Download Intercellular adhesion and cell separation in plants

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2003? 2003
26?977989
Original Article
Intercellular adhesion and cell separation in plants
M. C. Jarvis
et al.
Plant, Cell and Environment (2003) 26, 977–989
REVIEW
Intercellular adhesion and cell separation in plants
M. C. JARVIS1, S. P. H. BRIGGS1 & J. P. KNOX2
1
Department of Chemistry, Glasgow University, Glasgow G12 8QQ, Scotland, UK and 2Centre for Plant Sciences, University
of Leeds, Leeds LS2 9JT, England, UK
ABSTRACT
INTRODUCTION
Adhesion between plant cells is a fundamental feature of
plant growth and development, and an essential part of the
strategy by which growing plants achieve mechanical
strength. Turgor pressure provides non-woody plant tissues
with mechanical rigidity and the driving force for growth,
but at the same time it generates large forces tending to
separate cells. These are resisted by reinforcing zones
located precisely at the points of maximum stress. In dicots
the reinforcing zones are occupied by networks of specific
pectic polymers. The mechanisms by which these networks
cohere vary and are not fully understood. In the Poaceae
their place is taken by phenolic cross-linking of arabinoxylans. Whatever the reinforcing polymers, a targeting mechanism is necessary to ensure that they become immobilized
at the appropriate location, and there are secretory mutants
that appear to have defects in this mechanism and hence
are defective in cell adhesion. At the outer surface of most
plant parts, the tendency of cells to cohere is blocked,
apparently by the cuticle. Mutants with lesions in the biosynthesis of cuticular lipids show aberrant surface adhesion
and other developmental abnormalities. When plant cells
separate, the polymer networks that join them are locally
dismantled with surgical precision. This occurs during the
development of intercellular spaces; during the abscission
of leaves and floral organs; during the release of seeds and
pollen; during differentiation of root cap cells; and during
fruit ripening. Each of these cell separation processes has
its own distinctive features. Cell separation can also be
induced during cooking or processing of fruit and vegetables, and the degree to which it occurs is a significant quality
characteristic in potatoes, pulses, tomatoes, apples and
other fruit. Control over these technological characteristics
will be facilitated by understanding the role of cell adhesion
and separation in the life of plants.
Adhesion between cells is a fundamental characteristic of
multicellular organisms, and a central aspect of both plant
and animal morphogenesis. However, most plant cells
adhere to one another in a quite different way from animal
cells (Knox 1992), and the way in which intercellular adhesion influences cell development and is regulated during
plant growth has no direct parallel in the animal kingdom.
In short, the adhesion between plant cells that is established
as cells are formed during cytokinesis is generally maintained, and the active adhesion of existing cell walls takes
place only very rarely. Cell separation takes place in a limited and controlled manner and plant growth involves the
co-ordinated and directed expansion of adherent cells.
Recent advances have extended our understanding of
the molecular framework for cell adhesion in plants. There
has also been much progress towards understanding how
the structures responsible for cell adhesion can be locally
dismantled, often with considerable specificity, to allow the
limited separation of plant cells under developmental control. Examples of this are leaf and floral abscission, dehiscence in seed pods, opening of intercellular spaces, loss of
root cap cells and fruit ripening. Abscission and dehiscence
processes, which are interesting models for cell separation
in general, have been reviewed by Roberts et al. (2000),
Roberts, Elliot & Gonzalez-Carranza (2002) and Patterson
(2001). These processes will be considered here in only
enough detail to place them into the context of cell separation in general. With the characterization, in Arabidopsis
and other species, of mutations affecting cell adhesion,
some of which result in dramatic alterations to the phenotype (Delarue et al. 1997; Rhee & Somerville 1998; Thompson et al. 1999; Zinkl & Preuss 2000), our understanding of
how plant cells adhere and separate is now poised for rapid
advances.
