1 Institute of Plant Sciences, University of Berne, Switzerland
2 Institute of Applied Physics, University of Berne, Switzerland
* Author for correspondence (e-mail: cris.kuhlemeier{at}ips.unibe.ch)
Accepted 7 May 2003
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SUMMARY |
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Key words: Laser ablation, Meristem, WUSCHEL, Central zone, Peripheral zone, Meristem layer, L1 layer, Lycopersicon esculentum
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INTRODUCTION |
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Genetic analysis in Arabidopsis and Petunia has
identified the WUSCHEL (WUS) and CLAVATA
(CLV) genes as key players in the specification and maintenance of
stem cells (Clark et al., 1997;
Mayer et al., 1998
;
Fletcher et al., 1999
;
Brand et al., 2000
;
Stuurman et al., 2002
).
WUS is expressed in a cell cluster in the CZ, several cell layers
below the summit. WUS function induces stem cell identity in the
overlying cells of the CZ. Because of this inductive role, the
WUS-expressing cell cluster is referred to as the organising centre
(OC) of the meristem (Mayer et al.,
1998
). A negative feedback loop limits WUS expression,
thereby preventing accumulation of excess stem cells (for reviews, see
Simon, 2001
;
Fletcher, 2002
;
Gross-Hardt and Laux, 2003
).
This negative regulation requires the function of the CLAVATA (CLV) signalling
pathway. CLV3 peptide ligand produced by the stem cells, is perceived by the
CLV1 receptor kinase which is expressed in the cells below the stem cells.
Superimposed on the functional subdivision in CZ and PZ, the meristem is
organised in layers (Steeves and Sussex,
1989; Lyndon,
1998
). The external L1 layer covers the subepidermal
L2 layer and the remaining internal tissues, referred to as
L3. The layered organisation of the meristem is maintained by
stereotyped cell division patterns in L1 and L2. This
leads to separated cell lineages that can be maintained for years
(Tilney-Basset, 1986
).
Although the layered organisation of the meristem is found in virtually all
angiosperms, its functional relevance is still unclear. Since the three
meristem layers cooperate in organ formation, some sort of communication is
required to coordinate their development
(Szymkowiak and Sussex, 1996
).
Most information on layer interactions comes from analysis of periclinal
chimeras in which one or two of the meristem layers are mutant, and the
remaining layer(s) wild type. Such studies have revealed extensive
communication between the layers. For example, in tomato flowers the number
and the size of organs is largely determined by the interior layers
(Szymkowiak and Sussex, 1992
;
Szymkowiak and Sussex, 1993
).
Conversely, in flowers of an Antirrhinum periclinal chimera, the
wild-type L1 layer could restore the mutant phenotype in the
interior layers of floricaula mutants
(Hantke et al., 1995
). In this
case, the L1 layer induced the complete developmental program in
L2 and L3 to give rise to fertile flowers. Such
inductive interactions between meristem layers are reminiscent of induction
between the three germ layers in animal embryogenesis
(Gilbert, 2000
).
Mutants have provided a wealth of information on intercellular interactions
in the meristem (Simon, 2001;
Fletcher, 2002
;
Gross-Hardt and Laux, 2003
).
However, in many cases, the phenotypes of meristem mutants are pleiotropic,
and in some mutants such as wuschel or shoot meristemless, a
normal meristem is never established (Long
et al., 1996
; Mayer et al.,
1998
). This limits the use of such mutants for studies on dynamic
intercellular interactions. In such cases, it is helpful to induce controlled
lesions that are limited temporally and spatially. Physical ablation of cells
has successfully been employed to reveal cell-to-cell communication in the
root meristem (van den Berg et al.,
1995
; van den Berg et al.,
1997
). The advantage of such experiments is that they start from a
normal meristem which can, after experimental interference, reorganise itself
according to the natural regulatory mechanisms.
