Download Leaf initiation: the integration of growth and cell division

Ó Springer 2006
Plant Molecular Biology (2006) 60:905–914
DOI 10.1007/s11103-005-7703-9
Leaf initiation: the integration of growth and cell division
Andrew J. Fleming
Department of Animal and Plant Sciences University of Sheffield, Western Bank, S10 2TN, Sheffield, UK
(author for correspondence; e-mail a.fleming@sheffield.ac.uk)
Received 26 January 2005; accepted in revised form 21 May 2005
Key words: cell division, cell growth, development, leaf, morphogenesis
Abstract
The shoot apical meristem of higher plants is characterized by a conserved pattern of cell division, the
functional significance of which is unclear. Although a causal role for cell division frequency and orientation in morphogenesis has been suggested, supporting data are limited. An alternative interpretation
laying stress on the control of growth vector and its integration with networks of transcription factors and
hormonal signals is discussed in this review.
Introduction
The shoot apical meristem (SAM) is distinguished
by its position within the plant, its ontogeny and
its cellular organization. Histological observations
in a number of plants show that the SAM is
characterized by the following cellular pattern: an
outer layer (or layers) of cells in which cell division
is predominantly anticlinal (termed the tunica) and
an inner body of tissue (corpus) in which cell
division orientation is more random (i.e., not
restricted to anticlinal) (reviewed in Steeves and
Sussex, 1989). An example of this organization is
shown for a tobacco SAM in Figure 1A, B. This
commonality of form raises the questions: how
does such an organization arise and what is its
functional significance? In this review, I will
discuss both these issues and attempt to integrate
knowledge obtained from recent approaches
manipulating parameters of cell division and
growth into an understanding of how the SAM
functions as a centre of morphogenesis in the
plant. In particular, I will take the viewpoint that
aspects of biophysics can be used to interpret
many of the observations that have been made.
The acquisition of the tunica/corpus organization in
the SAM
The SAM first becomes identifiable at the late
globular stage of embryogenesis via expression of
the STM marker gene (encoding a homeodomain
protein) in a region between the developing
cotyledons (Long et al., 1996). As development
proceeds, the small dome-shaped structure characteristic of the SAM becomes apparent and this
occurs concomitantly with the cellular patterning
diagnostic for the tunica/corpus organization.
During this process, various gene expression patterns are established within the SAM which are
indicative of and required for normal meristem
function (reviewed in Veit, 2004). How or whether
these SAM-specific patterns of gene expression
influence the cellular patterning characteristic of
the SAM is unclear, although (as will be discussed
later) some workers have made interesting correlations between the expression of specific homeodomain proteins and the flexibility (or otherwise)
of cell division patterns. One alternative possibility
is that the specific cellular patterning observed in
the SAM is not a direct outcome of the specific
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Figure 1. The shoot apical meristem shows a pattern of cell division. (A) Longitudinal section through a tobacco SAM. (B) The
cellular outlines of the SAM in (A) have been couloured to indicate the tunica layer (red) in which cell division is anticlinal and
the corpus (blue) in which cell division is not restricted to an anticlinal orientation. (C) The same cellular outline has been coloured to indicate the clonal nature of the different cell layers in the SAM. The pattern of anticlinal cell divisions in the outer layers means that tissue derived from the LI layer (red) the LII layer (blue) and the LIII layer (white) are clonally distinct. (D)
Longitudinal section through a tobacco SAM in which cell division orientation has been locally disrupted by overexpression of a
gene encoding phragmoplastin. (E) Cellular outline of the SAM shown in (D). (F) As in (E) but the outer cell layers have been
coloured to highlight the abnormal pattern of cell division, in particular the difficulty of assigning the mauve coloured cells to a
particular layer (compare with (C)). (Data adapted from Wyrzykowska and Fleming, 2003).
patterns of gene expression but rather a consequence of the specific growth form instigated by
these gene products.
