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Journal of Experimental Botany, Vol. 51, No. 347, pp. 971–983, June 2000
REVIEW ARTICLE
Polarity and signalling in plant embryogenesis
Martin Souter and Keith Lindsey1
Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, UK
Received 11 February 2000; Accepted 24 February 2000
Abstract tion is severe, since reproductive success depends abso-
lutely upon the ability of individual plants to acquire
The establishment of the apical–basal axis is a critical
these. The germination of seeds beneath the soil elicits a
event in plant embryogenesis, evident from the earliest
complexity of phytochrome-dependent and COP/DET
stages onwards. Polarity is evident in the embryo sac,
gene-dependent signalling pathways to ensure rapid cell
egg cell, zygote, and embryo–suspensor complex. In
expansion along the apical-basal axis, to reach the light
the embryo-proper, two functionally distinct meris-
(Deng, 1994; Chory, 1997). Post-germinative growth is
tems form at each pole, through the localized expres-
most successful for those individuals able to out-compete
sion of key genes. A number of mutants, notably of
their neighbours for available light through the shade
the model genetic organism Arabidopsis thaliana, have
avoidance response (Ballare
´
, 1999), leading to rapid cell
revealed new gene functions that are required for pat-
division and expansion in the hypocotyl and stem. The
terning of the apical–basal axis. There is now increas-
acquisition of water and nutrients from the soil requires
ing evidence that two particular modes of signalling,
the modulation of root meristem activity and cell expan-
via auxin and cell wall components, play important
sion in the opposite direction, that is, downwards. Taller
roles in co-ordinating the gene expression pro-
stems also facilitate spore and seed dispersal, promoting
grammes that define determinative roles in the estab-
reproductive success through the exploitation of more
lishment of polarity.
distant ecosystems. The seedling can therefore be viewed
as a polar structure, with each pole exhibiting different
Key words: Embryogenesis, polarity, intercellular signal-
activities; both of which must have been critical for the
ling, auxin, cell wall components, Arabidopsis thaliana.
early success of the higher land plants.
The focus of this review is to examine the cellular and
Introduction
molecular basis of axialization in higher plants, with an
emphasis on studies in dicotyledonous species, using
The growth and development of higher plants can be
Arabidopsis as a model. In particular, the origins of
considered to be characterized by the execution of cell
apical-basal polarity in the embryo, its genetic control,
division, expansion and differentiation along two axes:
and the signalling systems that regulate the expression of
the apical-basal axis and the radial axis. The radial axis
relevant genes will be examined. The first part of the
is most clearly evident in dicotyledonous species as the
article will focus on how polarity is established and then
concentric rings of cell layers in the seedling stem, hypo-
fixed, whilst the second part will look at the different
cotyl and root, and an increase in size across this axis
signalling systems involved in maintaining this polarity
can arise from the generation of new cell layers following
and using it to enable the correct elaboration of the
divisions in the vascular cambium in the older plant. The
apical-basal pattern.
apical-basal axis can be defined by the patterning of
functionally distinct structures, rather than cell layers,
from the shoot apical meristem, to the hypocotyl and
Polarity originates early in development
stem, to the root apical meristem. In evolutionary terms,
the apical-basal axis of development can be considered In Arabidopsis, and other species such as Capsella bursa-
pastoris that have been studied in much detail, it is clearto have a strong selective advantage based upon plant
competition for light, water and nutrients. Such competi- that apical-basal polarity is evident even before the first
1 To whom correspondence should be addressed. Fax: +44 191 374 2417. E-mail: Keith.Lindsey@durham.ac.uk
© Oxford University Press 2000
972 Souter and Lindsey
zygotic division within the egg cell itself (Schulz and localization of F-actin at the rhizodermis; an asymmetric
distribution of RNA molecules in the zygote (thoughJensen, 1968; Mansfield and Briarty, 1991). It is, further-
more, the case that the embryo sac itself also exhibits actin mRNA interestingly accumulates at the opposite
pole to F-actin protein; Bouget et al., 1996); and apolar organization, with the egg cell and synergids adja-
cent to the micropyle, while the antipodal cells are found polarized secretion of Golgi-derived cell wall components
towards the ‘basal’ region from which the rhizoid cell willat the opposite chalazal end. Polarity in the egg cell is
seen anatomically as the location of a large vacuole at its develop. Experimental disruption of this secretion by
brefeldin A disrupts axis fixation and polarized growthmicropylar end, while the chalazal end is relatively cyto-
plasmic (Fig. 1). In some species, polarity in the egg cell (Shaw and Quatrano, 1996).
and, subsequently, the zygote is exaggerated by a reorgan-
ization of cytoplasmic components (Natesh and Rau,
Cell fate decisions: embryo-proper versus
1984; Schulz and Jensen, 1968). However, the molecular
suspensor
mechanisms that generate this polarity are still obscure,
and fall far behind current understanding of polarization The observed apical-basal polarity in the zygote of
Arabidopsis and Fucus presages polar development duringwithin, for example, the Drosophila egg (Gonzales-Reyes
et al., 1997). embryogenesis. In each species, the zygote undergoes an
asymmetric transverse division to generate two daughterThe brown alga Fucus offers some experimental features
that greatly facilitate the study of early events of zygote cells that are of unequal size and follow distinct develop-
mental pathways. In Arabidopsis one cell, the basal cellpolarization. Free-living egg cells and zygotes can be
harvested, manipulated and observed under the micro- which is the larger of the two, derives from the vacuolar
region of the zygote, while the smaller upper cell derivesscope, and some elegant recent work has provided new
insight into polarity generation early in plant develop- from the cytoplasmic region (Fig. 1). The upper cell then
divides to form the embryo proper, while the basal cellment. In the fucoid zygote, polarization events can be
triggered by a range of stimuli, including unidirectional forms a single file of typically six to nine cells, the
suspensor. Only the uppermost cell of the suspensor, thelight and fertilization (Hable and Kropf, 2000). For a
short period after the induction of polarization axis hypophysis, contributes to the embryo proper as part of
the root meristem (Dolan et al., 1993; Scheres et al.,formation is reversible, but subsequently irreversible
(Quatrano and Shaw, 1997). Associated with axis forma- 1994). The suspensor appears to have a number of
different functions: it physically projects the embryo intotion there is an observed localization or redistribution of
plasma membrane components, including ion channels; a the endosperm, and provides both a conduit and a source
of hormones and nutrients for the developing embryo.redistribution of calcium to the basal shaded end; a
Perhaps the most clear difference in fate between the
embryo-proper and suspensor is seen as the programmed
cell death of the suspensor when the embryo reaches the
torpedo-stage of development (Yeung and Meinke, 1993).
