of callus formation (Fig. 2K). By contrast, culture of explants on CIM containing only kinetin did not lead to callus proliferation and CUC2 reporter expression was faint and did not expand outside the vasculature of the primary root (Fig. 2L). Expression of the auxin-responsive DR5 reporter also did not expand on this medium (data not shown).
Callus induction is associated with the proliferation of multipotent cell types such as cells of the root meristems, lateral root progenitors and pericycle cells. To test the hypothesis that these cells were capable of responding to respecification cues without an intermediate culture on CIM, we cultured root explants directly on cytokinin-rich shoot inducing medium (SIM). After 5 weeks, an average of 3.9±0.2 shoots were induced per 2 cm root explant, compared to an average of 5.1±0.3 after 2 weeks of culture on auxin-rich CIM followed by a subsequent 4-week induction on SIM. In addition, we observed that shoots arose from proliferating cells originating from lateral root meristems labeled by the CUC2 reporter.
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;
Sieber et al., 2007), we
investigated the spatial distribution of CUC2 protein. The expression of a
translational CUC2-VENUS fluorescent protein fusion driven by CUC2
upstream regulatory sequences, pCUC2::CUC2-VENUS was detected at low
levels within shoot progenitor cells in a similar expression pattern to that
of the CUC2 transcriptional reporter (red in Fig. S1C-E in the
supplementary material).
Downregulation of the CUC2 reporter from non-progenitor cells lead
us to question if these cells had changed identity, marked by concomitant
activation of other gene regulators. RT-PCR and oligonucleotide arrays have
previously shown that WUS expression is upregulated in callus after 3
days induction on SIM (Cary et al.,
2002). We documented the expression of a transgene containing
WUS regulatory sequences driving GFP expression localized to the
endoplasmic reticulum (ER), pWUS::mGFP-ER. The WUS reporter
was upregulated after 3 days on SIM and its expression spread throughout large
domains of callus by 5 days of induction
(Fig. 3E, green), and declined
after 10 days culture. We observed that the WUS reporter was
initially expressed in cells peripheral to shoot meristem progenitor cells but
was later upregulated within the center of the phyllotactic shoot meristems
(Fig. 3F,G). We investigated
the relative expression domains of CUC2 and WUS activity
using a pCUC2::3XVENUS-N7; pWUS::DsRed-N7 marker line. These markers
formed non-overlapping domains of activity within callus
(Fig. 3H,I). As described
above, small rapidly dividing cells labeled by the CUC2 reporter
(green) gave rise to shoot meristem progenitor cells whereas the WUS
reporter (red) was expressed in peripheral cells that did not rapidly divide
(see Fig. S1F in the supplementary material). At later stages, the
CUC2 reporter was expressed in a radial pattern and the WUS
marker was upregulated in the future rib zone of the developing shoot
promeristem (Fig. 3J).
Our results show that induction on cytokinin-rich SIM leads to partitioning of cell identity and cell behavior within callus. We next questioned if hormonal response was partitioned within the callus in similar fashion. The cytokinin responsive ARR5 reporter was expressed in areas of shoot meristem initiation and within developing shoot meristems, but was downregulated in organ primordia (Fig. 4A, green). ARR5 reporter expression was absent from areas of callus that initiated root tissues or that did not regenerate at all, but strongly labeled regenerating root meristems (see Fig. S1G in the supplementary material). By contrast, auxin-responsive DR5 reporter signal (red, Fig. 4B) was low or undetectable in areas of shoot initiation, but marked surrounding regions of callus that did not initiate shoot tissues. In areas of low DR5 reporter signal, shoot meristems and shoot promeristems were marked by expression of a PIN1 reporter (green), consisting of PIN1 regulatory sequences driving expression of a PIN1-GFP fusion protein. Higher magnification images show that PIN1-GFP expression (green) initiates in cells with low DR5 activity (blue, Fig. 4C). DR5 signal was observed within the developing shoot meristem after PIN1 reporter upregulation at future sites of leaf primordium formation (Fig. 4D). As primordia grew outward DR5 signal increased at the primordium tip (Fig. 4E).