The classical SAM is a dome structure composed of cells which are undergoing consecutive
rounds of growth and cell division. For a given
increase in meristem mass, cells located at the
surface are liable to undergo growth preferentially
perpendicular to the radius of the dome to provide
the increased surface area required to maintain the
meristem geometry. Maintenance of cell division
in an anticlinal orientation would allow this to
occur, providing a supply of new cells to undergo
expansion to generate area. This concept invokes
the ability of plant tissue to respond to specific
patterns of growth-associated physical stress by
adjusting the preferred orientation of cell division.
There are indeed data from in vitro cultured and
intact tissue that cell division orientation can be
modulated by the vector of impinging forces (e.g.,
Linthilac and Vescky, 1984; Wyner et al., 1996).
However, the most convincing data showing that
biophysical stress pattern can initiate biologically
relevant responses in term of both cellular organization and gene expression come from the
animal field. Thus, using microfabricated matrices
which allow the controlled, measured application
of force to cultured cells and, simultaneously, the
measurement of cellular response in terms of
cytoskeletal organization and gene expression, it
has been shown that applied force can elicit
specific, reproducible outcomes (Tan et al., 2003;
Ingber, 2003). Whether plant cells/tissues show
similar responses remains to be demonstrated, but
the application of such approaches in plant biology is overdue.
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In biophysical terms, in the example given
above the growing meristem surface would take up
a minimal surface energy conformation (Green,
1992). Although there are few experimental data to
support the idea that such minimal energy conformations are causally involved in plant tissue
architecture, it is interesting to note that recent
research in the field of animal biology indicates
that, at least in some circumstances, the formation
of cellular arrangement does indeed seem to be
driven by such processes. For example, the drosophila compound eye is composed of many
repeating units, termed ommatidia. At the centre
of each ommatidium lies a group of four cells
arranged in a characteristic pattern. Modelling of
minimal surface energy conformations shows that
the endogenous pattern reflects the optimum
solution to reducing tensile stress between the
four cells. Mutations exist which result in fewer or
more cells in this region and this leads to new
cellular pattern. The patterns produced varies
depending on the number of cells involved, but
any specific pattern formed is found to be that
predicted to result in minimal surface energy
within the group of cells (Hayashi and Carthew,
2004). Such data suggest that groups of cells can
respond to and alter their relative arrangement to
form particular patterns of physical stress. Again,
investigation of the situation in plant tissues has
been limited.
The functional significance of the tunica/corpus
organization
Irrespective of the mechanism by which the tunica/
corpus organization is achieved, it has a significant
outcome on the destination of cells derived from
the SAM. The layered structure leads to a constraint on the final position that daughter cells can
take up in the plant body. Thus, cells in the plant
epidermis are derived from the outer layer of the
SAM (LI), cells in the sub-epidermal layers from
the inner tunica layer of the SAM (LII) and the
innermost tissue of the plant from the innermost
tissue of the SAM (LIII) (Figure 1C). Does this
distinct clonal origin of different cell layers have a
significance for the functioning of the plant? The
evidence is mixed.
Early work using genetic chimeras created by
grafting techniques indicated that the genotype of
particular layers of the plant could influence the
phenotype of whole organs. For example, in
flowers of Camellia the presence of stamens and
pistils seemed to be dictated by the genotype of the
epidermal LI layer (reviewed in Szymkowiak and
Sussex, 1996). Such experiments supported biophysical views of the importance of the epidermis
in controlling plant growth. As will be explained
later in this article, theoretical considerations of
the physical stress patterns generated in growing
plant tissue suggest that the outer cell layers of any
young organ are likely to be under physical
tension. Regulation of tissue response to this
tension would afford a mechanism for the control
of growth and would place special emphasis on the
biophysical/molecular parameters of the outer cell
layers of the plant. However, experiments looking
at other characteristics in chimeras (such as floral
organ number) indicated that often the inner layer
genotypes (LII, LIII) played a major role in
determining phenotype (Szymkowiak and Sussex,
1992). The interpretation of some of these analyses
is complicated by the fact that it is not always clear
to what extent each layer contributed to the organs
under analysis, but it is clear that it is not always
the LI layer genotype that controls plant phenotype. The possible importance of the LI layer in
morphogenesis will be returned to later in this
review.