The technological implications of a fuller understanding
of plant cell adhesion and separation are considerable. Premature dehiscence or pod shatter is a problem that was
overcome during the domestication of most seed crops but
which is still troublesome in modern varieties of legumes
and oilseed rape. The texture and processing characteristics
of tomatoes, apples and a number of other fruit species
depend on spontaneous cell separation during ripening.
Texture in potatoes and pulses reflects the extent of cell
separation induced by cooking or processing.
Key-words: cell adhesion; cell separation; intercellular
spaces; pectin; plant morphogenesis; texture.
Correspondence: M. C. Jarvis. Fax: +44 (0)141 3304888; e-mail:
[email protected]
© 2003 Blackwell Publishing Ltd
977
978 M. C. Jarvis et al.
INTERCELLULAR ADHESION
Adhesion between growing cells
Intercellular adhesion in plants arises in a different way
from adhesion between animal cells. With exceptions such
as pollen on the stigmatic surface (Zinkl & Preuss 2000),
most plant cells do not come together and stick: they are
Cell 3
formed in an adherent state and remain so throughout their
lifetime. After meristematic cells divide, the growth of the
daughter cells is a concerted process in which neighbouring
cells expand in a co-ordinated manner and adherent walls
remain fused along the line of the middle lamella as they
expand (Knox 1992). A schematic view of cell plate formation, plant cell adhesion and its loss is shown in Fig. 1.
Figure 1. Schematic diagrams showing
plant cell cytokinesis and intercellular space
formation. A membrane-bound cell plate
grows from the centre of a cell to fuse with
the plasma membrane/primary cell wall to
produce two daughter cells. As primary cell
wall is deposited on either side of the cell
plate it becomes a middle lamella and region
of intercellular attachment. For a intercellular space to form a region of the primary wall
of the parent cell must be degraded allowing
the new middle lamella to link up with the
older middle lamella of the parent cell. This
space may be filled with material or open up
to form an intercellular air space (a distinctive feature of parenchyma tissues).
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 977–989
Intercellular adhesion and cell separation in plants 979
It is not clear how the extension of the two adherent cell
walls is co-ordinated. A simple biophysical feedback loop
may be all that is required: if one of the two walls expands
less it will then carry more of the load and be further above
the threshold stress for expansion, provided that the middle
lamella resists shear. That is not to say that no mechanism
exists for the co-ordinated development of walls of adjacent
cells. The absence of shear at the middle lamella is necessary to prevent rupture of plasmodesmatal connections
between the adjacent cells during growth. The textured wall
structure and patterns of pectic epitopes observed around
plasmodesmata on both plasma-membrane faces of adherent tomato pericarp cell walls (Orfila & Knox 2000)
may reflect some, as yet unknown, mechanism of the coordination of cell wall extension in the region of pit fields.
Isotropic expansion of tissues is the exception rather
than the rule. Any anisotropy in growth means that two
walls of a single cell must extend at different rates, even
though each wall is co-ordinated in its extension with the
adherent wall of an adjacent cell. It may be easier to think
of the directional expansion of plant tissues as being quantized not into single cells, but into pairs of adherent cell
walls.
Do plant cells always adhere to the same cells?
It might be thought that intrusive growth is an exception to
the rule that plant cells do not slide past one another.
Examples of intrusive growth are the penetration of the
pollen tube through stylar tissue (Jauh & Lord 1996), the
differentiation of tracheids (Kalev & Aloni 1998) and laticifers (Serpe, Muir & Keidel 2001) and the elongation of flax
fibre cells into the cortical parenchyma (Roland et al. 199).
However, the intrusive cell expands between the invaded
cells by tip growth (Yang 1998; Ryan et al. 1998), so it is
only the intruding apex that actually moves, separating the
walls of the cells on either side. Along all the rest of its
length the intrusive cell remains firmly attached to its new
neighbours. At the growing tip therefore the original cell–
cell adhesion is dismantled and a new cell–cell bond is
established. Although intrusive growth is not a case of
whole cell movement it is a case of the formation of new
cell contacts that are not formed at cytokinesis.