There is also increasing evidence that the embryo and
suspensor express distinct gene expression programmes.
While a number of embryonic mutations, such as knolle
(Lukowitz et al., 1996), fass (Berleth and Ju
¨
rgens, 1993),
gnom/emb30 (Mayer et al., 1993), and hobbit ( Willemsen
et al., 1998) affect the cellular organization and/or division
activity of the embryo, hypophysis and suspensor, other
mutants, such as hydra1, show embryo-specific defects
(Topping et al., 1997), suggesting that the HYDRA1 gene
is expressed in the embryo, but not in the suspensor.
Direct evidence for different gene expression profiles in
embryo and suspensor comes from promoter trap analysis
in Arabidopsis, which has led to the identification of genes
that are specific to the embryo-proper (Topping et al.,
1994; Topping and Lindsey, 1997) and to the suspensor
(P Gallois, unpublished results). Differences in gene
expression between the apical and basal cell following the
first zygotic division have also been identified. For
example, the apical cell has been shown to accumulate
Fig. 1. Schematic representation of the zygotic division and subsequent
cell patterning in Arabidopsis thaliana.
the ARABIDOPSIS THALIANA MERISTEM LAYER
Polarity and signalling in plant embryogenesis 973
1 (AtML1) gene transcript, which is not detected in the with particular cell types ( Knox et al., 1991; Pennell
et al., 1991, 1995). The antibodies recognize componentsbasal cell (Lu et al., 1996).
of the pectin matrix of the wall, specifically arabino-
galactan moieties attached to proteins in the plasma mem-
A role for cell wall components
brane, the so-called arabinogalactan proteins (AGPs).
Of particular interest is the nature of the molecular
Interestingly, there are differences in AGP localization
mechanisms that regulate cell fate determination and the
during brassica embryogenesis. For example, the JIM8
associated gene expression programmes. In pollen devel-
antibody reveals cell differences between embryo-proper
opment, the formation of the structurally and functionally
and suspensor, binding only to the cells whose future fate
distinct vegetative and generative cells, and the expression
is as the suspensor (Pennell et al., 1991).
of genes within those cells, has been shown by in vitro
Not only have AGPs been identified that are differenti-
techniques to depend on the asymmetry of the formative
ally expressed during zygotic embryogenesis, but they are
cell division, pollen mitosis I (Eady et al., 1995). More
also differentially expressed during somatic embryogen-
recently, van den Berg et al. have used laser ablation
esis. Somatic embryos develop, not from fertilized egg
techniques to demonstrate the role of short-range signal-
cells, but from somatic (non-reproductive) cells that have
ling between cells to direct their fates (van den Berg et al.,
been tissue-cultured. These cells are induced to become
1995, 1997). The most productive approach to date in
structurally disorganized, and lose the characteristics of
addressing such questions in embryogenesis is the genetic
the differentiated state of the tissue from which they
approach, which involves screening for mutants in which
derive. However, they can reorganize if given appropriate
cell fate control is defective.
hormonal signals (usually a removal of auxin from the
In Fucus, the cell differentiation event leading to the
culture medium). Despite the fact that they are not in
generation of the thallus and rhizoid cells, respectively, is
contact with the maternal influences of the seed, they are
preceded by an asymmetric cell division, with the larger
able to develop in a polar way, to generate embryoidal
upper cell forming the thallus cell, which in turn forms
structures that are similar to zygotic embryos, and indeed
the laminate thallus structures of the mature alga. The
can go on to ‘germinate’ into plants.
smaller basal cell forms the rhizoid that undergoes polar-
The classical system to study somatic embryogenesis is
ized growth. Genestein, an inhibitor of tyrosine phos-
in cultured cells of carrot. In this system, meristematic,
phorylation, inhibits axis formation in the dark and in
relatively undifferentiated cells are grown in liquid
light-grown zygotes if applied early. Compelling evidence
medium in the presence of auxin as globular cell clusters:
has also been found to demonstrate a role for the differen-
these have been designated proembryonic masses (PEMs).
tial secretion of cell wall components in determining the
These probably represent preglobular-stage embryos,
subsequent identities of the rhizoid and basal cells. Here,
arrested in their further development by the presence of
wall fragments from thallus and rhizoid cells, respectively,
auxin. But when transferred to an auxin-free medium,
can direct the fate of protoplasts of either cell (Berger
cells of the PEMs become organized to form adventitious
et al., 1994), and a system of intercellular communication
embryos ( Krikorian and Smith, 1992). It is also possible
defines positional information to regulate cell fate (Bouget
to induce single cells of carrot to form embryos directly
et al., 1998). Candidate regulatory molecules within the
by manipulating auxin–cytokinin concentrations in the
cell wall of Fucus are sulphonated polysaccharides; inter-
culture medium (Nomura and Komamine, 1985; Pennell
estingly, their secretion is inhibited by genestein (Corellou
et al., 1995).
et al., 2000). Gradually, then, evidence is emerging for
In relation to the question of a role for AGPs in
the molecular basis of polarity generation in the Fucus
polarity, the single cell embryogenic system is of interest.
zygote.