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Pattern formation within the shoot promeristem
PIN1 reporter upregulation was associated with the morphogenesis
and patterning of phyllotactic shoot meristems from mounds of promeristem
cells. We therefore investigated PIN1 expression relative to
expression of other developmental genes: CUC2, REV, FIL, STM, CLAVATA
3 (CLV3). We documented simultaneous PIN1 and
CUC2 reporter activity using pPIN1::PIN1-GFP;
pCUC2::3XVENUS-N7 transgenic plants. PIN1 (green) was
upregulated in the superficial layers of shoot promeristem labeled by the
CUC2 reporter (red, Fig.
5A). Higher levels of PIN1-GFP were observed at future sites of
primordium initiation. As primordia initiated growth, the CUC2
reporter was expressed in the primordial-meristem boundary
(Fig. 5B). Using plants
transgenic for pPIN1::PIN1-GFP; pWUS::DsRed-N7 reporters we observed
that PIN1-GFP protein (green) was localized within the cell membrane directed
towards the apical tip of the shoot promeristem and away from non-progenitor
cells (blue, Fig. 5C).
At the periphery of the Arabidopsis meristem, organ primordia are specified with adaxial and/or abaxial polarity with respect to the shoot meristem, in part by the HD-ZIP gene REV and the YABBY transcription factor FIL. Early PIN1 reporter expression (green) was closely followed by upregulation of a pREV::REV-VENUS reporter (red) in a subset of internal cells of the developing shoot meristem (Fig. 5D). After 24 hours, as PIN1 reporter expression was upregulated at sites of primordium initiation, REV expression extended toward the adaxial side of initiating primordia (Fig. 5E) and later was observed in the adaxial side of leaf primordia (Fig. 5F). Fluorescent signal from a FIL reporter (red), pFIL::DsRed-N7, was first observed at the periphery of the early PIN1 domain and clearly demarcated early primordia (Fig. 5G), and its expression was later maintained in the abaxial sides of primordia (Fig. 5H,I).
Upregulation of PIN1 reporter expression within shoot promeristems was also associated with upregulation of the STM gene. Time-lapse imaging of transgenic plants containing reporters for PIN1, STM and WUS showed that the STM reporter (blue) is upregulated during the onset of PIN1 reporter expression (green). STM was expressed in a ring of cells surrounding the shoot promeristem and a subset of cells within the promeristem as the PIN1 reporter was upregulated in primordia initials (I1 and I2; Fig. 5J and see Fig. S1H-S in the supplementary material). After 24 hours, the PIN1 reporter marked growing primordia (P1 and P2) while the STM reporter became upregulated through the center of the shoot promeristem (Fig. 5K) and was maintained in this domain through 48 hours of observation (Fig. 5L).
Stem cells of the shoot reside at the apical tip of the meristem, marked by
expression of the CLV3 gene
(Fletcher et al., 1999;
Reddy and Meyerowitz, 2005).
We observed that CLV3 expression (green), was absent from shoot
progenitor cells, which were marked by the pPIN1::PIN1-CFP reporter
(white) in plants transgenic for pCLV3::GFP-ER, pPIN1::PIN1-CFP and
pWUS::DsRed-N7 reporters (Fig.
5M). CLV3 reporter expression appeared during
upregulation of WUS reporter expression (red) within the center of
the new meristem and the initiation of primordia (P1 and
P2) from the meristem periphery
(Fig. 5N). CLV3
reporter activity was confirmed in plants bearing a p35S::YFP 29-1
transgene (yellow), which express membrane-localized YFP within all cells of
the mature meristem (Fig.
5O).
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) and is restricted to the protodermal layer at the 16-cell
stage onwards (Lu et al.,
1996). We used a transgenic line containing
pATML1::GFP-ER and pCUC2::3XVENUS-N7 reporters in order to
understand relative timing of L1 cell-type specification with regards to
meristem organization. The ATML1 reporter was restricted to a subset
of superficial cells within the shoot promeristem marked by the CUC2
reporter (Fig. 6A,B). By
contrast, the ATML1 reporter was often not L1 specific when expressed
in callus (Fig. 6C). Primordium
initiation began after approximately 72 hours of development and was
associated with homogenous expression of the ATML1 reporter within
the protoderm (Fig. 6E). We further followed the shoot regeneration process in a pPIN1::PIN1-GFP; pSTM::STM-VENUS; pRIBO::2XCFP-N7 marker line. The pRIBO::2XCFP-N7 marker labeled all cells within a callus, enabling us to observe that shoot promeristems were composed of variable numbers of cells (Fig. 6G). Shoot promeristems composed of smaller numbers of cells developed into shoot meristems with fewer initial leaf primordia compared to larger promeristems (Fig. 6J).