More recent molecular and cell biological
approaches have revealed a potential mechanism
to explain some of these classical observations on
chimeras. The key to these new approaches has
been the realization that macromolecules (most
notably transcription factors) have the potential to
move from cell to cell via plasmodesmata in a
regulated fashion (Gillespie and Oparka, 2005).
The first indications that such a mechanism of
intercellular communication is possible came from
work on the homeodomain protein KNOTTED-1.
Observation of RNA and protein localization in
the SAM revealed a discrepancy which suggested
that the protein might move between cells. Later
microinjections studies and experiments in which
transgene expression was directed to different cell
layers indicated that indeed the KN1 protein can
move between layers in the SAM (Kim et al., 2002,
2003; Lucas et al., 1995). These data provide a
neat explanation for earlier work on this gene
which demonstrated that although the phenotype
involved altered cellular patterning in the maize
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leaf epidermis, the phenotype depended on the
genotype of sub-epidermal cells (Hake and Freeling, 1986). Following on from these initial studies,
work on other transcription factors has revealed
that inter-cellular movement is not specific to the
KN1 protein. For example, analysis of chimeras of
Antirrhinum in which the MADS box gene DEFICIENS (DEF) is expressed only in the LII and
LIII layers led to DEF protein accumulation in all
three layers of the floral meristem, implying
protein movement. Interestingly, reciprocal chimeras in which the DEF gene was only expressed
in the LI layer did not lead to DEF protein
accumulation in the internal layers, suggesting a
restriction in polarity of DEF protein movement
(Perbal et al., 1996). On the other hand, a careful
analysis of the LEAFY (LFY) transcription factor
revealed that the protein could move from the LI
to the inner cells of the SAM, indicating that there
is no general restriction on polarity of movement
of proteins within the SAM (Sessions et al., 2000;
Wu et al., 2003). Indeed, the work on the LFY
protein suggested that movement of this transcription factor occurred passively throughout the
SAM with no particular domain involved in, for
example, targeting of the protein. This contrasts
with investigation of the KN1 protein which
indicated that specific regions of the protein were
involved in intercellular movement.
Taken together, the overall picture of intercellular protein movement in the SAM is still
somewhat confused. Generally, the data support
the concept that plasmodesmatal (PD) connections
can act as an intercellular pathway for information
flow and, moreover, that this flow can be regulated
(Foster et al., 2002). However, the mechanism
involved and whether different proteins might
move via different specific pathways remains to be
elucidated.
Ignoring this issue of mechanism, one main
question relating to the observed potential of
intercellular communication between cells and
layers within the SAM is its biological relevance.
Does it provide the plant with a precise way of
channeling information? Alternatively, is it a
backup system which ensures that groups of cells
contain the same information, thus ensuring that
groups of cells take on identities delineated by the
regional expression of specific combinations of
transcription factors? Or are the observations that
have been made in some way artefacts of the
experimental approaches taken, i.e., they indicate
a potential which is only of significance in the
experimental context inflicted on the plant? At
present, the jury is still out. However, if the
concept of a PD-mediated information ‘‘superhighway’’ within the SAM is true, then the
observed conserved cellular patterning in the
SAM would have immediate impact. Primary
PDs are only formed in the newly formed cell
plate. Therefore, in a layer of cells dividing
anticlinally, all cells in the layer are connected by
primary PDs whereas symplastic connection with
an underlying layer requires the formation of
secondary PD. Since primary and secondary PD
are probably not functionally equivalent (Oparka
et al., 1999), the different ontogeny of the PD
connections between cells in the SAM could
provide a mechanism by which different groups
of cells form different potential networks of
communication. Two final points in this discussion
(which may not clarify but rather add to the
debate) are the observations that auxin flux within
the SAM (involved in patterning of leaf initiation)
occurs predominantly in the outer cell layers
(although most probably not via PDs) (Reinhardt
et al., 2003a) and that physical ablation of the LI
layer blocks leaf initiation (Reinhardt et al.,
2003b). These data again suggest that the layered
cellular structure of the SAM has functional
significance.