Classical microsurgical experiments have shown that needle pricking of the
CZ did not lead to meristem arrest
(Pilkington, 1929;
Loiseau, 1959
;
Sussex, 1964
). In all these
studies, some kind of regeneration was reported; however, the results and
their interpretation differed considerably. Pilkington mentions briefly that
after pricking of the centre `regeneration followed in nearly every case'
(Pilkington, 1929
). Loiseau
gives a detailed description of the peripheral expansion, the regeneration of
several new meristem centres and of fasciations after destruction of the CZ
(Loiseau, 1959
). He takes
these results as evidence for the importance of the PZ, and for the
dispensability of the CZ (`La destruction des cellules apicales n'interrompt
pas le fonctionnement de l'apex; ces cellules ne sont donc pas
indispensables'). Finally, Sussex reports that after puncturing of the apex
`axial growth ceased and one of the apical flanks grew out as the new
meristem' (Sussex, 1964
).
Despite the initial differences in interpretation, these classical studies
have led to the widely accepted notion that destruction of the meristem centre
leads to the establishment of one or more new growth centres at the periphery
(Steeves and Sussex, 1989). It
is of considerable interest to interpret the surgical experiments in the
framework of the recent molecular models, and vice versa. With this in mind,
we revisited the classical surgical experiments. We used technical innovations
from the last half-century, such as tissue culture, high-resolution stereo
light and scanning electron microscopy, and laser-based ablation techniques to
increase the temporal and spatial resolution of the experiments. In addition,
a number of control experiments support the notion that the effects induced by
the ablations are not a general stress response but specifically shed light on
endogenous developmental processes. Moreover, we monitored the expression of
key developmental genes after the microsurgical manipulations and thereby
enable a link to be made between the two types of experimental approaches.
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MATERIALS AND METHODS |
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Laser ablations
Ablation of the meristem was conducted with a Q-switched Er:YAG laser that
emits infrared radiation at a wavelength of 2.94 µm. Er:YAG laser radiation
shows a high ablation efficiency and precision, which, by virtue of the high
absorption coefficient in water (absorption coefficient is about 10,000
cm-1, corresponding to an optical penetration depth of
approximately 1 µm), leads to thermally damaged zones adjacent to the
ablation side that are restricted to a few micrometers
(Frenz et al., 1996).
Q-switching was performed by a FTIR-modulator as described previously
(Könz et al., 1993
). The
pulse duration was 60 nseconds. The laser was operated at a repetition rate of
2 Hz. The radiation was guided from the laser to the operating microscope
through an optical sapphire fibre with a core diameter of 125 µm and
focused with a lens system to a spot of approximately 40 µm in diameter on
the surface of the meristem. The pulse energy used was 0.3 mJ for ablation of
the L1 layer (one pulse applied), and 1.5 mJ for deeper ablations
(1 pulse for the ablation of the stem cells, and 10 consecutive pulses for the
ablation of the entire CZ).
Cloning of LeWUS
A cDNA library was constructed using mRNA isolated from tomato meristems
and the SMART library kit (Clontech). A 350 bp fragment of the LeWUS
gene was obtained by polymerase chain reaction (PCR) on DNA from the meristem
library using Triplex F primer (Clontech) and the WUS4R primer
(5'-GCCTTCAATCTTTCCGTACTGTCT-3'), which matches the conserved
region of the Petunia PhWUS gene
(Stuurman et al., 2002)
(GenBank accession number AF481951). Based on sequence information from the
350 bp fragment, we designed the WUS10F primer
(5'-CAACACAACATAGAAGATGGTGG-3'). A 1200 bp fragment amplified with
the primers WUS10F and Triplex R (Clonetech) was cloned into pBluescript and
used for generating 35S-labelled riboprobes. The sequence of
LeWUS was deposited in GenBank (accession number AJ538329).
In situ hybridisation and microscopy
In situ hybridisations were carried out either with 35S-labelled
riboprobes as described by Reinhardt et al.
(Reinhardt et al., 1998), or
with dig-labelled riboprobes according to the method of Vernoux et al.