The single cells divide, and the products of the division
How far can there be extrapolation from the Fucus
have separate fates: one cell becomes an embryonic initial,
studies to developmental mechanisms in higher plants? A
which undergoes further divisions to form an embryo;
prerequisite for a model in which cell wall components
while the other cell fails to divide further. The original
carry positional information would be that there are
single cell expresses one particular AGP epitope, recog-
detectable differences in such components between cells.
nized by JIM8: and this is indicative of a cell with
What is the evidence that such differences exist? Much of
embryonic potential, shown by video tracking (McCabe
the evidence for cell wall differences that are cell type- or
et al., 1997). When this cell divides, the cell that becomes
tissue-specific comes from work in which monoclonal
the ‘embryo initial’ switches off the JIM8 epitope, while
antibodies have been raised in response to immunizations
the second cell (the ‘nurse cell’) continues to express that
with complex mixtures of plant cell material. By labelling
protein. This is reminiscent of the suspensor cell expres-
these antibodies and localizing their binding sites in
sion pattern of JIM8 in the zygotic embryo (Pennell et al.,
plants, a series of probes has been generated that each 1991), and the two division products of the single cell are
analogous to the zygotic apical and basal cell.recognize cell surface polysaccharide epitopes associated
974 Souter and Lindsey
But is there evidence that the JIM8 target actually a C16 or C18 fatty acid group attached to the non-
reducing end. They are known to act as important signalsregulates cell fate? To investigate this, McCabe et al.
purified JIM8-positive or JIM8-negative cells, and col- in the nodulation process following Rhizobium interaction
with legume roots, and have been designated Nod factorslected cell wall components released from the walls of
each. JIM8-negative cell wall components, lacking that (Schultze and Kondorosi, 1996). Purified Nod factors
have a wide range of effects on the roots of legumes:epitope, were found not to continue to divide and form
embryos (McCabe et al., 1997). However, if the JIM8 some effects are very rapid, some over a period of days
or weeks.epitope, collected from the ‘nurse’ cells is added to the
‘initial’ cells, they will go on to form embryos; however, The most rapid response is transient depolarization of
the plasma membrane, occurring within 15 s. This leadsthey require JIM8-positive cell- conditioned medium in
order to do so. to an increase in intracellular pH, and a spiked oscillation
in intracellular calcium levels (reviewed by Schultze andThis indicates that the JIM8 epitope can be used to
identify cells which have a role in cell–cell communication Kondorosi, 1996). This may represent an activation of
an intracellular signal transduction pathway, but a causaland early cell fate specification in carrot somatic embryo-
genesis. Indeed, the JIM8 epitope may itself be involved relationship has not yet been demonstrated. Synthetic
Nod factors can also induce division in tobacco proto-in early events of determination of cell fate in carrot
somatic embryogenesis, and also in maintaining activity plasts in the absence of auxins and cytokinins and the
fatty acid structure has been shown to be important inof division of the embryo: i.e. it may signal to the initial
cells to keep dividing. Further support for an inductive this activity (Ro
¨
hrig et al., 1995). So a common role for
lipo-oligosaccharides in somatic embryogenesis and rooteffect of AGPs in somatic embryogenesis comes from
some earlier work ( Kreuger and van Holst, 1993, 1995). nodule formation may be as stimulators of cell division,
and at concentrations as low as 10−15 M.These authors found that the addition of AGPs from an
embryogenic carrot cell line to a non-embryogenic line One speculative view of the molecular mechanisms of
targeted secretion of wall components, and subsequentcaused an induction of embryogenic capacity of those
cells. A functional role for AGPs has been further sup- role in higher plant embryogenesis, derives from the
observation that the GNOM (GN ) protein of Arabidopsis,ported ( Willats and Knox, 1996). By treating seedlings
of Arabidopsis with Yariv reagent, which binds specifically which is believed to play a role in Golgi vesicle transport/
trafficking protein, is susceptible to brefeldin A inhibitionwith AGPs, they observed a reduced overall growth of
shoot and root. In roots, this correlated with a reduced (Steinmann et al., 1999). It was seen earlier how brefeldin
A can inhibit targeted wall secretion and polar axislongitudinal cell expansion and increased radial expan-
sion. These data suggest that Yariv binding to AGPs fixation in Fucus (Shaw and Quatrano, 1996), and, sim-
ilarly, gn mutants, defective in GN protein function, areinhibits their biological activity, which may include a role
in the control of cell expansion and organogenesis. also defective in establishing the asymmetry of the first
zygotic division and subsequent apical-basal patterning.Yet further evidence for the importance of cell wall
components in development comes from work with the Golgi vesicle transport proteins such as Sec7 of yeast,
which has similarities to GN, have roles in cell wallcarrot somatic embryogenesis system. One mutant cell
line, ts11, has been identified that fails to undergo embryo- elongation and in cell division, delivering important pre-
cursors for both the plasma membrane and the cell wall,genesis when grown at an elevated temperature, even
under conditions which are inductive for non-mutant as well as other proteins that require directional delivery
to the cell membrane or wall (Shevell et al., 1994). Suchlines (i.e. auxin-free). At elevated temperatures (32 °C),
ts11 embryos arrest at the globular stage. However, it processes require directed and precise delivery of the
vesicle. The work of Pennell et al. (Pennell et al., 1991)was found that developmental arrest at elevated temper-
atures could be bypassed by the addition of culture demonstrates the differential distribution of the JIM8
epitope along the apical-basal axis of the brassica embryo-medium in which fully embryogenic lines had been grown.
The secreted molecule was identified as a 32 kDa protein suspensor complex, and the results of McCabe et al. show
similarly its targeted and polar distribution in the bicellu-with homology to an endochitinase (de Jong et al., 1992).
In search of a substrate for this enzyme, a range of lar embryo–nurse cell complex in the carrot system
(McCabe et al., 1997). It is therefore possible that cellmolecules containing N-acetylglucosamine moities were
added to ts11 cells to find compounds which also rescue wall components such as the JIM8 epitope are crucial for
imparting positional information at the earliest stages ofthe mutant and so might represent natural substrates or
products of the chitinase. Interestingly, it was found that apical-basal axis formation, and require GNOM protein
function for their correct spatial distribution.the mutant could be rescued by the application of lipo-
oligosaccharides to the culture (de Jong et al., 1993). This These results therefore suggest a role for cell wall-
related molecules in regulating important aspects ofclass of molecule consists of an oligosaccharide backbone
of 4 or 5 b-1,4-linked N-acetyl-glucosamine residues with embryogenesis and polarity. Whether fertilization induces
Polarity and signalling in plant embryogenesis 975
targeted secretion of wall-localized regulatory molecules and shows supernumerary suspensor cells (Hobbie et al.,
2000), also lends support to this model. The likely rolein higher plants is still unknown, but is an intriguing
possibility. There will be a return to the relationship of auxin in embryonic patterning will be discussed later.
between targeted secretion, hormonal signalling and
polarity later.