Manipulation of cell division pattern in the SAM
Although the SAM displays a characteristic
pattern of cell division, the pattern is not constant.
In particular, it has long been noted that at (or just
prior) to leaf initiation, a change in pattern is
observed at the site of presumptive leaf formation
(reviewed in Lyndon, 1990). Careful analysis
indicated that this altered pattern reflected not so
much an increased frequency of cell division,
rather a relaxation of the restriction of anticlinal
cell division in the inner tunica layer(s). These
observations raise the question of whether the
altered cell division pattern associated with leaf
initiation plays a causal role in the process. A
number of data suggest that this is not the case.
For example, mutants in which cell division
orientation throughout the plant is altered can
lead to a relatively mild phenotype (Smith et al.,
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1996). Even when growth is severely disrupted by
mutations which alter cell division orientation, the
plant can still generate a structure in which
morphogenesis is recognizably occurring (Traas
et al., 1995). These data suggest that if cell division
orientation is normally involved in leaf initiation,
the plant contains some mechanism for coping
with its disruption. Experiments in which cell
proliferation has either been promoted or
repressed throughout the plant also indicate that
leaf initiation, and morphogenesis in general, is
not dependent on a particular set pattern of cell
division. For example, promotion of cell proliferation by overexpression of a cyclinD2 led to an
increased growth rate but plant form was normal
(Cockcoft et al., 2000). Repression of cell proliferation can lead to the generation of smaller plants
(e.g., by overexpression of E2F/DP factors or
CDK inhibitors (De Veylder et al, 2002) or the
formation of plants of normal size (e.g., by
overexpression of a dominant negative form of
CDK (Hemerley et al., 1995)), however in all cases
plant morphology is hardly affected. The average
size of the constituent cells of these plants can be
influenced by these manipulations, but the overall
form of the organs produced is not drastically
changed, and the initiation of the organs appears
to be totally normal.
Most of these manipulations have involved
changing cell division rates and patterns throughout the plant at all stages of development, raising
the question of whether gradients of cell proliferation were maintained against an altered background level of cell division. Experiments performed in my own group addressed this issue by
locally and transiently promoting cell proliferation
in the SAM (by overexpression of a cyclinA and a
cdc25 protein) (Wyrzykowska et al., 2002). Leaf
initiation was neither promoted nor inhibited by
this manipulation. Similarly, disruption of the
anticlinal pattern of cell division in the tunica layer
via overexpression of a gene encoding phragmoplastin (which disrupts phragmoplast formation)
did not alter the ability of the SAM to form
normal leaves (Figure 1D–F) (Wyrzykowska and
Fleming, 2003).
It should be noted, however, that some data
show that altered expression of cell cycle genes
does alter plant morphogenesis. For example,
when a cyclinD3 gene was over expressed throughout the plant abnormal leaves were generated
(Riou-Khamlichi et al., 1999). Since cyclinD proteins lie at the interface of various inputs to the cell
cycle, it is possible that manipulation of expression
of some of these proteins might feed back to these
inputs. Indeed, the manipulation of cyclinD3
expression described above was shown to influence
plant response to the growth regulator cytokinin.
Such effects might lead to numerous outcomes on
SAM function in addition to those directly related
to cell proliferation. For example, recent data have
implicated cytokinin in the control of meristem
size and function (Giulini et al., 2004), thus any
manipulation altering cytokinin perception is
likely to have a profound influence on meristem
function.