(Vernoux et al., 2000
). Silver
grain signal was visualised on a Zeiss LSM310 confocal microscope as described
previously (Reinhardt et al.,
1998
), and appears as yellow grains on a blue background. For
scanning electron microscopic analysis, apices were viewed with an S-3500N
variable pressure scanning electron microscope from Hitachi (Tokyo, Japan),
equipped with a cool stage. In digital images lanolin paste was pseudocoloured
for clarity. For live imaging, developing tomato apices were cultured on
plates and repeatedly photographed with a Sony DKC-5000 digital camera mounted
on a Nikon SMZ-U stereoskope. Plastic sections were prepared as described
previously (Loreto et al.,
2001
) with one modification: OsO4 was omitted. Semithin
sections (5 µm) were viewed on a Zeiss Axioskop2 equipped with an Axiocam
camera.
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RESULTS |
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Ablation of the central zone does not affect organ formation but
leads to the establishment of a new meristem centre
To reveal the function of the CZ in organ formation and meristem
maintenance, we generated lesions in the meristem centre that were
approximately 40 µm wide and 100 µm deep (compare to a meristem diameter
of approximately 150 µm). (Fig.
2A,C,D). These lesions eliminated the CZ including the
LeWUS-expressing cells in the L3 layer, which is located
approximately 50 µm below the summit of the meristem
(Fig. 2B, compare with
Fig. 3A,B). After such
ablations, leaf formation continued without delay
(Fig. 2E,G,I). Primordium
initiation rate may even have been slightly higher than in control apices,
i.e. after 3 days, apices with lesions had formed 1.95±0.4 (s.d.) new
primordia (n=15) compared to 1.54±0.29 (s.d.) new primordia in
controls (n=7). Also, the position of new primordia was normal, i.e.
new primordia diverged from the next older primordia by approximately
137°. In general, the hole closed within 2 days
(Fig. 2F). Later, the lesions
were, in most cases, gradually displaced from the meristem centre (16 out of
22; Fig. 2G,H,I). We presume
that this displacement was caused by the activation of a new meristem centre
at the flank. In 3 out of 22 cases, two new centres were initiated
concomitantly at opposite sides of the lesion, resulting in the split of the
meristem (Fig. 2J). In such
cases, leaf position sometimes becomes irregular
(Fig. 2J;
Fig. 3G). In the remaining
cases (3 out of 22), the lesion remained on the meristem. These results show
that after elimination of the entire CZ, including the
LeWUS-expressing cells, organ formation continued without an obvious
lag.
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Organ formation continued after ablations of the CZ, indicating that basic
meristem functions in the PZ were not affected. In order to confirm the
maintenance of meristem identity in the PZ, we analysed the expression of the
LeT6 homeobox gene (also referred to as TKn2), a marker for
meristem identity (Chen et al.,
1997; Parnis et al.,
1997
). In untreated controls, LeT6 was consistently
expressed in the CZ and the PZ but down-regulated in the leaf primordia and
the site of incipient leaf formation (I1)
(Fig. 3H,I), in a manner
similar to the homologous genes KNOTTED1 in maize
(Jackson et al., 1994
) and
SHOOT MERISTEMLESS in Arabidopsis
(Long and Barton, 2000
). This
is in contrast to previous reports that found LeT6 to be expressed
across the meristem, with only moderate reduction in leaf primordia
(Chen et al., 1997
;
Parnis et al., 1997
). After
ablation of the CZ, LeT6 continued to be expressed in the remaining
cells at levels comparable to those in the controls
(Fig. 3J). In parallel with the
re-establishment of one or two new meristem centres, LeT6-expressing
cells increased either on one (Fig.
3K), or on two opposite sides of the lesion
(Fig. 3L).