A further exception to the rule that plant cells remain in
contact with the same cells throughout development is the
behaviour of pollen on the stigma surface. Here adhesion
occurs de novo. It can take different forms. In species such
as Arabidopsis with dry stigmas, the initial adhesion event
is rapid (seconds), species-specific and mediated by lipophilic molecules so that it can function in the absence of
free water (Zinkl et al. 1999). Pollen hydration and alterations to the underlying stigmatic cells then follow. Compatible pollen tubes growing down the hollow style of lily
flowers adhere to its inner surface through the co-operative
action of a number of water-soluble macromolecules
including a pectic fraction and a small lipid transfer protein
(Park et al. 2000; Mollet et al. 2000)
Why do epidermal cells not adhere on contact?
When two leaves or stems of the same plant come into
chance contact they do not fuse, but remain distinct. Likewise pollen tubes that germinate on an epidermal surface
other than the stigma do not interact with it. However
during flower formation in many species, fusion of floral
organs such as carpels is part of the normal process of
development. It also seems that some form of adhesion of
parenchymatous tissues is an early event in the formation
of a graft union between cut faces of two compatible plants.
This stage of the grafting process has been little studied
because the key events determining compatibility occur
later when vascular continuity is becoming established
(Jeffree & Yeoman 1983). Thus the ability of a plant to
delineate its own surface, the boundary between itself and
non-self, seems to reside in the epidermis but to be capable
of being inactivated under developmental control in special
circumstances such as the fusion of floral parts. Some
understanding of the molecular articulation of these processes has recently emerged from experiments on mutant
plants with defects in cuticle formation.
In certain of these mutants, plant surfaces fuse that would
not be expected to do so (ectopic organ fusion) and pollen
can become hydrated on these surfaces when normally it
hydrates only on the receptive stigma surface (ectopic pollen hydration). This fusion of normally separate juvenile
organs is a feature of the eceriferum (cer) mutants in Arabidopsis and the glossy mutants in maize, which were originally identified from defects in cuticular wax formation
(Post-Beittenmiller 1998). The FIDDLEHEAD gene in
Arabidopsis, identified originally from ectopic organ fusion
and ectopic pollen hydration in the mutant fdh1 phenotype,
encodes a probable b-ketoacyl CoA synthase involved in
chain lengthening of fatty acids, a requirement for cuticular
lipid synthesis (Yephremov et al. 1999; Pruitt et al. 2000).
Altered cuticle formation was likewise found in the
adherent1 mutant in maize, previously recognized by postgenital organ fusion and pollen hydration like fdh1 (Sinha
& Lynch 1998).
With some exceptions, the mutated genes encode
enzymes involved in long-chain lipid synthesis (Xu et al.
1997; Fiebig et al. 2000, Rashotte, Jenks & Feldmann 2001).
CER2 encodes a novel, possibly regulatory protein (Xia,
Nikolau & Schnable 1997), although the cer2 phenotype
shows aberrant cuticular wax composition as in other cer
mutants (Negruk et al. 1996). The Arabidopsis ALE1 gene,
necessary both for normal cuticle formation in embryos and
to prevent organ fusion, encodes a probable serine protease
(Tanaka et al. 2001). It might be suggested that ectopic
organ fusion is normally prevented by some non-cuticular
function of long-chain lipids, for example in signalling. This
is unlikely in view of the observation that transgenic Arabidopsis plants expressing a fungal cutinase (Sieber et al.
2000) showed strong postgenital organ fusion despite having no abnormality in the synthesis of lipid monomers.
How does the cuticle maintain the distinctness of vegetative tissues in contact with one another? It is not simply
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 977–989
980 M. C. Jarvis et al.
a matter of preventing water movement (Kerstiens 1996).
