Download The co-ordination of cell division, differentiation and morphogenesis

Journal of Experimental Botany Advance Access published November 29, 2005
Journal of Experimental Botany, Page 1 of 8
doi:10.1093/jxb/eri268
FOCUS PAPER
The co-ordination of cell division, differentiation and
morphogenesis in the shoot apical meristem: a perspective
Andrew J. Fleming*
Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
Received 13 May 2005; Accepted 14 July 2005
Whether morphogenesis is cell division-driven or
organismal-based has been a long-running debate in
plant biology. This article is a summary of a series of
experiments aimed at distinguishing these alternate
views by local manipulation of parameters of cell
division frequency, orientation, and growth within the
shoot apical meristem. These data, put in the context
of other investigations in this area, support an organismal view of plant morphogenesis and support the
idea that the cell wall plays a key role in the mechanism by which this is achieved. At the same time, the
data indicate that the intimate but variable relationship
between cell growth and division within the organism
means that cell proliferation can indirectly influence
this process, leading to a context-dependent influence
on morphogenesis. Finally, cell growth and proliferation are intimately related with the process of differentiation as cells exit the meristem. In the final part of
the article the molecular mechanism by which these
basic cellular parameters are intertwined is discussed.
Key words: Cell division, cell wall, leaf, meristem, microinduction, morphogenesis.
Introduction
The first description of the cellular nature of biological
material was made over 300 years ago by Robert Hooke.
Although the concept that plant material consisted of
discrete compartments became rapidly accepted, it was
not until the middle of the 19th century that it became clear
that animals too were multicellular in nature. Since then the
cellular basis of life has become a central dogma which
underpins our understanding of biology. It is, therefore,
perhaps surprising that there are still some basic questions
about cells which remain open to debate. In particular, it
remains very unclear how cell size and number are integrated into the whole organism and what precise role cells
play in driving growth and morphogenesis (Day and
Lawrence, 2000; Nijhout, 2003; Rudra and Warner, 2004).
In plants, new cells can be generated essentially throughout the organism but specific organs (the meristems) are
sites of high rates of cell proliferation. More importantly,
the cells of the meristem are pluripotent so that their
progeny can become committed to a spectrum of developmental fates. With respect to the shoot apical meristem
(which will be the focus of this article) there is the added
complication that cells derived from the meristem do not
simply undergo particular patterns of differentiation, they
may also become incorporated into new structures, the
leaves. Thus, the process of differentiation must be coordinated with the process of morphogenesis if a fully
functional organism is to be produced. The relationship
between cell proliferation and differentiation, and the
connection between cell proliferation and morphogenesis,
will be discussed in the remainder of this article.
Cell proliferation and morphogenesis:
is there a causal link?
From simply looking at a section through an apical
meristem, the mechanism involved in generating a new
leaf primordium seems obvious (Fig. 1A). Surely cell proliferation simply increases at the presumptive site of leaf
formation and this leads to the formation of a new organ?
From this simple viewpoint cell division is driving morphogenesis. However, although cell proliferation is certainly associated with leaf formation (Laufs et al., 1998),
a number of data indicate that things are not that simple. For
example, transgenic plants in which cell division rate has
been altered generally do not display massive changes in
* Fax: +44 (0)114 222 0002. E-mail: a.fleming@sheffield.ac.uk
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Abstract
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Fig. 1. (A) Longitudinal section through the meristem of a tobacco plant. The meristem consists of an orderly array of cells. (B) Hand section through
the shoot apical meristem of a Tet::GUS plant which has been micro-induced. This leads to local induction of gene expression, as visualized by blue
signal. (C) Section (7 lm) through an apex micro-induced as in (B). Blue signal is restricted to one flank of the meristem and encompasses several cell
layers. (D) Diagram of the shoot apex to demonstrate the micro-induction experimental strategy. The meristem (m) generates leaf primordia (P2, P1) in
a sequence so that the position and timing of the next incipient primordium (I1) can be predicted. Micro-inductions (red dots) were performed at the
opposite side of the meristem (I2) where leaf formation/morphogenesis is not expected until after that at I1. (E) Section (7 lm) through the meristem
of a Tet::CycA3;2 plant in which Nicta;CycA3;2 gene expression has been induced on one flank of the meristem (red dot). An accumulation of smaller
cells is observed on this flank, but no morphogenesis. (F) Scanning electron micrograph of the apex of a Tet::Expansin plant in which expansin gene
expression has been induced at the I2 position (red dot) between primordia P2 and P1. Morphogenesis has occurred to form a bulge at this position.
(G) Section (7 lm) through the apex of a wild-type tobacco plant which has been hybridized with an anti-sense probe for NTH15, encoding a KNOX1like protein. Signal (purple) is seen throughout the meristem (including the I2 position) but is excluded from the I1 position. (H) Longitudinal section
through the apex of a Tet::phragmoplastin tobacco plant which has hybridized with an anti-sense probe for NTH15. Micro-induction was previously
performed at the I2 position of the intact meristem. Signal (purple) is absent from the meristem at both the I2 and I1 positions. (I) Leaf of
a Tet::CyclinA3;2 plant in which the primordium was micro-induced on one flank (red dot). Growth of the lamina is inhibited at this position in the leaf.