Taken together, we have shown that after ablation of the CZ, LeWUS is induced in cells at the flank within 1 day. This increase in LeWUS expression is unlikely to be caused by proliferation of a few LeWUS-expressing cells that might have escaped destruction. Also, the new LeWUS signal always occurred at some distance from the lesion. Therefore, the LeWUS mRNA at the flank appears to result from ectopic transcriptional induction of LeWUS in cells that did not express WUS before the ablation. LeWUS induction preceded the establishment of a new meristem centre by approximately 2 days. In parallel with ongoing organogenesis, the meristem marker gene LeT6 continued to be expressed at the periphery, even after ablation of all LeWUS-expressing cells.
Ablation in the PZ and stress treatments do not affect the function
of the CZ
A major concern with all surgical ablations is that they may cause wound or
stress responses that complicate or even invalidate the conclusions from the
experiments. For example, it could be argued that ectopic induction of
WUS, or establishment of a new growth centre, may be influenced not
only by the loss of the CZ cells, but also by wound effects from the lesions,
or by secondary stress signals. In order to exclude such effects, we performed
a number of control experiments.
First, we performed laser ablations of similar magnitude not in the CZ but in the PZ at the site of incipient leaf formation (Fig. 4A). Such ablations affected the positioning of new leaves in two ways. Either the site of the ablation was skipped and the next primordium was initiated at the next expected position (Fig. 4B,C), or the primordia were displaced to either side of the lesion (Fig. 4D,E), resulting in a smaller or larger divergence angle than expected. If ablations were performed in the centre of the youngest primordium (P1), it became split (Fig. 4F). However, we never observed a reorientation of the growth axis after ablations at the PZ, indicating that despite the strong local effects on organ formation, the ablations did not affect the neighbouring CZ or lead to establishment of a new meristem centre. To confirm this, we analysed LeWUS expression after ablations at the PZ (Fig. 4G). LeWUS continued to be expressed in an area similar to that in control meristems although the expression level tended to decrease after ablation (Fig. 4H). The fact that ectopic induction of LeWUS was not found after peripheral ablations shows that wounding per se is not sufficient to induce ectopic LeWUS expression.
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From these control treatments, it can be concluded that ectopic induction of LeWUS and initiation of a new meristem centre after ablations of the CZ is a specific response to the removal of the CZ, and not a general stress response to wounding or secondary stress signals. It follows that under normal conditions, LeWUS expression at the flank is repressed by cells in the CZ, and that this block is released by the ablations.
Ablation of the distal part of the CZ does not lead to rapid ectopic
induction of LeWUS
The ablations discussed in the previous sections removed the entire CZ
including the LeWUS-expressing cells (Figs
2,
3). To test the effect of
removal of only the distal portion of the CZ, we performed ablations at the
meristem centre that consisted of about 8 cells in diameter and reached
approximately 4-5 cell layers deep. Such ablations removed most of the cells
distal to the LeWUS-expressing cells, presumably including all stem
cells, while leaving the LeWUS-expressing cells intact
(Fig. 5A,B, compare with
Fig. 2B). We assume that the
rapid induction of LeWUS after elimination of the entire CZ is due to
the release of inhibition from cells in the centre. If this inhibition
originates from the distal cells of the CZ, the result of deep and partial
ablations would be expected to be similar, because in both cases, the distal
cells are removed. If however, cells in deeper layers were responsible for
LeWUS limitation, then the outcome of the complete and partial CZ
ablations would be expected to be different.
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The role of the L1 layer in meristem function
The previous experiments, which involved complete and partial ablations of
zones of the meristem (CZ and PZ), showed the high efficiency with which the
remaining tissues compensated for the loss by adjusting their fates. In
another series of experiments, we asked what the role of L1 is in
meristem development, and whether the layers of the meristem are equally
flexible as the zones. By single low energy pulses of the laser directed at
the summit of the meristem, small patches of cells in the centre of the
L1 layer were ablated (Fig.