Transmission of molecular signals across the divide is certainly involved in both pollen hydration and the normal
fusion of floral organs (Lolle et al. 1997). Only at a later
stage in floral organ fusion are symplastic connections
formed (). It has been suggested that small polar molecules
are transmitted across the epidermal walls to signal contact
between floral organs that will fuse (Siegel & Verbeke
1989), and that the permeability of the cuticle may be tuned
to control their passage (Lolle et al. 1997). Whatever the
signal molecules whose passage from cell to cell is prevented by the cuticle, its presence seems to be the key
factor that prevents adhesion between plant cells when
none is programmed to occur.
In addition to the phenomenon of organ fusion in lipid
elongation mutants, some of them show other developmental defects. One such mutant, fdh1 in Arabidopsis,
is affected in trichome differentiation (Yephremov
et al. 1999), whereas hic mutants in Arabidopsis (Gray
et al. 2000) and a range of cer mutants in barley (PostBeittenmiller 1998) are altered in stomatal abundance.
These observations prompted suggestions that the transport of signal molecules involved in differentiation is influenced by cuticular composition. The HIC gene (Gray et al.
2000) is particularly interesting because its product may
form part of a signal cascade sensing ambient CO2 concentrations and controlling stomatal abundance in response
(Retallack 2001). Since the cuticle is also a CO2 barrier, a
reasonable hypothesis might be that a shared CO2 sensing
mechanism forms part of a feedback loop controlling the
development of the cuticle itself.
Biomechanics of intercellular adhesion
Although adhesion between plant cells restricts the capacity for individual cell movement, cell adhesion makes it
possible for plants to grow into structures of greater height
and mechanical robustness than anything in the animal
kingdom. Plant tissues become mechanically strong by two
strategies, both of which are fundamentally dependent on
adhesion between cells (Jarvis & McCann 2000).
One strategy is typical of wood, in which compressive as
well as tensile stresses are carried by the cell walls. Because
of scaling factors this strategy is the only one possible in a
tree, but it requires a much greater investment of material
in thick, rigid secondary cell walls that can withstand compressive buckling. Bending stresses on a tree are also translated into shear stresses between adjacent files of cells, and
these are resisted by lignification of the pectic middle
lamella (Hafren, Daniel & Westermark 2000). Wood under
heavy bending loads frequently splits at this point (Thuvander & Berglund 2000). However, in this review we shall
not concern ourselves further with intercellular adhesion in
wood.
In the other strategy, typical of soft plant tissues in rapid
growth, turgor pressure carries all compressive stresses on
the tissue and the primary cell walls are kept permanently
in tension. This makes it possible for the cell walls to be
thin and flexible, allowing economy in structural material.
However, because a sphere is the shape of lowest energy
for a flexible-walled, pressurized cell, turgor generates secondary stresses that tend to tear the cell away from its
neighbours at each corner, pulling it towards a spherical
shape. The magnitude of these cell separation stresses is
comparable with the tensile stress within each cell wall, and
to withstand them considerable intercellular adhesion
strength is necessary at the cell corners (Jarvis 1998).
It is common to think of the middle lamella as a line of
adhesive gluing the cells together, and certainly the question of intercellular adhesion is linked to the structure of
the middle lamella. However the idea that the middle
lamella glues two cells together is misleading in more than
one respect. We use glue to stick together surfaces that
were initially apart, but two plant cells joined along the
middle lamella have never been apart. Furthermore, as
explained above, the stresses that tend to separate cells
are not distributed evenly over the cell surface but are
concentrated at the cell corners (tricellular junctions) and
at the corners of intercellular spaces (Jarvis 1998). Precisely
at these points there are reinforcing zones which can be
distinguished under the electron microscope and which, as
will be seen later, differ in polymer composition from
both the primary cell walls and the middle lamella. It is
these reinforcing zones that carry the turgor-imposed
stress and are the first line of defence against cell separation
(Parker et al. 2001). We must focus on their mechanical
properties if we wish to understand intercellular
adhesion and separation from a biomechanical point of
view. It follows that the bulk composition of the cell wall
will normally tell us little about the strength of intercellular
adhesion.