Taken together, the simplest interpretation of
the data available is that cell division frequency
and orientation in the SAM are not causally
involved in leaf formation. Does this mean that
cell division is simply a means by which the plant
volume is split into compartments to allow for
biophysical integrity and for cellular differentiation (Kaplan, 2001)? A number of data suggest
that this is not the case. Instead, it seems that cell
division pattern during the early stages of leaf
initiation may be closely related to the patterns of
transcription factor gene expression that occur at
the onset of organogenesis and that the downstream targets of these transcription factors could
act to set or promote particular patterns of growth
and cellular patterning required for appropriate
differentiation within the leaf.
One of the most conspicuous patterns of gene
expression observed in the SAM is the absence of
transcripts encoding members of the KNOX
(homeodomain-encoding) gene family at the presumptive site of leaf formation (Jackson et al.,
1994). This coincides with the region of altered cell
division pattern describe above. Based on a series
of observations of cellular patterning in leaves
following ectopic expression of KNOX genes,
Matsouka and colleagues suggested that such
homeodomain proteins might play a regulatory
role in maintaining the flexibility of cell division
within organs (Sato et al., 1996; Tamaoki et al.,
1997). Moreover, further work demonstrated that
certain KNOX proteins can directly interact with
the promoter region (and repress expression) of a
gene encoding a GA20-oxidase, which catalyses a
key step in biosynthesis of gibberellic acid (GA)
(Sakamoto et al., 2001). A significant body of
910
evidence indicates that GA can influence the plant
cell cytoskeleton and, thus, the vector of plant cell
expansion, although there is some discussion on
the mechanism involved (Wasteneys, 2000). Thus,
it is highly plausible that KNOX proteins can
influence cell division pattern via modulation of
GA levels (Hay et al., 2002). In this scenario, the
KNOX proteins would not dictate the orientation
of cell division directly, rather they (or their
absence) would provide the permissive signal to
allow GA-mediated cell expansion to occur. The
actual outcome of elevated GA on cell division/
expansion is likely to be highly context dependent,
i.e., different tissues would respond differently to
the same input of altered KNOX/GA switching. In
the context of the SAM, two questions arise: Why
does the expression of particular KNOX genes
decrease at the site of presumptive leaf formation?
Why does the permissive condition for GAmediated expansion in the SAM lead to growth
in a new direction (i.e., perpendicular to the
surface of the SAM)? With respect to the decrease
in KNOX gene expression, the likeliest explanation is that these genes are targets for the flux of
auxin predicted to occur at the site of presumptive
leaf formation, although this has yet to be shown
(Reinhardt et al., 2003a). With respect to the new
vector of growth, a number of data indicate that
the architecture of the cell wall plays a key role, as
discussed in the next section.
The role of the cell wall in providing the growth
vector for leaf initiation
Plant growth is essentially a matter of biophysics
(Green 1992, 1994, 1999). It depends on the
equilibrium of internally generated turgor pressure
and tensile forces within the cell wall which
restrain the tendency of the cell to expand and
(eventually) explode. This is shown diagrammatically in Figure 2. Each cell within the SAM
generates a turgor pressure which acts equally in
all directions. Cells within the SAM thus both
exert and experience force on all sides. Due to
symplastic connections between cells, this hydrostatic pressure will tend to equalize itself throughout the body of the SAM. However, cells located
at the epidermis (LI layer) lack cellular neighbours
towards the outside. To balance the outward
acting forces, a tensile stress must develop within
the outer cell walls to counteract and balance the
internally generated force. It is noticeable that in
real SAMs the outer cell wall of the LI layer is
thickened, as expected of a structure designed to
contain or withstand relatively high tensile stress.
In the diagram in Figure 2A the tissue is in
equilibrium, i.e., the total outwardly acting forces
are balanced by the tensile stress in the outer cell
walls. If, however, the architecture of this wall
were locally altered so that the walls in this region
became weaker (less resistant to the tensile force
developed within it), then the tissue would tend to
bow outwards as a consequence of the internallygenerated outwardly acting forces (Figure 2B).