6A,B). Similar to ablations of the entire CZ
(Fig. 2), such lesions did not
perturb organ formation at the periphery
(Fig. 6C-E). After 5 days, the
meristems had formed 3.28±0.93 (s.d.) new leaf primordia
(n=73), compared to controls that had formed 2.86±0.66 (s.d.)
leaf primordia (n=14), and the primordia were initiated at the normal
positions. However, the development of the cells just below the lesion was
altered (Fig. 6F). Instead of
dividing anticlinally to propagate the continuous layers of the meristem, they
started to divide predominantly periclinally, leading to the formation of cell
stacks that grew out perpendicularly to the surface
(Fig. 6F). This indicates that
the L1 layer normally exerts a restriction to such cell divisions.
Later, superficial lesions were displaced from the meristem, indicating that
the growth centre of the meristem was displaced to the flank (data not shown).
However, this shift of the growth centre appeared to be delayed compared to
the establishment of a new growth centre after ablations of the entire CZ.
After 6 days, only 51 out of 73 superficial lesions were displaced from the
meristem (70%), whereas in the case of ablations of the entire CZ after 4
days, i.e. 2 days earlier, already 19 out of 22 lesions were displaced from
the meristem (86%).
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The fact that organ formation was never observed at an area from which the L1 layer was ablated points to a special role of this layer in organ formation. However, since the loss of L1 also led to the loss of meristem characteristics in subtending cells, the defect in organogenesis could also be indirectly caused by the loss of meristem identity. To test this possibility, we followed the expression of the meristem marker LeT6. This gene continued to be expressed for several days after L1 ablation (Fig. 7K-M), but concomitantly with vacuolization and periclinal divisions, LeT6 expression faded away in the upper layers (Fig. 7M). Thus the loss of meristem identity developed more slowly than the immediate block of organ formation at the ablated site.
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DISCUSSION |
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Another important modern tool is molecular markers, which allowed us to establish tissue identities. We note, however, that the number of markers available in tomato is rather limited. Similarly, it would have been very useful to perform these manipulations in mutant backgrounds. It is unfortunate that its small and inaccessible meristem makes Arabidopsis completely unsuitable for this type of experiments. Micromanipulation experiments, such as the ones presented here, can only provide indications of novel dynamic interactions in the meristem. They can direct the further genetic and biochemical experiments that are required to build definitive molecular models.
Ablation of the CZ leads to the establishment of a new meristem
centre from the PZ but has no direct effect on organogenesis
Clonal analysis has demonstrated that the postembryonic leaves originate
from a few stem cells in the CZ (Stewart
and Dermen, 1970), emphasising the pivotal function of the CZ. The
importance of the CZ is also supported by a wealth of genetic data. For
instance, expression of the WUS gene in the CZ is necessary and
sufficient for stem cell induction and maintenance
(Mayer et al., 1998
;
Schoof et al., 2000
).
Confirming and extending classical ablation experiments, we show here that
after removal of the CZ, including the LeWUS-expressing cells by
infrared laser ablation, a new functional meristem centre is rapidly and
efficiently established from cells in the PZ. This indicates that ablated stem
cells can be replaced by cells at the periphery.
After ablation of the CZ, leaves continued to be initiated without the
slightest lag. Notably, several new leaves were formed before the new growth
centre became evident, indicating that despite the lack of stem cells, the
pool of meristematic cells was large enough to sustain organogenesis for
several plastochrons. This temporal sequence of events clearly indicates that
the CZ has no direct role in organ formation and patterning of the apex,
except as the ultimate source of cells
(Steeves and Sussex, 1989).
Mutants with perturbed CZ frequently exhibit defects in organogenesis,
however, this effect is indirect. For instance, the cessation of leaf
formation in the wus mutant is an indirect effect of stem cell
depletion (Mayer et al.,
1998
). Similarly, the irregular phyllotaxis in the
clavata mutants is likely to be an indirect effect of irregular
enlargement of the apex (Clark et al.,
1993
; Clark et al.,
1995
).
LeWUS induction in the PZ precedes initiation of a new
meristem centre
Differences in the cells of the CZ and PZ have been identified by several
different means, e.g. cytological markers, gene expression profiles, cell
division activity, organogenic capacity, etc. Consequently, the establishment
of a new meristem centre from the periphery involves the reprogramming of
cells. After ablation of the CZ, the tomato WUS homologue
LeWUS was rapidly induced in the PZ, before a new meristem centre
became apparent (compare Fig.