Apical-basal patterning: the embryo-proper and
seedling
Genetic control of embryo-suspensor cell fate determination
The apical-basal pattern is defined by the positioning of
The fates of the apical and basal cells, following zygotic
the shoot meristem and cotyledons, the hypocotyl and
division in Arabidopsis, are clearly distinct. Direct evid-
the root and root meristem. The study of mutants has
ence for a genetic control of suspensor cell identity derives
led to the theory that the embryonic axis is therefore
from studies of mutants in which the suspensor undergoes
partitioned into three main regions; apical, central and
abnormal patterns of cell division, most commonly
basal (Mayer et al., 1991). The shoot meristem and the
ectopic division. In the abnormal suspensor (Schwartz
majority of the cotyledons originate in the apical region,
et al., 1994) and raspberry (Yadegari et al., 1994) mutants
while the central region contributes to the majority of the
of Arabidopsis, the embryo-proper arrests and the sus-
rest of the axis, namely the shoulder of the cotyledons,
pensor subsequently enters into a series of inappropriate
the hypocotyl, the embryonic root, and the vascular,
divisions. Significantly, the modified suspensor takes on
cortex and endodermal root initials of the root meristem.
a variety of characteristics of the embryo-proper.
It is only the quiescent centre, the columella initials and
Ultrastructural analysis has revealed that, in the case of
the central root cap that arise from the clonally separate
the sus mutants, for example, accumulation of storage
hypophyseal cell, the uppermost suspensor cell, whilst the
protein bodies, lipid bodies and starch grains occurs in
rest of the pattern is derived from the embryo-proper
both the embryo-proper and, unusually, the suspensor
(Scheres et al., 1994; Mayer and Ju
¨
rgens, 1998). Despite
(Schwartz et al., 1994). It has also been observed that
the temptation to consider the formation of each of the
AtLTP, which encodes an Arabidopsis homologue of the
three regions as independently regulated events, it will
carrot EP2 lipid transfer protein (Sterk et al., 1991;
become clear that interactions between tissues in each
Thoma et al., 1994), is strongly expressed in the protod-
region are essential for the correct integrated patterning
erm/epidermis of embryos and seedlings but is not
of the whole seedling. For convenience, however, relevant
expressed in the wild-type suspensor. However, it is
features of each of the three regions, respectively, will be
expressed in the peripheral cells of the raspberry embryo-
examined.
proper and suspensor (Yadegari et al., 1994). Even more
Each region follows its own programme of cell divisions
spectacular is the re-differentiation of suspensor cells in
once they have been established, all three being present
the twin (twn) mutants. Here, the suspensor cells reorgan-
by the octant stage. The formation of the O∞ boundary
ize into secondary embryos, following arrest of the
at the quadrant stage creates the upper and lower tiers,
embryo-proper (Vernon and Meinke, 1994). The TWN2
corresponding to the apical and central regions, respect-
gene has now been cloned, and encodes a valyl-tRNA-
ively, whilst the hypophyseal cell is formed by divisions
synthase, though its mode of action remains unclear
in the suspensor. The apical region divides without prefe-
(Zhang and Sommerville, 1997).
rential orientation, while divisions that are perpendicular
It has been suggested that the wild-type embryo-proper
to the axis create the cell files that characterize the central
signals to the suspensor to maintain its differentiated
region. Within the basal region a more stereotyped set of
state, and in the case of the sus and raspberry mutants,
divisions is required to create the root meristem and
this signal is blocked or not produced, and the suspensor
central root cap, such that the fate of any cell in that
embarks on a default pathway of embryonic development
region can be predicted with high probability (Scheres
(Schwartz et al., 1994). In this laboratory a novel mutant
et al., 1994).
of Arabidopsis, designated asf1 (for altered suspensor fate
Through studying the development of each of these
1) that exhibits a novel pattern of inappropriate cell
regions in both wild-type and mutant backgrounds, the
division in the suspensor, and exhibits a reprogramming
different signalling mechanisms involved are becoming
of gene expression and cell differentiation (Fig. 2) has
clearer. Much progress has come from the application of
been identified. Activation of auxin-inducible genes in the
a strategy of mutagenesis and the progressive isolation
modified suspensor leads us to propose a model in which
and characterization of genes that are specifically involved
the mutant phenotype is mediated by the de-regulated
in embryonic pattern formation. It is worthwhile to note
partitioning of auxin between embryo-proper and sus-
that, as the embryonic pattern is reiterated through the
pensor, to activate the observed ectopic cell division
meristems during post-embryonic development, many
(Horne, 1998; Horne and Lindsey, in preparation). The defects that originate in the embryo are often identifiable
in seedling mutant screens.recently described axr6 mutant, which is auxin-resistant
976 Souter and Lindsey
Fig. 2. Histological section through the asf1 mutant embryo of Arabidopsis. Note the supernumerary divisions of the suspensor.
What then are the mechanisms that generate positional GURKE gene of Arabidopsis is also required for the
information to promote region-specific gene expression
correct organization of the shoot apical region (Torres-
patterns? In the remainder of this review article the genes
Ruiz et al., 1996). Strong mutant alleles are unable to
that specify cell fate within the Arabidopsis apical-basal
construct the entire apical region, and even part of the
axis will be examined and evidence for the signalling
hypocotyl, while weaker alleles produce abnormally
events involved considered.
shaped leaves and flowers. The root and radial patterning
is apparently unaffected, even in strong mutant alleles.
The apical region of the embryo
The defect can be traced back to the transition-stage
embryo.