(J) Model to interpret the phenotype observed in (I). Cells on the flank of the leaf primordium (left-hand schematic) normally undergo a switch from
meristematic (red cells) to expansion growth (blue cells), as shown in the middle schematic. Micro-induction of cyclin gene expression (red dots in righthand schematic) leads to some cells retaining meristematic growth, while adjacent cells undergo the appropriate switch to expansion growth. Differential
growth is produced in the tissue, leading to morphogenesis. (K) Hand-section through the apex of a tomato plant transgenic for an RBCS Pr::GUS
construct. GUS activity (yellow) has been visualized with a fluorescent substrate. Signal is absent from the meristem dome (m) but appears as the
leaf primordium is formed. (L) Section through the apex of a tobacco plant transgenic for an RBCS Pr::GUS construct. Gus signal has been developed
using a histogenic substrate and visualized using dark-field optics so that signal appears red. Signal is absent from the meristem (m) but is visible in the
adjacent stem and leaf tissue. Parts (A), (G), and (H) are adapted from Wyrzykowska and Fleming (2003); parts (B), (C), and (F) from Pien et al. (2001);
parts (E) and (I) from Wyrzykowska et al. (2002); part (K) from Fleming et al. (1996) (copyright 1996 Blackwell Publishing Ltd and the Society for
Experimental Biology); part (L) is Fig. 5B from Mandel T, Fleming AJ, Krahenbunl R, Kuhlemeier C. 1995. Definition of constitutive gene expression in
plants: the translation initiation factor 4A gene as a model. Plant Molecular Biology 29, 995–1004, and is reprinted with kind permission of Springer
Science and Business Media.
Cellular parameters controlling morphogenesis
This approach involved the dissection and exposure of the
shoot apical meristem, precluding the use of the model
plant Arabidopsis on technical grounds. Therefore tobacco
was chosen as a compromise system in which dissection
of the apical meristem was feasible and in which a reliable tetracycline-inducible promoter system was established
(Gatz et al., 1992). Experiments using a GUS reporter gene
construct under tetracycline-inducible transcriptional regulation (Tet::GUS) showed that the technique worked and
that, depending on the inducer concentration and size of
lanolin bead used, it was possible to induce transgene
expression within a fraction of an apical meristem (Fig. 1B,
C; Pien et al., 2001). The fact that this approach led to the
induction of gene expression in a reasonable fraction of the
apical meristem was important, since the view was taken
that altered gene expression in only a single cell or a few
cells would be unlikely to alter the developmental cellular
and growth dynamics of the meristem as a whole.
Having established the technique, the question remained
as to which gene products to manipulate. With respect to
cell proliferation there was already a significant body of
data (reviewed in Potuschak and Doerner, 2001; Inze,
2005). The plant cell cycle is, in essence, very similar to
that described for other eukaryotes, depending as it does on
a controlled sequence of phosphorylation and dephosphorylation events to drive the various phases of the cycle.
Importantly, it had been shown that overexpression of
specific regulators of the cell cycle could (in a given
context) promote cells to divide faster or over a longer time
period than they normally would. For example, Shen’s
group in Strasbourg had shown that misexpression of
a cyclin (Nicta;CycA3;2) gene in tobacco led (at the whole
plant level) to a phenotype consistent with the encoded
protein promoting cell proliferation (Yu et al., 2003). In
addition, Francis and colleagues in the UK had created
a series of transgenic tobacco plants in which a yeast cdc25
was under inducible transcriptional regulation and had
shown that in the root the elevated expression of this
transgene promoted cell proliferation (McKibbin et al.,
1998). Since these two genes were relatively well characterized, had been cloned into a plant system compatible
with the micro-induction approach, and had been shown to
display phenotypes consistent with a promotion of cell
proliferation, efforts were focused on local induction of
these genes within the apical meristem. An important part
of this strategy was the attempt to promote cell division in
cells that were already primed to enter the cell cycle, i.e. an
attempt was being made to persuade cells to do something
that they are already predisposed to do. Another important
aspect of the experimental strategy was the fact that the
apical meristem is a pattern-generating machine in which
leaf primordia are produced at regular intervals of time and
space. This enabled the inductions to be focused on the
region of the meristem (I2) that, normally, would not form
a new leaf primordium within the next 48–72 h (Fig. 1D).