2E and Fig. 3D). We
propose that LeWUS expression in the PZ induced the overlying cells
to regenerate a new growth centre.
In Arabidopsis, limitation of the WUS-expressing OC is
mediated by the CLV3 signal from the overlying stem cells
(Simon, 2001;
Fletcher, 2002
;
Gross-Hardt and Laux, 2003
).
The observation of ectopic WUS induction after ablations of the CZ is
compatible with an inhibitory signal coming from the centre and acting on the
periphery. However, on the basis of this experiment, it cannot be decided
which cells in the CZ are responsible for WUS suppression. To address
this question we ablated only the distal portion of the CZ, approximately
eight cells wide and approximately four to five cell diameters deep
(Fig. 5). This treatment left
the LeWUS domain intact (compare
Fig. 2B and
Fig. 5A; 5B), but is likely to
have destroyed most if not all of the overlaying stem cells. Three days after
ablation of the distal cells, the LeWUS zone had remained
approximately the same size (Fig.
5), hence, ectopic induction of LeWUS was not observed.
It is conceivable that the partial ablations led to a slower and more moderate
induction of LeWUS or a gradual shift of the OC, which escaped
detection by in situ analysis. Alternatively, considering the large difference
between the responses to superficial and deep ablations, the cells in deeper
layers may play a special role in preventing ectopic LeWUS induction.
For instance, the LeWUS-expressing cells could inhibit LeWUS
expression in neighbouring cells by a mechanism analogous to lateral
inhibition. It is also conceivable that, as yet, unknown signals are involved.
This type of micromanipulation experiments can provide useful indications of
dynamic interactions that may remain hidden in genetic approaches. However, we
emphasise again that with the paucity of mutants and molecular markers in
tomato it is hard to arrive at conclusive molecular models.
The L1 layer controls cell division orientation and
meristem maintenance
Ablations of the L1 layer led to local changes in cell division
patterns from anticlinal to periclinal in the subtending cell layers (Figs
6,
7). This was the case
irrespective of whether the ablations affected only a limited area or the
entire meristem surface. This regular cell division pattern was clearly
different from irregular callus-like proliferation at the base of cut
primordia (Fig. 7J), indicating
that it is a characteristic feature of meristem cells. Since similar
aberrations in cell division patterns were not found in any other ablation, we
propose that this response is not a general wound response, but is
specifically due to the loss of L1. Genetic perturbation of the
embryo protodermal layer (corresponding to the L1 layer) by
L1-specific expression of a cytotoxic gene led to defects in
subtending cell layers of Arabidopsis root
(Baroux et al., 2001). In
particular, the cell division pattern was affected, resulting in supernumerary
cell tiers in the embryonic root tip, similar to the development after our
L1 ablations. Thus, the L1 layer controls cell division
patterns in subtending cell layers, and prevents periclinal cell
divisions.
Secondly, we observed a gradual loss of meristem identity, as judged by increasing cell expansion and decreasing LeT6 expression. This occurred only when most of the L1 layer was ablated. In combination with periclinal cell divisions (see above), this resulted in stacks of vacuolated cells that resembled differentiating stem cortex tissue. Therefore, L1 not only controls cell division, but also prevents cell differentiation in lower cell layers.