Quantification of regeneration in wus-1 and pin1-4
To determine if WUS and PIN1 are necessary for efficient
initiation of new shoot meristems, we quantified the number of shoots formed
from 2 cm callus explants in the strong wus-1 and pin1-4
mutants after 4 weeks of growth on SIM. The average number of shoots formed in
the wus-1 mutant (n=91) decreased to 5% of wild-type number
of shoots (n=106; 0.25±0.08 versus 5.06±0.04), whereas
the average number of shoots formed in the pin1-4 mutant
(n=174) decreased to approximately 20% of wild type numbers
(n=166; 0.90±0.07 versus 5.16±0.24;
Fig. 7A).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-naphthalene acetic acid (NAA), induce
lateral root growth in wild-type root explants, but callus-like proliferation
in mutants for pin auxin efflux carriers
(Benkova et al., 2003).
Furthermore, a reporter for CUC3 expression was expanded in roots
simultaneously treated with IAA and the auxin transport inhibitor NPA. 2,4-D
is an auxin analog that is poorly transported by the auxin efflux system
(Delbarre et al., 1996). Our
data shows that CIM containing 2,4-D as the sole added hormone in the growth
medium is sufficient to induce callus formation, which involves proliferation
of multipotent cell types including root pericycle cells, lateral root
progenitors and cells of the root meristems. Combined, these findings suggest
that callus induction is due to an inability of root tissue to regulate auxin
distribution, leading to unrestrained proliferation of multipotent cells of
the root.
Recently, it has been shown that pericycle cells uniquely continue division
through the elongation and differentiation zones of the root after exit from
the root meristem (Dubrovsky et al.,
2000). Later, a subset of these cells gives rise to lateral root
primordia. The ability of these cells to continue division may be linked with
their enhanced response to environmental stimuli, such as the availability of
hormones. Consistent with this model, we observe that most cells initiating
and proliferating as callus are marked by expression of the auxin-responsive
DR5 and cytokinin-responsive ARR5 reporters. The enhanced
capacity to divide in response to hormone induction and the ability to give
rise to multiple cell types may explain the preferential proliferation of
these cells on CIM and their plasticity during induction of shoot tissues when
transferred to a high cytokinin environment.
The different quantitative requirements for auxin and cytokinin in order to
induce various tissues in culture is probably in part due to different
endogenous concentrations of these hormones within explants
(Skoog, 1950). Root meristems
are sites of endogenous cytokinin production
(Aloni et al., 2005;
Nordstrom et al., 2004). The
upregulation of the cytokinin responsive ARR5 reporter within callus
forming on CIM containing 2,4-D but no exogenous cytokinin suggests that
callus induced from root meristems may endogenously produce cytokinin.
Partition of cell identity and hormone response within callus during shoot meristem initiation
Previous studies have shown that mosaic overexpression of either of the
redundant transcription factors CUC1 or CUC2 is sufficient to enhance the
number of shoots initiated in culture whereas the respective mutants are
deficient in this process (Daimon et al.,
2003). Another recent study has shown that broad expression of a
CUC1 enhancer trap on auxin-rich CIM is progressively restricted
within callus upon transfer to cytokinin-rich SIM
(Cary et al., 2002). We show
similar dynamics for the partially redundant gene CUC2. In addition,
we show that CUC2 downregulation within cells during induction on
cytokinin-rich SIM is synchronized with upregulation of WUS
expression, leading to a partition of cell identity and behavior within callus
(i.e. progenitor/not progenitor). We, therefore, propose that the dynamic
partitioning of CUC2 and WUS expression may underlie the
gradual localization and promotion of shoot meristem cell fate within callus
tissue.
|
). However, CUC2 has been shown to be downregulated
in mutants defective in auxin transport (PIN1) and auxin-regulated
gene activation (MONOPTEROS) (Aida
et al., 2002; Leibfried et
al., 2005). Indeed, we observed that a CUC2
transcriptional reporter is upregulated on auxin-rich CIM medium and
downregulated on cytokinin-rich SIM medium. Furthermore, expression of
CUC2 is maintained in shoot promeristems which express PIN1-GFP,
polarized such that it is predicted to transport auxin into the shoot
promeristem from surrounding cells. By contrast, WUS is induced only
after culture on cytokinin-rich SIM medium and the WUS reporter forms
gradients of expression relative to the CUC2 reporter in
non-overlapping domains. We show that shoot meristems initiate in areas of low
auxin and high cytokinin response. Our data is therefore consistent with a
model in which gradients of auxin and cytokinin specify cell identities within
callus through induction of gene regulators.