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plant form, i.e. the basic elements of plant structure
are elaborated (Hemerly et al., 1995; Cockcroft et al.,
2000; Wang et al., 2000; De Veylder et al., 2001), although
in some cases the total size of the plant may be reduced (De
Veylder et al., 2002). Similarly, mutants in which the cell
division pattern is disrupted still form functional plants
(Traas et al., 1995; Smith et al., 1996). Such observations
can be interpreted as supporting an organismal theory of
plant development in which some endogenous (unspecified) mechanism sets the size and shape of an organ or
organism independent of cell division processes (Kaplan,
2001). In this interpretation, the observed cell divisions that
accompany morphogenesis are simply a means by which
the increased volume accompanying morphogenesis is
chopped up into usable units (cells) which allow compartmentation and specialization of function within the plant
body. As to the nature of this unspecified size- and shapesetting mechanism, although the molecular details are
obscure, many workers in this field would incorporate an
emphasis on the cell wall. Plant growth and morphogenesis
(which is essentially differential growth) are dependent on
physical characteristics of the cell wall, so any size- or
shape-setting mechanism which is cell division independent is likely to include control of cell-wall parameters
(Green, 1999; Cosgrove, 2000; Fleming, 2002). Efforts
to reconcile these different views on the mechanism of
plant morphogenesis have been discussed in a number of
articles (Meyerowitz, 1996; Doonan, 2000; Fleming, 2002;
Tsukaya, 2003).
To investigate these two distinct interpretations of the
role of cell division in morphogenesis, a series of experiments was designed to alter parameters of cell proliferation
and growth (as controlled by cell-wall parameters) in local
regions of the shoot apical meristem. The question posed
was: Is there a difference in the efficacy of these parameters
in influencing plant morphogenesis? Although the use of
specific promoter elements to direct gene expression to
specific regions of the meristem is a viable approach to this
problem (Deveaux et al., 2003), at the time the experiments
were started the availability of such promoter elements was
very limited. Even now, the regions of the meristem that
can be precisely targeted by such methods remain restricted. Therefore a novel technique was developed to
manipulate gene expression in the apical meristem. The
approach (which was termed micro-induction) was inspired
by work from the Tickle laboratory working on chick limb
development (Cohn et al., 1995). This group had shown by
the insertion of FGF (fibroblast growth factor)-loaded beads
into the flanks of developing embryos that a localized
source of this growth factor was sufficient to induce limb
formation. At the time, a number of chemically inducible
promoters had been described in plants and it was reasoned
that if one could manipulate a local source of inducer on
to the surface of a meristem then a local gradient of inducer (and, thus, of transgene expression) would follow.
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Cell wall extensibility and morphogenesis:
is there a causal link?
Although key details on their mechanism of action are still
obscure, expansins have the ability when supplied exogenously to increase cell-wall extensibility (McQueen-Mason
et al., 1992). A large number of expansin genes have been
characterized and two general aspects have emerged (reviewed in Cosgrove, 2000; Lee et al., 2001). Firstly, in the
in vitro assays used to determine expansin activity, different
expansin proteins from the same species seem to have
similar activity profiles. At the same time, different expansin genes often show very distinct expression patterns.
Although not proven, these generalizations suggest that a
key to expansin function is when and where the genes are
expressed rather than any particular functional differences
between the proteins. Thus, targeting ectopic foreign
expansin gene expression to the apical meristem seemed
a reasonable strategy to influence tissue growth in this
organ. A key aspect to this strategy was that cells in the
meristem are poised to undergo massive expansion. It was
reasoned that these cells might be primed to respond to an
increase in expansin gene expression by an increase in
growth, i.e. again the cells would be made to do what they
were predisposed to do.
Having created and characterized a series of transgenic
plants in which expansin gene expression was inducible
(Tet::Expansin plants), local inductions were performed
at the I2 position of the apical meristem. This manipulation was sufficient to induce morphogenesis (Fig. 1F; Pien
et al., 2001). Moreover, the bulges induced grew out to
form phenotypically normal leaves. These data support the
organismal view of morphogenesis, i.e. that local altered
growth characteristics are likely to be the key to initiating
morphogenesis, with cell division in the meristem being
coupled to growth so that the increased volume generated
is split into appropriate compartments or cells. The observed cell division is not driving growth. The nature of
this coupling is an aspect of the research currently being
undertaken that will be discussed later in this article.
Although the strategy described above did not assume
that expansins represented an endogenous part of the
programme of leaf initiation (i.e. interest was mainly in
using expansin as a tool), expression analysis has revealed
that specific expansin genes are expressed at the appropriate
time and place to play a role in leaf initiation (Cho and
Kende, 1998; Reinhardt et al., 1998). However, due to the
size and complexity of the expansin gene family in model
organisms such as Arabidopsis, molecular genetic data
showing that expansin gene expression is essential for the
normal process of leaf initiation are still lacking.
The role of cell proliferation in morphogenesis
is context dependent
Do the data described above mean that cell division is not
required for morphogenesis? The answer is no. There is
a tight (but variable) relationship between cell growth and
division. If division is blocked then growth will eventually
be limited and, since, in plants, growth and morphogenesis
are intimately linked, there will be a point at which
morphogenesis will not occur. Moreover, in addition to
this basic requirement for cell proliferation if multicellularbased morphogenesis is to occur, the tight but variable
relationship between cell growth and proliferation can lead
to rather complex and, indeed, non-intuitive linkages
between cell proliferation and morphogenesis. An example
of this was revealed by a series of experiments that were
performed using inducible expression of cell cycle-related
genes in young leaf primordia.