Ontogeny of cell junctions in plants
In general terms, one of two possible histories can be
ascribed to an adherent pair of cell walls occurring in a
plant. These contrasting origins are exemplified in epidermal and cortical cells where anticlinal cell walls (at right
angles to the plant surface) originate in cell division and the
periclinal cell walls (parallel to the plant surface) originate
largely from cell wall assembly during cell expansion. When
the cell plate is formed within a dividing cell, it expands
radially by the accretion of material at the edges until it
reaches the existing cell walls (Heese, Mayer & Jurgens
1998). At the edges of the expanding cell plate, nascent
microfibrils and matrix polymers arriving from both daughter cells have the opportunity to mingle before becoming
consolidated, in ways that are still unclear, into the completed structure of the newly inserted wall (Heese et al.
1998; Otegui & Staehelin 2000; Verma 2001). However,
when two adherent cell walls expand together, the noncellulosic polymers that will provide the increased area of
middle lamella must diffuse through the interstices in the
outermost microfibril layer of each of the cell walls.
We may anticipate that where a new cross-wall meets a
pre-existing wall, the adhesion of walls at the tricellular
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 977–989
Intercellular adhesion and cell separation in plants 981
junction may have different characteristics. Structural elements of the existing wall will remain intact across the
edges of the cross-wall unless modified biochemically at the
junction point, and these existing and new cross walls may
have different adhesion characteristics. This is indeed
observed: enzymic cell separation in elongating tissues,
such as hypocotyls, releases long files of cells that have
separated along longitudinal walls but remain attached to
one another at the ends by means of transverse walls
(Cocking 1960) and see also Fig. 2.
a
b
c
Figure 2. Regulation of plant cell separation. (a, b) In parenchyma systems, cells separate to a limited extent producing intercellular space at tricellular junctions (indicated by arrows). (a) A
transverse section of a pea stem immunolabelled with antipectin
monoclonal antibody JIM5 indicating all primary cell walls. (b) An
equivalent section immunolabelled with antipectin monoclonal
antibody LM7 indicates a pectic homogalacturonan epitope that is
restricted to cell walls lining intercellular space and particularly the
corners of tricellular junctions at points of cell adhesion/separation. Micrographs courtesy of Dr Bill Willats. For details see Willats et al. (2001). (c) Three cells from a cell file of mung bean
hypocotyl isolated by treatment with pectin lyase (0.9 U mL-1)
remain adhered at transverse cell walls (arrows). Scale bars (a,
b) = 100 mm. (c) = 250 mm.
Structural polymers responsible for
intercellular adhesion
Except in graminaceous plants the principal macromolecules of the middle lamella are pectins, accompanied by
proteins (Jarvis 1984; Carpita & Gibeaut 1993). It is possible to consider the middle lamella and reinforcing zones
simply as regions of the extracellular matrix from which
microfibrils are absent, and which therefore lack cellulose.
The microfibrils of the primary cell walls are themselves
arranged in a more or less lamellate structure (Carpita &
Gibeaut 1993) and it has been suggested that pectins attach
each of the microfibril layers to the next (Jarvis 1998). In
tobacco cells habituated to the herbicide dichlobenil, which
inhibits cellulose biosynthesis, conventional microfibrils
were absent and the middle lamella could not be distinguished ultrastructurally from the primary walls on either
side of it (Sabba, Durso & Vaughn 1999)
That is not to say that the polymers of the middle lamella
and those of the reinforcing zones, at tricellular junctions
and the corners of intercellular spaces, are identical with
the polymers of the interfibrillar matrix of the primary cell
wall. They are not, and the differences are characteristic. In
a wide range of dicotyledonous plant tissues linear, lowester pectic homogalacturonans are most abundant in the
middle lamella, and especially in the reinforcing zones (e.g.