Role of the L1 layer in organ formation
It has been proposed that biophysical forces in the L1 layer regulate organ
formation and meristem patterning, with no necessity for specific chemical
signals in the meristem (Green,
1996). Biophysical regulation is thought to be based on tensile
and compressive forces within the meristem. According to these models, such
forces result from the geometry and the growth of the apex and operate on the
meristem (including the L1) as a whole. Although computational
modelling can recreate natural phyllotactic patterns
(Green, 1992
;
Green, 1996
), experimental
evidence for the involvement of biophysics has proved difficult to gain. In
our experiments, ablations had only local effects on organ formation and
positioning, and none of the ablations had `systemic' effects on organ
formation in unperturbed parts of the meristem. Therefore, our results do not
support a role for biophysical mechanisms in meristem patterning. However, we
emphasise that once the site of organ formation is determined, the execution
of the organogenic programme is likely to involve biophysics, particularly the
modulation of cell wall properties (Fleming
et al., 1997
; Reinhardt et
al., 1998
; Pien et al.,
2001
).
An immediate effect of L1 ablations was the complete block of
organ formation at the ablated site, although the remaining L2 and
L3 cells were still able to divide and to expand (see above). Could
the lack of organ formation be due to the loss of meristem identity in
subtending layers? We do not think so, since the loss of meristem identity
developed only gradually, and after 5 days meristematic cells were still
evident in L3. In contrast, the block in organ formation was
immediate and complete, since an organ was never formed at a site devoid of
L1. Therefore, the block in organ formation is unlikely to be an
indirect consequence of meristem degeneration, but appears to be a direct
consequence of the loss of L1. The similarity of the defects caused
by genetic L1 ablations with the phenotypes of bodenlos
and monopteros mutants may indicate a role of the L1 layer
in auxin-related patterning of the embryonic root
(Baroux et al., 2001). Since
leaf formation at the shoot meristem is controlled by auxin
(Reinhardt et al., 2000
;
Kuhlemeier and Reinhardt,
2001
; Reinhardt and
Kuhlemeier, 2002
; Stieger et
al., 2002
), the fact that leaf formation was blocked at sites bare
of L1, could point also to an auxin-related role of L1
in this process. Since CZ ablations did not affect leaf formation, it is
likely that the auxin-based mechanism operates in the PZ, but not in the
CZ.
While leaf formation in the vegetative meristem requires the L1
layer (see above), there is an influence of the lower layers in flower
development. lateral suppressor (ls) mutants of tomato lack
petals. However, a periclinal chimera with an ls mutant L1
layer and wild type L2 and L3 layers has normal flowers
(Szymkowiak and Sussex, 1993),
indicating that in this case, the L1 responded to organogenic
signals from lower layers. It is conceivable that factors from the
L1 layers, as well as factors from lower layers, are required to
allow organ formation.
Regenerative capacities of plant cells
An important feature of L1 ablations was the lack of
regeneration. In contrast to the rapid regeneration of a new growth centre
after ablation of the CZ, the L1 could never be regenerated, even
after ablations of limited extent. Although single cells that are displaced to
the L1 from the L2 layer can adopt L1
identity (Tilney-Basset,
1986), this depends on the presence of L1 neighbours.
Without information from neighbouring L1 cells, L1
identity cannot be expressed in L2 cells, therefore, de novo
formation of L1 (or the epidermis) is not possible in plants
(Bruck and Walker, 1985
).
The re-establishment of a new CZ after ablation implies rapid and efficient
regeneration of functional stem cells from organogenic cells at the periphery.
This reveals a remarkable flexibility of plant cell fate compared to animals.
It has long been assumed that adult animal stem cells cannot be replaced, once
they are lost. However, evidence is now accumulating that under certain
experimental conditions, stem cells can be (re)generated by dedifferentiation
or transdifferentiation from cells with other (more differentiated) identities
(Blau et al., 2001). However,
this occurs at low frequency and may, in some cases, be due to activation of
hidden pluripotent stem cells rather than to plasticity of differentiated
cells (Weissman, 2000
). It is
therefore a matter of debate, to what extent regeneration of stem cells is
relevant for animal development (Holden
and Vogel, 2002
). In contrast, establishment of new stem cells in
plants is clearly part of normal development. Reinitiation of stem cells in
axillary meristems is the basis for the branched architecture of plants, and
routinely allows breeders to clonally propagate plants much more easily than
animals.
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ACKNOWLEDGMENTS |
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REFERENCES |
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