As described, the apical meristem generates new leaf
primordia. The question was therefore posed whether this
tissue (derived from and closely associated with the apical
meristem) showed a similar response to local manipulation
of cell proliferation and growth. With respect to induced
expansin gene expression the phenotype observed was
comparable with that seen in the meristem, i.e. local
expansin expression led to local increased growth coordinated with appropriate cell proliferation (although in
this context the induced growth had appropriate leaf
identity rather than the switching from meristem to leafidentity, as observed in the meristem; Pien et al., 2001).
The outcome of local promotion of cell proliferation in leaf
primordia was dramatic and counter-intuitive. During the
initial phase of induction an accumulation of cells at the site
of induction was observed. However, as the leaf grew, the
growth rate in the induced region was slower than in the
adjoining tissue, leading to an overall lack of lamina growth
in the area that had originally been induced to undergo cell
proliferation (Fig. 1I; Wyrzykowska et al., 2002).
The mechanism involved in this response is open to
discussion, but one possibility is that plant cells that are
undergoing proliferation have a lower overall rate of growth
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Therefore the question was posed whether promotion of
cell proliferation in this region was sufficient to induce premature morphogenesis. The results for both Nicta;CycA3;2
and cdc25 were clear; cell proliferation was locally promoted after the manipulation but meristem growth was
normal (Fig. 1E; Wyrzykowska et al., 2002). These results
support the hypothesis that cell proliferation on its own is
not sufficient to drive morphogenesis.
Since cell division did not seem to influence morphogenesis in the meristem, what about parameters affecting
tissue growth independent of any direct influence on cell
proliferation? To address this question a similar experimental approach to that described above, but using molecular tools to modify cell wall extensibility, was undertaken,
focusing in particular on a family of cell-wall proteins
termed expansins.
Cellular parameters controlling morphogenesis
Cell division pattern and morphogenesis:
is there a causal link?
In addition to the role of cell division rate on plant
morphogenesis, another long-held view is that the orientation of cell division influences plant growth. Plant cytokinesis involves the formation of the phragmoplast within
the dividing cell. This agglomeration of vesicles and cytoskeletal elements dictates the position of the new cell
wall which separates the daughter cells following division.
According to classical observations, orientation and position of the new cell wall dictates the relative size and shape
of the daughter cells, which therefore dictates the future
preferred orientation of growth. One can imagine that local
control of the orientation of cell division within the apical
meristem would control the direction of growth of the
daughter cells which, thus, would control leaf primordium
outgrowth. Indeed, histological observations reveal that one
of the first markers of the presumptive site of leaf initiation
is a switch in cell division orientation, although the
correlation is not absolute (Lyndon, 1990; Selker et al.,
1992). Since experiments simply increasing cell proliferation did not directly address this issue, a series of experiments was performed in which the dynamin-like protein
phragmoplastin was used as a tool to locally disrupt the cell
division pattern in the meristem. Previous work from the
Verma laboratory had shown that overexpression of phrag-
moplastin disrupted the cell division pattern in BY2 cells
and led to seedling lethality (Gu and Verma, 1997; GeislerLee et al., 2002). Overexpression of phragmoplastin seems
to lead to an overloading of the vesicle transport system to
the forming phragmoplast, preventing appropriate orientation. Using the micro-induction system, local overexpression of phragmoplastin disrupted the pattern of cell division
in the outer tunica layers of the meristem but no overt
outcome on leaf initiation or meristem function was
observed (Wyrzykowska and Fleming, 2003). These observations again support an organismal view of the meristem
in which altered cell division per se is not sufficient to drive
morphogenesis.