Knox et al. 1990; Roy, Vian & Roland 1992; Liners & Van
Cutsem 1992; Willats et al. 1999; Parker et al. 2001).
Recently the introduction of the monoclonal antibody
LM7 (Willats et al. 2001) has made it possible to distinguish
the pectic galacturonans of the reinforcing zones from
those of the middle lamella and primary cell wall. LM7
labelling in Pisum and other angiosperms was restricted to
the reinforcing zones and, at lower intensity, to the adjacent
cell wall surface lining intercellular spaces (see Fig. 2). LM7
binds to a partially methyl-esterified epitope of homogalacturonan that is abundant in samples with a random pattern
of methyl-esterification (Willats et al. 2001). Random esterification is characteristic of the process by which methyl
ester groups are added to pectins in the Golgi, whereas deesterification of galacturonans of higher initial ester content, by the action of most plant pectin methylesterases,
generates long galacturonan blocks completely free from
methyl ester groups (Goldberg et al. 1996). However, plant
pectin methyl esterases have varied action patterns
(Catoire et al. 1998; Goldberg et al. 2001) and the LM7
epitope may be generated in muro.
Pectins of the middle lamella and reinforcing zones are
also characterized by low or zero levels of the branched
polymer segments rhamnogalacturonan-I (RGI) (Jones,
Seymour & Knox 1997; Willats et al. 1999; McCartney et al.
2000) and rhamnogalacturonan-II (RGII) (Williams et al.
1996; Matoh et al. 1998). Of the wide variety of structural
and non-structural cell wall proteins, a number have been
located in the middle lamella or lining intercellular spaces
of specific plant tissues but not throughout cell walls. For
example, labelling of carrot roots with antibodies to the
extensin-2 class of hydroxyproline-rich glycoproteins indi-
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982 M. C. Jarvis et al.
cated that they were restricted to tricellular junctions in
certain instances (Swords & Staehelin 1993; Smallwood
et al. 1994; Smallwood, Martin & Knox 1995). This is also
the case for polyamine oxidases (Wisniewski et al. 2000;
Laurenzi et al. 2002). It is possible that these components
function together to generate specific cell wall matrix properties at cell junctions.
Cross-linking of pectic polysaccharides
There must be a mechanism for the middle-lamella and
reinforcing-zone polymers (contributed by both cells) to be
linked into a coherent network. Otherwise there would be
nothing to hold the two cells together. Whatever their pattern of esterification, low-ester pectic polymers have a relatively high affinity for calcium ions. Both the middle
lamella and, in particular, the reinforcing zones show elevated levels of calcium imaged by SIMS (Rihouey et al.
1995) or EELS microscopy (Huxham et al. 1999). Lowester galacturonans gel in vitro with calcium but the
mechanical properties and porosity of these gels depend
strongly on whether the esterification pattern is random or
blockwise, independent of the degree of esterification (Willats et al. 2001).
A reasonable hypothesis might be that calcium-linked
gels of low-ester pectins, rich in the LM7 homogalacturonan epitope, might be responsible for the cohesion of the
reinforcing zones and hence for intercellular adhesion.
However there is evidence that this is not the only mechanism involved. Extraction of dicot cell wall preparations
with calcium-chelating agents brings pectin into solution,
but normally much less than half of the total pectin present
(Goldberg et al. 1996), and only limited cell separation
occurs under these conditions (Cocking 1960; McCartney
& Knox 2002). The presence of further, presumably covalent, intermolecular linkages must therefore be proposed.
Borate diester links between RGII segments are unlikely
because RGII was not detected in these locations (Matoh
et al. 1998). Whatever the nature of these intermolecular
cross-links, the enzymes catalysing their formation must
reside in the middle lamella of expanding cells and in the
reinforcing zones as they are formed – although similar
cross-links and similar enzyme systems may also be found
in the primary cell wall.