The apical region forms the self-perpetuating shoot meris-
SHOOT MERISTEMLESS (STM ) expression is initi-
tem. A number of genes have been isolated which affect
ated at the late globular stage in the central region of the
the establishment and characteristics of the shoot meris-
embryo apex (Long et al., 1996), and is independent of
tem (Laux and Mayer, 1998). The four inner apical cells
WUS action (Mayer et al., 1998). stm mutants have fused
at the 16-cell stage Arabidopsis embryo start to express
organs originating from the shoot meristem, which indi-
the WUSCHEL (WUS) gene, which is an early marker
cates a role for STM in restricting cells with a shoot
of the shoot meristem cell fate (Mayer et al., 1998). WUS
meristem fate from participating in organ formation
is expressed through a number of asymmetric divisions
(Long et al., 1996; Long and Barton, 1998; Endrizzi et al.,
which also produce the future cotyledonary primordia,
1996). STM is expressed by only a specific set of cells
though expression now becomes restricted to the group
within the apex of the embryo, and has been shown to
of cells at the apex of the embryo which will become the
be a member of the KNOTTED homeodomain proteins
shoot meristem (Laux et al., 1996). The WUS gene has
(Long et al., 1996). AINTEGUMENTA (ANT ) mean-
been shown to encode a novel homeodomain protein
while is expressed by the two cell groups which flank the
(Mayer et al., 1998). A possible role for WUS is in
shoot meristem, and which will eventually form the
maintaining the pluripotent capacity of the shoot meris-
tem precursor cells (Lenhard and Laux, 1999). The cotyledons (Elliott et al., 1996).
Polarity and signalling in plant embryogenesis 977
CLAVATA1 (CLV1) is also expressed in the embryonic Hadfi et al. used this same B. juncea culture system to
look at the effects of auxin (IAA), an anti-auxin (PCIB),shoot apex, from the heart stage onwards. CLV1 has
been cloned and shown to encode a predicted membrane- and an auxin transport inhibitor (NPA) (Hadfi et al.,
1998). When auxin was supplied, ball-shaped or cucum-bound kinase receptor (Clark et al., 1996), which suggests
a role in signalling. CLV1 acts independently of STM ber-shaped embryos resulted, possibly because the
embryo, flooded with exogenous auxin, is unable to(Long and Barton, 1998), although it is thought that
they act competitively between each other to regulate the establish the auxin gradients which are essential for
morphogenesis. The anti-auxin PCIB inhibited cotyledonbalance between undifferentiated cells and organ forma-
tion in response to positional information (Clark et al., growth so that either only one or no cotyledons developed.
1996; Laux and Schoof, 1997). clv1 mutants have enlarged
Correct hypocotyl and radicle growth was also found to
meristems in post-embryonic development. PRIMORDIA
require auxin action and movement. Furthermore, when
TIMING (PT) also causes an enlargement of the shoot
globular-stage embryos were treated with exogenous
meristem, though it acts from the globular stage onwards.
NPA, axis duplication was seen, whilst a later application
From analysis of pt clv1 double mutants, it is clear that
produced split-collar or collar-like cotyledons. These
these two genes work in different pathways despite their
results confirm the findings of Liu et al. (Liu et al., 1993),
apparently similar roles. CLV1 is therefore probably not
and help clarify the model of auxin movement which they
involved, like PT, in early meristem formation processes,
first proposed: continuous auxin transport removes auxin
which is supported by the temporal differences in their
from the area between the two emerging cotyledons, and
phenotypes (Mordhorst et al., 1998).
supplies the auxin back to the cotyledonary primordia.
One gene which does interact with STM is ZWILLE
Auxin removal starts in the central apical region of the
(ZLL, Moussian et al., 1998). The zll mutant shoot
globular or early transition embryo, and continues asym-
meristem is initiated correctly, but STM expression is
metrically across the apex of the embryo.
either restricted or down-regulated, resulting in cells which
Inhibition of auxin transport therefore blurs the posi-
follow other development fates, possibly due to the influ-
tional information that is created by its normally precise
ence of other spatial cues. ZLL is therefore required to
redistribution, resulting in increased cell division through-
maintain meristem cell identity within the apex, possibly
out the shoot apex. These findings indicate that auxin
through acting as a translational control. The ZLL gene
translocation is a prerequisite for the radial globular
is expressed in the vascular precursor cells, situated just
embryo to progress to the bilaterally symmetrical heart
below the meristem primordia, from early stages until
stage embryo. Similar results were found by Fischer et al.
leaf primordia are established, when presumably the
for morphogenesis of the embryo of the monocot wheat
meristem can maintain itself.
(Fischer et al., 1997).
The analysis of these genes has shown that position-
dependent cell fate specification is achieved from the late
The central and basal regions of the embryo
globular stage onwards. It appears that meristem forma-
The central part of the embryo produces the majority of
tion occurs through the activation of genes which specify
the embryonic axis, and a number of mutants have been
cell fate in a spatially precise manner. A key area of
found that are defective not only in the generation of
research has been to identify possible signals that may
hypocotyl and root, but also the radial axis within this
activate and regulate the expression of the genes described
region. Indeed, the radial organization of the seedling is
above. One signal molecule which has proven particularly
established during embryogenesis, to define the cellular
interesting is auxin.
patterning that runs throughout the hypocotyl and the
Auxin has been proposed as a key signal molecule in
root (Scheres et al., 1995).
providing positional information within the apical region
The MONOPTEROS (MP) gene is required for the
of the embryo, particularly during the transition period
formation of the hypocotyl, root, root meristem, and root
from globular to heart stage. Liu et al. first reported the
cap; products of the central and basal regions of the
use of auxin transport inhibitors to study development in
embryo (Berleth and Ju
¨
rgens, 1993). The MP gene is also
cultured zygotic embryos of Brassica juncea (Liu et al.,
required for correct cell axialization and development of
1993). They showed that inhibition of auxin transport at
aligned vascular strands (Przemeck et al., 1996). The MP
the globular stage leads to the formation of embryos
gene has been cloned and found to encode a transcription
which lack bilateral symmetry at the heart stage. Bilateral
factor with nuclear localization sequences and a DNA
symmetry is established when the two cotyledons form
binding domain which is highly similar to a domain which
either side of the shoot meristem region. Instead of two
binds auxin-inducible promoters. In fact MP has
cotyledons, embryos developed with fused and collar-like
the same binding specificity as AUXIN RESPONSE
cotyledons, which interestingly phenocopied known auxin
FACTOR1 (ARF1; Ulmasov et al., 1997a), which is atransport-defective mutants pin1 (Okada et al., 1991) and
gnom (Steinmann et al., 1999). transcription factor that binds to auxin response elements
978 Souter and Lindsey
(AREs) within promoters of auxin-inducible genes. cause either a break in the auxin transport system, or a
diffusion of the auxin gradients and short-range signalsExpression of MP is initially in broad domains in the
embryo, becoming eventually confined to the procambial which maintain the correct gene expression patterns.