Gene expression influences cell division and cell
division influences gene expression
An interesting aspect of these experiments was revealed by
an analysis of marker gene expression in the induced
meristems. Research from a number of experimental
systems has led to the identification of genes whose
expression pattern defines particular regions of the meristem and which, moreover, encode proteins which play
vital roles in various aspects of meristem function (reviewed in Byrne, 2005). For example, KNOX genes (such
as NTH15 in tobacco) play an essential role in the
maintenance of meristem indeterminancy and are expressed
in a specific pattern so that transcripts within the meristem
are excluded from the presumptive site of leaf formation
(Fig. 1G; Sakamoto et al., 2001; Wyrzykowska and
Fleming, 2003). The expression pattern of KNOX genes is
intimately linked with that of ARP transcription factors
(such as NtPHAN) whose transcripts accumulate within the
developing primordium in a pattern complementary to that
shown by KNOX genes. Analysis of the expression patterns
of these marker genes revealed that, subsequent to local
induction of phragmoplastin, the patterns of NTH15 and
NtPHAN were disrupted, although no obvious effect on
meristem function was observed (Fig. 1H; Wyrzykowska
and Fleming, 2003). One cautionary point here is that there
is a significant body of data indicating that the proteins
encoded by many genes within the meristem (including
KNOX genes) are mobile so that the pattern of transcript
does not necessarily indicate the pattern of protein accumulation (Perbal et al., 1996; Sessions et al., 2000; Kim
et al., 2002; Wu et al., 2003). Nevertheless, the data
suggest that the pattern of cell division within the meristem
can influence the pattern of transcript accumulation of at
least some genes. The mechanism is unclear, but hypotheses invoking plasmodesmatal-gating of transcript and
protein movement within the meristem have been proposed
(Haywood et al., 2002), and an altered pattern of cell
division would be predicted to influence such symplastic
pathways. Thus, in addition to the canonical pathway of
gene expression influencing cell division, there is the
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than cells which are exiting the cell cycle to undergo
purely expansion growth. These two distinct phases of
plant cell development (proliferative- and non-proliferativeassociated growth) have been termed meristematic growth
and expansion growth (Chen et al., 2001). Recent observations on suspension culture cells support the hypothesis
that these two distinct phases of cell development do indeed
display different growth rates (Campanomi and Nick,
2005). Interpreted in the light of these data, it is speculated
that the cells on the induced flank of the primordium were
maintained in a proliferative growth phase, while adjacent
cells were entering a phase of expansion growth (Fig. 1J).
This would lead to a differential growth rate and, thus,
morphogenesis. Although the induced tissue might later
regain an appropriate growth rate as the cells underwent
a transition to expansion-type growth, due to the fixation of
plant cells relative to one another via the cell wall, the
relative positions of the tissues would become more or less
fixed in space (Fig. 1J).
Irrespective of whether this conjecture is correct, the data
demonstrate that the influence of cell proliferation on
morphogenesis is highly context dependent. One cannot
make simple predictions on what the outcome of altered
cell proliferation will be on morphogenesis, a view that
the literature in this area supports (Cockcroft et al., 2000;
Autran et al., 2002; Dewitte et al., 2003; reviewed
in Doonan, 2000; Tsukaya, 2003).
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possibility of a feedback mechanism by which an altered
cell division pattern can influence gene expression.
From meristem to non-meristem: the control
of differentiation
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The experiments described above were focused on unravelling the interaction of cell proliferation, growth, and
morphogenesis. As noted at the beginning of this article,
these parameters must also be integrated with the process of
differentiation if a fully functional organism is to be
formed. The micro-induction approach has also been used
to investigate this aspect of development, again focusing
on the shoot apical meristem.
Cells in the meristem are characterized by a number of
parameters. Although a relatively high rate of proliferation
is one of them, this is in no way exclusive to the meristem.
Cells outside the meristem in the young leaf and stem also
display relatively high rates of division, although this tends
gradually to decrease as the organs mature. By contrast, one
of the most dramatic shifts in cellular function between the
meristem and non-meristem cell state is the acquistion of
photosynthetic machinery. Meristem cells contain proplastids which lack the organized thylakoid structure of
functional chloroplasts. Moreover, the cells do not contain
chlorophyll or express the array of gene products required
for photosynthesis. However, within a very short time of
being displaced from the meristem, cells gain the capacity
to be photosynthetic, as shown, for example, by the
expression of genes encoding RBCS (small subunit, ribulose bisphosphate carboxylase) (Fig. 1K, L; Fleming et al.,
1996). Simple exposure of meristems to light is insufficient
to trigger this switch, indicating that it is an endogenous
programmed event and not simply a result of enclosure of
the meristem within the surrounding primordia to create
a darkened environment. At the same time as gaining
photosynthetic capacity, the cells that are displaced from
the meristem enlarge via vacuolation. Simultaneously the
rate of cell proliferation decreases. Thus, there is a correlation of decreased proliferation, acquisition of a specific
differentiation pathway, and an increase in cell size via
vacuolation. The molecular mechanism that controls these
decisive events is obscure. For example, modulation of cell
cycle genes influences the rate of growth but meristem
function is maintained (Hemerly et al., 1995; Cockcroft et al.,
2000; Wang et al., 2000; De Veylder et al., 2001, 2002). One
potential candidate that might influence this switch is the
retinoblastoma family of proteins.
Retinoblastoma protein (pRb) was first identified in
mammalian cells as a tumour suppressor. Since then it
has been characterized as a key player in the cell cycle
(Weinberg, 1995; Harbour and Dean, 2000). Specifically,
the paradigm has emerged that pRb acts as an intermediary
between cyclin/CDK complexes which promote entry into
the cell cycle and E2F/DP transcription factors which
promote the expression of genes associated with, and
required for, entry into the S-phase of the cell cycle. In its
hypo-phosphorylated form pRb binds to E2F/DP factors
and inhibits transcription of target genes required for entry
into the cell cycle. Phosphorylation of pRb by cyclin/CDK
complexes alleviates this restriction and allows the E2F/DP
transcription factors to initiate entry to the cell cycle.