Provided there was a mechanism by which the
architecture of the cell walls could re-establish its
previous stress-resistant quality, then (after a
certain amount of give in the material) a new
biophysical equilibrium would be established but
the tissue would have a new shape, i.e., morphogenesis would have occurred. In the context of the
SAM, such a mechanism would provide a new
vector of growth upon which the release of
KNOX-mediated repression of GA biosynthesis
could act to fix and amplify outward growth of a
new leaf primordium. However, does such a
biophysical mechanism exist in plants? A number
of data suggest that it does.
Although our understanding of the control of
plant cell wall architecture is still very much
incomplete, a number of investigators have identified components of the cell wall that can modulate its extensibility. The prime candidate as an
endogenous regulator of cell wall extensibility is
the protein expansin. Expansins are encoded by
relatively large gene families in all higher plants so
far investigated. Although their mechanism of
action remains obscure, they have the ability to
loosen the cell wall matrix to increase its extensibility (Cosgrove, 2000) and specific members of the
expansin gene family are expressed at the site of
presumptive site of leaf formation (Cho and
Kende, 1998; Reinhardt et al., 1998), i.e., they
are present at the appropriate time and place to act
at the very earliest stages of leaf formation.
Moreover, local overexpression of an expansin
gene is sufficient to induce morphogenesis at the
SAM in a manner consistent with the model
portrayed in Figure 2 (Pien et al., 2001). Although
data showing that lack of expansin gene expression
blocks leaf formation are lacking, the results so far
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(A)
LI
(B)
LI
(C)
LI
Figure 2. Local loosening of the SAM cell wall is sufficient to induce morphogenesis. (A) A theoretical group of cells towards the
edge of the SAM dome. Each cell generates an internal, force (turgor) indicated by the crossed-arrows. This acts outwards equally
in all directions. Cells located internally both exert and experience a force in all directions. Cells located in the outer layer (epidermis, LI) lack neighbours on one side. The outward acting force of these cells in this direction is balanced by the development of a
tensile force within the outer cell walls. The forces are in equilibrium and no growth occurs. (B) If the architecture of the cell wall
is locally altered (red colour) so as to loosen the cell wall, then the restraining tensile force within this region of the wall will be
dissipated. (C) As a consequence, providing the cells in this region have sufficient growth potential (e.g., sufficient energy and
materials for growth), the tissue will bulge outwards until a new biophysical equilibrium is achieved, i.e. sufficient tensile stress is
attained in the outer cell walls to restrict growth.
strongly suggest that modulation of cell wall
extensibility at the site of leaf formation (presumably downstream of the auxin patterning process)
provides the differential growth impulse for leaf
initiation.
One consequence of viewing the initial steps of
leaf morphogenesis from a biophysical perspective
is the observation that, since the orientation of cell
division in plant cells tends to occur perpendicular
to the principal axis of growth, the new vector of
growth associated with leaf formation will automatically lead to a new pattern of cell division. As
shown schematically in Figure 3, in the outer
layers of the SAM growth occurs preferentially
parallel to the surface plane of the tissue. As
discussed in the first part of this article, this might
simply be a consequence of the biophysical stress
patterns in the growing dome which necessitate
that the surface tissue maintains a high rate of
surface area increase to maintain the integrity of
the dome structure. This pattern of growth will
lead to the orientation of cell division in these
surface cells being preferentially anticlinal. If the
vector of growth switches to perpendicular to the
surface of the SAM, then the cells within the
growing bulge will now tend to divide in a
periclinal orientation (Figure 3B). Does this switch
in cell division orientation have an outcome for the
tissue in which it is occurring? As outlined above, a
change of cell division pattern is not necessary for,
nor does it disrupt, leaf initiation. However, such
new cellular patterns are likely to lead to new
patterns of PD connections. Although the functional significance of PD connections within the
SAM is still being unraveled (as discussed in a
previous section), this might present a mechanism
which ensures that groups of cells entering a
particular developmental pathway share relevant
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(A)
LI
(B)
LI
Figure 3. A new vector of growth can feed back onto the pattern of cell division and the potential pattern of intercellular
contacts. (A) A theoretical group of cells towards the edge of
the SAM dome. Growth is restricted parallel to the surface
plane of the tissue (large arrows). New cell walls are laid perpendicular to the vector of growth, thus transport through
primary plasmodesmata will be restricted within the cell layers (double-headed arrows). (B) As a consequence of the new
vector of growth which defines leaf formation (large arrow),
cell division orientation within the tissue is altered, i.e., periclinal divisions occur. Primary plasmodesmata within these
new cell walls allow new potential pathways of information
flux.