Does the central region signal to the basal region totissues (Hardtke and Berleth, 1998). This is similar to
PIN1 expression, although PIN1 has been shown not to enable the correct development of the latter? There is
growing evidence that signalling between embryonicrequire MP gene function (Steinmann et al., 1999; Palme
and Ga
¨
lweiler, 1999). MP is therefore required for correct domains establishes the positional information that allows
cells to activate fate-determining gene expressioncell axialization in the early embryo, and for correct
vascular development in the later stages of embryogenesis programmes.
The BODENLOS (BDL) gene of Arabidopsis has beenand during post-embryogenic development, through its
likely role in regulating the transcription of auxin respons- implicated in auxin-mediated apical-basal patterning pro-
cesses (Hamann et al., 1999). Development in bdl mutantsive genes.
Whether the central region of the mp mutant fails to is disrupted at the two-cell stage, when the apical cell
divides horizontally rather than vertically. Hypophysealrecover from its altered axialization and, therefore, cannot
recover hypocotyl and root formation, or if the basal development is subsequently compromised, leading to
mutants that lack an embryonic root (quiescent centreregion’s failure to generate the root meristem is because
of a lack of aligned vascular primordia, is not known. and central root cap). Hypocotyl development is also
affected in some mutant individuals. Interestingly, bdlThere is a large amount of evidence to indicate that auxin
is required for root formation (Boerjan et al., 1995; mutants show insensitivity to the synthetic auxin 2,4-D
within the same range as axr1 seedlings, which suggestsCelenza et al., 1995; Reed et al., 1998). However, if the
central section of the embryo does not develop correctly, that auxin-mediated signalling is required to specify the
fate of the basal region of the embryo. Furthermore, thethen the corollary of this for the basal region must be
considered. The MP gene is required for correct alignment BDL gene only affects the embryonic root, since bdl
seedlings can still form lateral root meristems. The modelof the vascular tissue, and cell axialization within the
hypocotyl (Przemeck et al., 1996). It is therefore open to put forward for the action of BDL suggests that auxin is
involved in determining hypophyseal cell fate at the octantsuggestion that the defective polar auxin transport system
may cause downstream effects on root development in stage. Later, at the heart stage, the quiescent centre
signals to the cells above it to block differentiation,the mp mutant.
Within the radially swollen fass and hydra mutants, conferring the fate of root meristem initials (Hamann
et al., 1999). Studies show that ablation of the quiescentmultinumerary cotyledons and apical meristem regions
develop ( Torres-Ruiz and Jurgens, 1994; Topping et al., centre in seedlings results in the differentiation of the
adjacent initial cells (van den Berg et al., 1997).1997). hydra mutants also exhibit a form of axis duplica-
tion through their hypocotyl region, which is radially auxin resistant6 (AXR6) mutant seedlings are arrested
in their development soon after germination, and lack aswollen and highlighted by separated vascular strands
running through the tissue (Topping et al., 1997). These root and hypocotyl (Hobbie et al., 2000). The stronger
axr6–1 allele has more severe vascular defects than thephenotypes may result as secondary effects from impaired
auxin transport and/or auxin action within these tissues. weaker axr6–2, and tends to produce only one cotyledon.
Mutants are also more resistant to auxin, undergoingHormonal studies of fass show that it has an average of
2.5 times more free auxin than wild-type plants (Fisher irregularly timed and oriented cell divisions, which are
first observed in the early embryo. Principally the sus-et al., 1996). It is possible that the high level of auxin
may trigger higher levels of ethylene—it has been demon- pensor is disrupted by cell divisions which create radial
layers rather than the characteristic single file of seven tostrated that transcripts encoding pea ACC synthase isoen-
zymes, for example, are rapidly induced by exogenous nine cells. As a result, the hypophyseal cell does not form
correctly, and the distinction between the embryo properIAA (Peck and Kende, 1998). Interestingly fass roots
elongate 2.5-fold more when removed from the plant and and the suspensor is lost. Within the central region the
vascular precursor cells fail to establish during the globu-cultured than when left intact on the plant. This suggests
that a signalling from the upper part of the plant inhibits lar stage, a defect which is also seen in monopteros
(Przemeck et al., 1996). AXR6 therefore represents afass root length. A shorter root phenotype is a common
response to exogenous ethylene. Like fass, hydra also has novel gene which causes defects in cell division patterns
within the embryo and the suspensor. It is feasible thata short root phenotype, which is rescued by treatment
with silver ions, inhibitors of ethylene action (M Souter the aberrant cell divisions occur because there are prob-
lems in auxin-mediated positional or cell-fate signalling.and K Lindsey, unpublished data). Clearly then, these
two mutants have hormonal imbalances which have led Indeed, the similarities between the phenotypes of the
mp, bdl and axr6 mutants suggests that they may functionto alterations in the number and size of pattern compon-
ents. The radially swollen apical and central regions may in similar pathways (Hobbie et al., 2000).