Although the main focus of research has been on the role of
pRb in cell proliferation, it has become clear that pRb can
also influence differentiation, although to what extent this is
an indirect effect of altered cell proliferation and how much
a direct influence of pRb has been somewhat obscure.
Recent genomic approaches studying the outcome of
altered pRb expression have revealed a number of potential
transcriptional responses to altered pRb expression which
are not overtly linked with the cell cycle, favouring the
view that pRb may also have a reasonably direct influence
on a spectrum of gene expression events linked with
differentiation (Dimova et al., 2003). pRb has, therefore,
emerged as a potential pivot in the cellular decision to
proliferate or enter a pathway of differentiation.
With respect to plants, the situation is unclear. Although
plants contain genes encoding retinoblastoma-related proteins (RBRs) their functional analysis has been limited (Xie
et al., 1996; Durfee, 2000). RBRs can certainly interact with
cyclin/CDKs (Huntley et al., 1998; Nakagami et al., 2002),
and experiments in which a truncated RBR cDNA was shot
ballistically into tissue culture cells inhibited cell division
(Gordon-Kamm et al., 2002). At the level of the whole
plant, RBR is an essential protein. Deletion of the single
copy RBR gene in Arabidopsis is gametophytic lethal,
leading to the formation of multi-nucleate and infertile eggs
(Ebel et al., 2004). This lethality precludes simple analysis
of the knock-out phenotype in the mature vegetative plants.
One approach to tackle this problem has been to use a viralsuppression strategy to down-regulate endogenous RBR
levels in developing leaves of tobacco (Park et al., 2005).
This strategy revealed that a decreased RBR transcript level
led to an inhibition of organ growth and the disruption of
epidermal cell characters, consistent with the paradigm of
RBR being required to suppress cell proliferation. The
function of RBR has also been investigated in the intact
plant using the micro-induction technique. This allowed
the focus to be on the response of the apical meristem to
manipulation of RBR protein levels. After transient induction of RBR gene expression it was observed that
meristem cells underwent at least the initial stages of
differentiation, including cell expansion and expression
of marker genes for photosynthesis (J Wyrzykowska,
M Schorderet, S Pien et al., unpublished results). This
outcome is very different from that observed when the
expression of other cell cycle genes (e.g. Nicta;CycA3;2) is
altered in the meristem, suggesting that RBR is affecting
other elements than simple cell proliferation and, moreover, that the observed outcome on differentiation is not
Cellular parameters controlling morphogenesis
simply related to a decrease in cell proliferation. These data
therefore support the idea that RBR can act as a pivot in the
cellular decision to divide or differentiate.
Conclusion
References
Autran D, Jonak C, Belcram K, Beemster GTS, Kronenberger J,
Grandjean O, Inzé D, Traas J. 2002. Cell numbers and leaf
development in Arabidopsis: a functional analysis. EMBO Journal
21, 6036–6049.
Byrne ME. 2005. Networks in leaf development. Current Opinion in
Plant Biology 8, 59–66.
Campanomi P, Nick P. 2005. Auxin-dependent cell division and cell
elongation: 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid activate different pathways. Plant Physiology 137,
939–948.
Chen JG, Ullah H, Young JC, Sussman MR, Jones AM. 2001.
ABP1 is required for organized cell elongation and division
in Arabidopsis embryogenesis. Genes and Development 15,
902–911.
Cho HT, Kende H. 1998. Tissue localization of expansins in deepwater rice. The Plant Journal 15, 805–812.
Cockcroft CE, den Boer BGW, Healy JMS, Murray JAH. 2000.
Cyclin D control of growth rate in plants. Nature 405, 575–579.
Cohn MJ, Izpisuabelmonte JC, Abud H, Heath JK, Tickle C.
1995. Fibroblast growth factors induce additional limb development from the flank of chick embryos. Cell 80, 739–746.
Cosgrove DJ. 2000. Loosening of plant cell walls by expansins.
Nature 407, 321–326.
Day S, Lawrence PA. 2000. Measuring dimensions: the regulation
of size and shape. Development 127, 2977–2987.
Deveaux Y, Peaucelle A, Roberts GR, Coen E, Simpon R,
Mizukami Y, Traas J, Murray JAH, Doonan JH, Laufs P.
2003. The ethanol switch: a tool for tissue-specific gene induction
during plant development. The Plant Journal 36, 918–930.
De Veylder L, Beeckman T, Beemster GTS, et al. 2002. Control
of proliferation, endoreduplication and differentiation by the
Arabidopsis E2Fa-DPa transcription factor. EMBO Journal 21,
1360–1368.
De Veylder L, Beeckman T, Beemster GTS, Krols L, Terras F,
Landrieu I, Van Der Schueren E, Maes S, Naudts M, Inzé D.
2001. Functional analysis of cyclin-dependent kinase inhibitors of
Arabidopsis. The Plant Cell 13, 1653–1667.