The three-dimensional cross-linked network formed in
this way could be purely pectic, or it might include pectic
chains covalently linked to other insoluble polysaccharides
on both sides of the middle lamella. The formation of either
of these kinds of network would also explain the insolubility of native pectins (‘protopectin’) in the primary walls of
dicot cells (Goldberg et al. 1996).
A covalent polymer network can be disrupted either by
breaking the cross-links between the chains or by cleaving
the chains themselves between crosslinks. Enzymic cleavage of pectic galacturonan chains is an efficient method for
pectin solubilization (Keegstra et al. 1973), and generally
separates dicot cells (Ramana & Taylor 1994; Zhang, Henriksson & Johansson 2000) Interestingly, in a detailed study
of polygalacturonase action on carrot cell walls, even
though endo-polygalacturonase degradation removed most
of the polymer material from the middle lamella of carrot
and left it greatly weakened, a sparse reticulum of unidentified material remained (Tamura & Senda 1992). Enzymic
cleavage of the galactan side-chains characteristic of some
pectins neither solubilized these pectins nor separated cells
with convincing efficiency (Redgwell & Harker 1995).
These observations support the central role of cross-linked
galacturonans in intercellular adhesion, but do not show
how they are cross-linked.
Mild alkaline extraction, under conditions suitable for
cleaving ester or labile amide linkages, solubilizes a substantial pectic fraction (Goldberg et al. 1996) and also separates some of the cells that are not separated by chelators.
The nature of the alkali-labile bonds is unclear. Galacturonoyl ester links to hydroxyl groups on other chains have
repeatedly been suggested (Jarvis 1982; Fry 1986; Kim &
Carpita 1992; Brown & Fry 1993; MacKinnon et al. 2002),
although such ester-linked fragments have not so far been
isolated and characterized. The total fraction of substituted
pectic carboxyl groups significantly exceeded the fraction
esterified with methanol in cell walls of maize (Kim &
Carpita 1992) and potato (Mackinnon et al. 2002).
Although these additional substituents have not been identified, other hydroxyls on the same galacturonate residue
(giving a lactone) can be ruled out on grounds of stability,
suggesting that an inter-residue or intermolecular linkage
is present. Amide linkages to peptides or polyamines could
also have suitable properties for cross-linking pectic chains
(Perrone et al. 1998). Bound polyamines are present in cell
walls (Geny et al. 1997) and inhibition of polyamine biosynthesis affects cell wall integrity and intercellular adhesion
(Berta et al. 1997), whether directly or indirectly.
All these observations are consistent with the idea that
dicot cells of many types adhere to one another through the
formation of a three-dimensional pectic gel network, which
has features in common with the pectic network of the
primary cell wall and is responsible for the insolubilization
of native pectins (Goldberg et al. 1996). This network
appears to contain cross-links of at least three types: chain
aggregates held together by calcium and possibly other cations; covalent links having the alkali lability of esters or
possibly amides; and alkali-resistant covalent links with
characteristics similar to glycosidic bonds.
Beet, spinach and related species differ from other dicots
in having an additional cross-linking mechanism in their
cell walls, which involves the peroxidase-catalysed formation of dimeric feruloyl esters between the pectic sidechains, stabilizing the cells against separation (Waldron
et al. 1997b) In grasses, cereals and related monocots the
abundance of pectins in the primary cell wall is relatively
low and cell separation by chelating agents is not generally
possible. Feruloyl and other hydroxycinnamoyl esters are
abundant, however. Their dimers cross-link cell wall
polymers, in this case arabinoxylans (Ng, Greenshields &
Waldron 1997), and fluorescence microscopy readily
demonstrates their presence at tricellular junctions, like
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 977–989
Intercellular adhesion and cell separation in plants 983
calcium-bound pectic galacturonans in dicots (Waldron
et al. 1997a).