Polarity and signalling in plant embryogenesis 979
The HOBBIT (HBT ) gene is required for correct hypo- carrier, whose cellular localization needs to be precise as
it might be expected to determine the course of auxin flow.physeal cell formation (Willemsen et al., 1998). hbt
embryos have incorrect hypophyseal cell development To date, seven PIN genes have been identified, whilst
more than ten different PIN homologues have been foundfrom the quadrant stage onwards, so that by the heart
stage activation and formation of the lateral root cap in Arabidopsis. PIN genes have also been identified in
maize, rice and poplar, with high conservation betweenlayer has not occurred. Mature embryos lack a quiescent
centre and columella root cap. Root meristem formation monocot and dicot species indicating a conserved function
for PIN proteins throughout the plant kingdomis not only defective in the embryonic root, but also in
the seedling, where secondary roots fail to form, even (K Palme, personal communication). In Arabidopsis mem-
bers of this family of transporters have different expres-when cultured. HBT, unlike BDL, is therefore required
for root meristem formation both embryonically and sion patterns within time and space, and so offer the
plant a means by which auxin can be transported pre-post-embryonically. It is unclear at present whether the
exact role of the HBT gene is to specify the basal region cisely. PIN1 has shown to be linked to the development
of vascular tissue, which follows Sach’s canalization hypo-or if it is required for the correct division programme
that the hypophysis must go through to produce the root thesis (Sachs, 1991). PIN1 is located at the basal end of
cells within the vascular stele (Ga
¨
lweiler et al., 1998).meristem and root cap.
The correct patterning of the root therefore would During embryogenesis, PIN1 becomes polarized in its
expression pattern at the mid-globular stage, before theappear to depend on signalling between the central and
basal regions of the embryo, as well as the cell-cell two cotyledons have started to develop. By the heart
stage the pattern very much resembles the pattern it takescommunication which is established once the root meris-
tem becomes active. throughout the rest of the plant’s post-embryonic develop-
ment, forming a characteristic Y shape from the two
cotyledons to the basal end of the embryo (Steinmann
et al., 1999). PIN1 expression in MP is not affected,
A synthesis: auxin as a positional and a patterning
which suggests that its targeting to the basal membrane
signal molecule
does not require the MP ARF; although correct axializ-
ation of vascular strands does. In contrast, PIN1 localiz-Clearly the results presented so far implicate auxin as
playing a major role in embryogenesis, providing posi- ation in the gnom background is severely affected,
indicating that directed vesicle secretion is required, astional information for the co-ordination of correct cellular
patterning from the globular stage onwards. Auxin has indicated above (Steinmann et al., 1999).
Recent direct evidence for the existence of auxin gradi-proved a difficult molecule to localize in tissues, being
highly diffusible and occurring in both active and inactive ents that correlate with a physiological response is
described by Uggla et al. ( Uggla et al., 1996, 1998). These(conjugated) forms (Normanly and Bartel, 1999). Shoot
meristems and leaf primordia are regarded as the main authors used the highly sensitive technique of GC-MS to
show the presence of a steep radial gradient of auxinsites of synthesis, with the polar auxin transport system
holding the key to many responses. Vascular tissue forma- across the vascular cambium in Pinus sylvestris (L.). This
lateral meristem contributes to the secondary growth oftion follows the flow of auxin (Aloni, 1987; Mattsson
et al., 1999), which is canalized into files of cells so that the plant which is activated at the start of each new
growing season. The gradient of auxin across the tissueconnected vascular strands form (Sachs, 1991). Auxin
controls much of post-embryonic development, especially appears to provide positional information for the develop-
ing tissue, with possibly other morphogen gradients orplant architecture, through the modulation of meristem
activity and cell expansion in response to environmental cell–cell communication systems determining the precise
cell division patterns and cell fates required to producefactors (Hobbie, 1998).
Auxin transport therefore holds a key to our under- the specific cell types that exist within this tissue. The
significance of this work lies in the fact that auxin appearsstanding of much of auxin’s role within the plant. The
chemiosmotic theory proposes that auxin requires an to be providing positional information to a developing
and patterning tissue.influx and efflux carrier in order to move through cells
and tissues. This requires anion symport (influx) and Studies on the POLARIS gene of Arabidopsis provide
further information on the role of auxin in definingefflux carrier proteins. AUX1 is a candidate for the influx
carrier (Bennett et al., 1996), whilst the PIN gene family position and cell activities during embryonic and seedling
root development. This gene was identified by promoterconstitutes the putative transport protein of the efflux
carrier complex. For a comprehensive review of auxin trapping, leading to the activation of GUS expression in
the basal region of the embryo, from heart-stage onwards;transport the reader is referred to Lomax et al. (Lomax
et al., 1995) and Palme and Ga
¨
lweiler (Palme and and subsequently in the seedling root tip (Topping et al.,
1994). It encodes a very short transcript that appears toGa
¨
lweiler, 1999). Here the focus will be on the efflux
980 Souter and Lindsey
regulate root sensitivity to ethylene, to modulate root Therefore, ascorbic acid oxidase may help to maintain
the meristem’s identity, whilst the auxin maximum allows
growth (S Casson, P Chilley, K Lindsey, unpublished
the maintenance of the meristem itself, which is the source
data). Although this GUS fusion gene was originally
of the pattern in the root. Directional signals are respons-
considered to be a root-meristem marker, it was found
ible for the cell fate specification within the root, with the
to be expressed in the appropriate position, i.e. in a
more differentiated cells within a cell file signalling to the
polarized pattern, even in mutants such as gnom, hydra
daughters of the meristem initials to initiate cell fate
and hobbit that either lack root meristems or have defect-
processes (van den Berg et al., 1995).
ive root meristem patterning (Topping and Lindsey, 1997;
Willemsen et al., 1998). The POLARIS gene promoter is
up-regulated by auxin very rapidly, within minutes, and
Conclusions
its spatial expression pattern represents a useful marker
Both intrinsic and extrinsic signals help to establish
of auxin localization in the root ( Topping and Lindsey
polarity in the early plant embryo. The asymmetric zygotic
1997, and unpublished data). Interestingly, correct spatial
division fixes polarity, which may rely on the asymmetric
patterning of POLARIS expression is disrupted signific-
delivery of cell wall components, possibly AGPs, and
antly only in the most severe, ball-shaped gnom seedlings,
which requires GN in order to execute it. The fate of the
suggesting that these individuals, but not the more con-
basal cell is now established, and is marked in species as
ical-shaped gnom seedlings, are defective in polar auxin
diverse as Brassica napus and carrot, by the expression of
transport ( Topping and Lindsey, 1997). This is consistent
JIM8-binding AGPs, which may provide cell fate informa-
with the observed defective PIN1 localization in gnom
tion to the suspensor.
embryos (Steinmann et al. 1999), and suggests that auxin
Once the Arabidopsis embryo has reached the globular
provides a chemical framework for the patterning of
stage, containing roughly 100 cells, the auxin transport
apical-basal gene expression and cellular activity in both
mediator PIN1 becomes polarized in its expression.
embryo and seedling.