Dewitte W, Riou-Khamlichi C, Scofield S, Healy JMS, Jacqmard
A, Kilby NJ, Murray, JAH. 2003. Altered cell cycle distribution,
hyperplasia and inhibited differentiation in Arabidopsis caused by
the D-type cyclin CYCD3. The Plant Cell 15, 79–92
Dimova DK, Stevaux O, Frolov MV, Dyson NJ. 2003. Cell cycledependent and cell cycle-independent control of transcription by
the Drosophila E2F/RB pathway. Genes and Development 17,
2308–2320.
Doonan J. 2000. Social controls on cell proliferation in plants.
Current Opinion in Plant Biology 3, 482–487.
Durfee T, Feiler H, Gruissem W. 2000. Retinoblastoma-related
proteins in plants: homologues or orthologues of their metazoan
counterparts? Plant Molecular Biology 43, 635–642.
Ebel C, Mariconti L, Gruissem W. 2004. Plant retinoblastoma
homologues control nuclear proliferation in the female gametophyte. Nature 429, 776–780.
Fleming AJ. 2002. The mechanism of leaf morphogenesis. Planta
216, 17–22.
Fleming AJ, Manzara T, Gruissem W, Kuhlemeier C. 1996.
Fluorescent imaging and RT-PCR analysis of gene expression in
the shoot apical meristem. The Plant Journal 10, 745–754.
Gatz C, Frohbergand C, Wendenburg R. 1992. Stringent repression and efficient tetracycline de-repression of expression from
a modified CaMV 35S promoter in intact tobacco plants. The Plant
Journal 2, 397–404.
Geisler-Lee CJ, Hong ZL, Verma DPS. 2002. Overexpression of
phragmoplastin results in increased level of callose deposition at
the forming cell plate. Plant Science 163, 33–42.
Green PB. 1999. Expression of pattern in plants: combining
molecular and calculus-based biophysical paradigms. American
Journal of Botany 86, 1059–1064.
Gordon-Kamm W, Dilkes BP, Lowe K, et al. 2002. Stimulation of
the cell cycle and maize transformation by disruption of the plant
retinoblastoma pathway. Proceedings of the National Academy of
Sciences, USA 99, 11975–11980.
Gu X, Verma DPS. 1997. Dynamics of phragmoplastin in living
cells during cell plate formation and uncoupling of cell elongation
from the plane of cell division. The Plant Cell 9, 157–169.
Harbour JW, Dean DC. 2000. Rb function in cell-cycle regulation
and apoptosis. Nature Cell Biology 2, E65–E67.
Haywood V, Kragler F, Lucas WJ. 2002. Plasmodesmata: pathways for protein and ribonucleo-protein signaling. The Plant Cell
supplement, 14, S303–S325.
Hemerly A, Engler JA, Bergounioux C, Van Montagu M, Engler
G, Inzé D, Ferreira P. 1995. Dominant negative mutants of the
Cdc2 kinase uncouple cell division from iterative plant development. EMBO Journal 14, 3925–3936.
Huntley R, Healy S, Freeman D, et al. 1998. The maize retinoblastoma protein homologue ZmRb-1 is regulated during leaf
development and displays conserved interactions with G1/S
regulators and plant cyclin D (cycD) proteins. Plant Molecular
Biology 37, 155–169.
Inzé D. 2005. Green light for the cell cycle. EMBO Journal 24,
657–662.
Kaplan DR. 2001. Fundamental concepts of leaf morphology and
morphogenesis: a contribution to the interpretation of molecular
genetic mutants. International Journal of Plant Science 162,
465–474.
Kim JY, Yuan Z, Cilia M, Khalfan-Jagani Z, Jackson D. 2002.
Intercellular trafficking of a KNOTTED1 green fluorescent protein
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 26, 2014
Cells are the basic unit of plants, yet fundamental questions
still remain as to how cell proliferation is integrated into the
whole organism and how this process is itself related to
basic aspects of differentiation. One approach has been to
target a very specific and essential organ for plant development (the shoot apical meristem) to observe how the
system responds to specific, transient perturbations of parameters of cell division frequency, orientation, and growth.
Using gene products as tools, it has been possible to
decipher some ground rules of how the meristem (and the
leaf primordia derived from it) function as multicellular
organs. The challenge is now to identify the endogenous
processes/genes controlling the cellular and tissue mechanisms by which morphogenesis occurs, to understand how
these end-processes are integrated with the transcriptional
networks controlling them, and how these end-processes
feed back to these same networks.
7 of 8
8 of 8 Fleming
of leaf formation in the tomato meristem. The Plant Cell 10,
1427–1437.
Rudra D, Warner JR. 2004. What better measure than ribosome
synthesis? Genes and Development 18, 2431–2436.
Sakamoto T, Kamiya N, Ueguchi-Tanaka M, Iwahori S,
Matsuoka M. 2001. KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene in the
tobacco shoot apical meristem. Genes and Development 15,
581–590.
Selker JL, Steucek GL, Green PB. 1992. Biophysical mechanisms
for morphogenetic progressions at the shoot apex. Developmental
Biology 153, 29–43.