WUS and WOX genes in diverse regeneration processes
Our observations of WUS reporter expression in callus is
consistent with previous studies which have described ectopic induction of
WUS during cell respecification after cell ablations in the shoot
meristem (Reinhardt et al.,
2003). Furthermore, the WUSCHEL related homeobox 5 gene
(WOX5), normally active in the quiescent center (QC), is ectopically
induced in surrounding cells after QC ablation in the root meristem
(Haecker et al., 2004;
Xu et al., 2006). In addition,
mosaic over-expression of WUS has been shown to induce shoot tissues
directly from root explants (Gallois et
al., 2004). Thus it appears that broad induction of WUS
and related WOX genes may be a general phenomenon associated with
regeneration of specific tissues in plants.
Necessity of WUS and PIN1 function for proper shoot formation
The strong wus-1 mutant regenerated only 5% of the number of
shoots observed in wild-type samples and WUS expression was required
for initiation of wild-type numbers of shoot promeristems, marked by
coexpression of the PIN1 and STM markers. These data support
a model in which early WUS expression within callus is required to
promote shoot meristem progenitor cell identity, and late WUS
expression is required for further shoot development. However, once shoot
promeristems are initiated, they are largely autonomous in their development
and express PIN1 and STM in a pattern that is initially
similar to that of wild type. Other factors may compensate for loss of
WUS function to initiate shoot promeristem development, such as
members of the WOX gene family or ENHANCER OF SHOOT
REGENERATION (ESR1), which confers cytokinin-independent shoot
regeneration (Banno et al.,
2001). The pin1-4 mutant was also deficient in shoot
regeneration, though this was not as severe as in wus-1 mutant
tissue. The pin1-4 deficiency produced a phenotype that was similar
to that previously reported for stm-1 mutant tissue
(Barton and Poethig, 1993).
PIN1 activity may be more dispensable for shoot induction than
WUS, because of its greater redundancy including other PIN proteins
(Vieten et al., 2005),
consistent with higher levels of NPA-blocking shoot regeneration
(Christianson and Warnick,
1984; Murashige,
1965) and redundancy of PIN family members during
embryogenesis (Friml et al.,
2003).
|
). We,
therefore, can break the shoot organization process into distinct events:
callus induction, cytokinin-induced partition of cell identity within the
callus, radial patterning within shoot progenitors, and meristem morphogenesis
(Fig. 8).
Classical tissue culture methods for studying developmental patterning
Over a century ago, Haberlandt noted the possible utility of tissue and
cell culture for understanding development. He pointed out that cell culture
was particularly well suited to determine the potential of individual cells as
well as their reciprocal influences on each other
(Haberlandt, 1902). Our study
represents an early step towards realizing this potential. In vitro culture
experiments support the idea that cell identity in plants is largely governed
by positional cues mediated by specific hormones
(Steward et al., 1964). We
propose a model in which partition of cell identity within a callus on SIM is
mediated through non-homogeneous distributions of auxin and cytokinin, which
are initially broadly distributed and therefore induce broad CUC2 and
WUS expression, respectively. The expression of these genes may
further feed back on hormone synthesis, transport or perception, to enhance
gradients of hormone signaling, which then alters CUC2 and
WUS expression. This feedback could lead to self-organizing patterns
observed during de novo shoot meristem initiation. If this is the case, the
primary difference between shoot meristem initiation in planta and shoot
meristem induction in culture is the initial distribution of auxin and
cytokinin. Auxin and cytokinin distribution is tightly controlled at all
stages during development in planta, whereas this distribution must be
gradually reorganized from disrupted initial conditions during shoot induction
in culture. In vivo imaging of this dynamic process during gene and hormone
perturbations should test the validity of this model.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/19/3539/DC1
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ACKNOWLEDGMENTS |
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