Again, directional vesicle transport, via GN, is required
Kerk and Feldman have proposed a biochemical model
for the correct localization of the protein within the cell
for auxin’s role in initiating and maintaining the quiescent
membrane, which is expressed in a polar pattern at the
centre of the maize root meristem ( Kerk and Feldman,
basal end of the cell. The establishment of the auxin
1995). The quiescent centre is located at the distal part
transport system is a prerequisite for patterning events in
of the root, and is also the most distant tissue from the
the apical region of the embryo at the beginning of the
path of polar auxin transport. Ascorbic acid is a com-
transition from globular to heart stage embryo. Later in
pound which is necessary for the transition from G
1
to S
development it is required for hypocotyl and root forma-
phase in the cell cycle, and which is broken down by
tion and maintenance, with auxin responsiveness essential
ascorbic acid oxidase (AAO). AAO mRNA is increased
in order for the positional information provided by the
in response to auxin, which was shown to have higher
polar transport of auxin to be interpreted into pattern
levels in the quiescent centre than surrounding cells,
elements. Short-range cell–cell communication is required
determined by immunolocalization of auxin in the root
for many of the cell fate decisions, but these clearly
tip. These results suggest that auxin is influencing AAO
depend on the presence of information indicating their
levels within the root meristem, and that this ensures the
position within the apical-basal axis. Regional signalling,
continued stem cell ability of the quiescent centre.
involving genes such as BDL and other auxin response
The influence of auxin on the activity of the root
pathways such as AXR6 and MP, is also crucial to the
meristem is also elegantly demonstrated through studies
correct cell division patterns and cell fate decisions which
by Sabatini et al. (Sabatini et al., 1999). The authors
need to occur in the central and basal regions. Analysis
utilized a synthetic auxin-responsive promoter construct,
of mutants such as asf1 and axr6 suggests strongly that
termed DR5, which consists of seven tandem repeats of
auxin signalling is required for the correct cell divisions
a auxin-responsive element fused to the b-glucuronidase
and cell fate of the suspensor to be established.
(GUS) reporter gene ( Ulmasov et al., 1997b). The DR5
Once the meristems in the root and shoot have been
reporter is activated rapidly by auxins within the
established, their self-maintaining ability is determined by
10−8–10−4 M range. Expression of this gene fusion shows
the expression of a number of recently discovered genes,
a ‘maximum’ in the distal root meristem region, in the
although the signalling systems that regulate their expres-
columella initials of wild-type seedlings. By studying the
sion are far from fully understood. Germination activates
effect of known mutations on the position of the auxin
the meristems to reiterate the programmes of patterning
maximum, they suggest that pattern and polarity in the
initiated in the embryo, programmes which can be altered
Arabidopsis root is mediated by an auxin-dependent
by the inhibition or antagonism of auxin. There are some
organizer, which is established by the auxin maximum
differences in the gene expression programmes that specify
embryonic and post-embryonic patterning, as the differentlocated distal to the vascular tissue boundary.
Polarity and signalling in plant embryogenesis 981
polarization and embryo patterning in Fucus using genistein,
temporal patterns of CLAVATA1 and PRIMORDIA
a potent inhibitor of protein tyrosine kinase. Developmental
TIMING clearly highlight.
Biology (in press).
A number of studies of the molecular mechanism of
de Jong AJ, Cordewener J, Loschiavo F, Terzi M,
auxin in the seedling have been highlighted. However, it
Vandekerchove J, van Kammen A, de Vries S. 1992. A carrot
somatic embryo mutant is rescued by chitinase. The Plant
is important to note that these mechanisms are established
Cell 4, 425–433.
in the embryo, and their interruption or disturbance at
de Jong AJ, Heidstra R, Spaink HP, Hartog MV, Meijer EA,
this early stage cannot always be corrected during post-
Hendriks T, Lo Schiavo F, Terzi M, Bisseling T, Van
embryonic development. Continued study of the mechan-
Kammen A, De Vries SC. 1993. Rhizobium lipooligosacchar-
isms that control the movement and action of auxin, and
ides rescue a carrot somatic embryo mutant. The Plant Cell
5, 615–620.
its possible relationship with cell wall contruction and
Deng X-W. 1994. Fresh view of light signal transduction in
composition, can be expected to lead to the discovery of
plants. Cell 76, 423–426.
more upstream events and downstream targets which are
Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S,
required for patterning in plant embryogenesis.
Roberts K, Scheres B. 1993. Cellular organization of the
Arabidopsis thaliana root. Development 119, 71–84.
Eady C, Lindsey K, Twell D. 1995). The significance of
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Acknowledgements
transcription and generative cell differentiation. The Plant
We gratefully acknowledge financial support for our work on
Cell 7, 65–74.
embryogenesis from BBSRC, EC (FPIV contract BIO 4 CT
Elliott RC, Betzner AS, Huttner E, Oakes MP, Tucker WQJ,
960217) and The Gatsby Charitable Foundation. MS is
Gerentes D, Perez P, Smyth DR. 1996. AINTEGUMENTA,
supported by a BBSRC CASE studentship in association with
an APETALA2-like gene of Arabidopsis with pleiotropic roles
Shell Forestry.
in ovule development and floral organ growth. The Plant
Cell 8, 155–168.
Endrizzi K, Moussian B, Haecker A, Levin JZ, Laux T. 1996.
The SHOOT MERISTEMLESS gene is required for mainten-
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