Sessions A, Yanofsky MF, Weigel D. 2000. Cell-cell signalling and
movement by the floral transcription factors LEAFY and
APETALA1. Science 289, 779–781.
Smith LG, Hake S, Sylvester AW. 1996. The tangled1 mutation
alters cell division orientations throughout maize leaf development
without altering leaf shape. Development 122, 481–489.
Traas J, Bellini C, Nacry P, Kronenberger J, Bouchez D,
Caboche M. 1995. Normal differentiation patterns in plants
lacking microtubular preprophase bands. Nature 375, 676–677.
Tsukaya H. 2003. Organ shape and size: a lesson from studies of leaf
morphogenesis. Current Opinion in Plant Biology 6, 57–62.
Wang H, Zhou Y, Gilmer S, Whitwill S, Fowke LC. 2000.
Expression of the plant cyclin-dependent kinase inhibitor ICK1
affects cell division, plant growth and morphology. The Plant
Journal 24, 613–623.
Weinberg RA. 1995. The retinoblastoma protein and cell cycle
control. Cell 81, 323–330.
Wu X, Dinneny JR, Crawford KM, Rhee Y, Citovsky V,
Zambryski PC, Weigel D. 2003. Modes of intercellular transcription factor movement in the Arabidopsis apex. Development
130, 3735–3745.
Wyrzykowska J, Fleming AJ. 2003. Cell division pattern influences
gene expression in the shoot apical meristem. Proceedings of the
National Academy of Sciences, USA 100, 5561–5566.
Wyrzykowska J, Pien S, Shen W-H, Fleming AJ. 2002. Manipulation of leaf shape by modulation of cell division. Development
129, 957–964.
Xie Q, Sanz-Burgos AP, Hannon GJ, Gutierrez C. 1996. Plant
cells contain a novel member of the retinoblastoma family of
growth regulatory proteins. EMBO Journal 15, 4900–4908.
Yu Y, Steinmetz A, Meyer D, Brown S, Shen W-H. 2003. The
tobacco A-type cyclin, Nicta;CYCA3;2, at the nexus of cell
division and differentiation. The Plant Cell 15, 2763–2777.
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 26, 2014
fusion in the leaf and shoot meristem of Arabidopsis. Proceedings
of the National Academy of Sciences, USA 99, 4103–4108.
Laufs P, Grandjean O, Jonak C, Kieu K, Traas J. 1998. Cellular
parameters of the shoot apical meristem in Arabidopsis. The Plant
Cell 10, 1375–1389.
Lee Y, Choi D, Kende H. 2001. Expansins: ever expanding numbers
and functions. Current Opinion in Plant Biology 4, 527–532.
Lyndon RF. 1990. Plant development: the cellular basis. London:
Unwin Hyman.
Mandel T, Fleming AJ, Krahenbuhl R, Kuhlemeier C. 1995.
Definition of constitutive gene expression in plants: the translation
initiation factor 4A gene as a model. Plant Molecular Biology
29, 995–1004.
McKibbin RS, Halford NG, Francis D. 1998. Expression of fission
yeast cdc25 alters the frequency of lateral root formation in
transgenic tobacco. Plant Molecular Biology 36, 601–612.
McQueen-Mason S, Durachko DM, Cosgrove DJ. 1992. Two
endogenous proteins that induce cell-wall extension in plants.
The Plant Cell 4, 1425–1433.
Meyerowitz EM. 1996. Plant development: local control, global
patterning. Current Opinion in Genetics Development 6, 475–479.
Nakagami H, Kawamura K, Sugisaka K, Sekine M, Shinmyo A.
2002. Phosphorylation of retinoblastoma-related protein by the
cyclinD/cyclin-dependent kinase complex is activated at the
G1/S-phase transition in tobacco. The Plant Cell 14, 1847–1857.
Nijhout HF. 2003. The control of growth. Development 130,
5863–5867.
Park J-A, Ahn J-W, Kim Y-K, Kim SJ, Kim WT, Pai H-S. 2005.
Retinoblastoma protein regulates cell proliferation, differentiation,
and endoreduplication in plants. The Plant Journal 42, 153–163.
Perbal MC, Haughn G, Saedler H, Schwarz-Sommer Z. 1996.
Non-cell-autonomous function of the Antirrhinum floral homeotic
proteins DEFICIENS and GLOBOSA is exerted by their polar
cell-to-cell trafficking. Development 122, 3433–3441.
Pien S, Wryzykowska J, McQueen-Mason S, Smart C, Fleming
AJ. 2001. Local induction of expansin is sufficient to induce the
entire process of leaf development and to modify leaf shape.
Proceedings of the National Academy of Sciences, USA 98,
11812–11817.
Potuschak T, Doerner P. 2001. Cell cycle controls: genome-wide
analysis in Arabidopsis. Current Opinion in Plant Biology 4,
501–506.
Reinhardt D, Wittwer F, Mandel T, Kuhlemeier C. 1998.
Localised upregulation of a new expansin gene predicts the site