transcriptional information. Thus, the switch in
cell division pattern which must accompany leaf
initiation could provide a fool-proof mechanism to
ensure that the cells within the new organ form a
PD network-defined entity separate from the
SAM. Actual evidence that altered cell division
in the SAM has a consequence on gene expression
pattern has come from work in my own group
(Wyrzykowska and Fleming, 2003). Disruption of
anticlinal cell divisions in the tunica via local
overexpression of phragmoplastin led to a local
decrease in transcripts encoding a Knotted-like
transcription factor (NTH15) and, simultaneously,
accumulation of transcripts encoding a Phantastica-like transcription factor implicated in leaf
formation. Similar changes were also observed
when cell proliferation was locally promoted in the
SAM. The mechanism by which such altered
patterns of cell division and gene expression are
linked remain totally unclear, as is their functional
significance (since in both instances meristem
growth and leaf formation appeared unimpaired).
Nevertheless, these data demonstrate the potential
for information flow not only from the gene to cell
division but also from cell division pattern to the
gene.
The above model lays special emphasis on the
outer layers of the SAM and recent data based on
micro-ablation of cells within the SAM have
provided evidence that the physical integrity of
the LI layer does indeed play a special role in
organogenesis and that the LI layer influences cell
division pattern in the internal layers. Thus,
physical removal of the LI layer prevents leaf
initiation at that site and, at the same time, leads to
growth in that region which is accompanied by
periclinal cell divisions (Reinhardt et al., 2003b).
These data are consistent with the idea that the LI
layer is somehow intimately involved in the
process of leaf morphogenesis, that it normally
acts to restrict outward growth, and that increased
periclinal cell divisions are linked to and are a
consequence of outward growth in the SAM. In
the context of the model put forward in Figure 2,
removal of the outer cell layer would lead to a loss
of the physical restraint on growth provided by
this layer, thus outward growth is the predicted
(and observed) result. In the context of the model
shown in Figure 3, such outward growth should
lead to the occurrence of periclinal divisions, again
as observed. As to the question of why a leaf
primordium does not result from this initial
impetus for outward growth by layer ablation,
then it seems that the LI layer plays an additional
role to that of a biophysical restraining wall, with
epidermal-localised auxin flux the prime suspect
(Reinhardt et al., 2003a; Fleming, 2005).
Summary and future directions
Classical views of the SAM have focussed on the
role of cell division in the SAM. More recently,
molecular genetic studies have identified many of
the components involved in regulating meristem
function in terms of control of meristem growth
and leaf formation. In many of these studies, cell
division has been assumed or indirectly implicated
as the downstream target of these regulatory
systems. In reality, many of the observations can
be better interpreted as a consequence of altered
growth parameters in the SAM. Although growth
and cell proliferation are tightly coupled in the
913
SAM, they are not synonymous terms. Indeed, as
outlined in this review, causal data showing that
cell division frequency or orientation is directly
linked to the process of morphogenesis and leaf
formation are very limited. An alternative interpretation laying stress on the control of the vector
of growth provides a simpler explanation for the
observed data. Unfortunately, our understanding
of the molecular control and integration of this
basic parameter remains astonishingly limited,
both in plants and animals (Rudra and Warner,
2004). Research directed at deciphering the linkage
between transcriptional or hormonal regulators of
SAM function and non-cell division based target
processes may provide the key insights required
for future progress.
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