The spatial pattern of polymer cross-linking matches the
location of mechanical stresses imposed by turgor pressure
(Jarvis 1998). In many tissues of most dicots that have been
studied, LM7-binding pectins and associated calcium ions
are concentrated exactly where the stress is greatest, at the
tricellular junctions and the corners of the intercellular
spaces. In beets a similar spatial pattern is shown by ferulate esters (M. Marry, unpublished results). The small
amount of low-ester pectic galacturonan in oat roots and
the abundant ferulate in water chestnut (Cyperaceae, with
cell walls like a cereal) also show this spatial distribution
(Waldron et al. 1997a). This evidence is consistent with the
idea that intercellular adhesion depends on the formation
of a specific type of cross-linked pectic network at a specific,
mechanicallly stressed extracellular location, the reinforcing zones. This is the case even though the polysaccharides
concerned differ between dicots and graminaceous plants,
and though the enzymes responsible for covalent crosslinking must also differ.
Reduced intercellular adhesion is a common feature of a
group of Arabidopsis mutants in which cytokinin responses
are disrupted (Delarue et al. 1997; Faure et al. 1998). It is
also found in the tumorous shoot development (tsd) mutants
that have defects in what appears to be an intercellular
signalling system involving class I knox homeobox genes
and cytokinin sensitivity (Frank et al. 2002). In some tissues
of these mutants the reduced intercellular adhesion results
in ‘vitreous’ texture as enlarged intercellular spaces become
filled with apoplastic liquid, an effect that can be phenocopied by excess cytokinin (Faure et al. 1998). In the tsd
mutants the organization of the shoot apical meristem is
drastically disrupted, apparently due to failure of interlayer
signalling. It was pointed out by Frank et al. (2002) that the
developmental disorganization and the loss of intercellular
adhesion seemed to be connected, and Shevell et al. (2000)
suggested that normal cell adhesion was required for the
intercellular signalling needed to co-ordinate morphogenesis (Gisel et al. 1999). However the chain of cause and
effect in the complex emb30 and tsd phenotypes is not clear.
CELL SEPARATION
Intercellular adhesion, secretion and
plant morphogenesis
It seems reasonable to infer the existence of a mechanism
for secreting polysaccharides and proteins to precisely the
required locations through the plasma membrane and the
separate barrier of the primary cell wall. How this happens
is unknown. The Arabidopsis gene EMB30 (GNOM)
encodes a protein required for the targeting of secretion to
specific parts of the cell surface. Mutants in EMB30 are
defective in cell adhesion as well as in polar cell expansion
and the control of plant form (Shevell, Kunkel & Chua
2000). The defect in cell adhesion was associated with accumulation of pectic polysaccharides in the intercellular
spaces, as if the pectins themselves were secreted normally
but a factor that retained them in the reinforcing zones of
the wild-type was absent (Shevell et al. 2000).
In the tomato mutant Cnr, which shows defective cell
adhesion in the pericarp of ripe fruit, altered spatial patterns of galacturonan esterification were accompanied by
blocked secretion, across the plasma membrane, of a pectic
polymer rich in a(1,5)-L-arabinan (Orfila et al. 2001). A
Nicotiana callus with reduced cell adhesion similarly
showed low levels of pectic arabinan, and lost galacturonan
from the middle lamella into the culture medium (Iwai,
Ishii & Satoh 2001) There was no suggestion that the
tomato arabinan was directly involved in cell adhesion as
the LM6 epitope was restricted to the primary cell wall, and
was absent from the middle lamella and reinforcing zones,
in the wild type (Orfila et al. 2001). This epitope is characteristic of the primary walls of proliferating cells, rather
than walls that have expanded (Willats et al. 1999; McCartney et al. 2000; Bush et al. 2001). However it is possible that
the secretory defect also affected other polymers more
directly involved in network formation at the points of
maximum stress.
As outlined above, there are a number of circumstances in
which plant cells with primary cell walls separate in a highly
controlled way to lead to the separation of organs, the
formation of single cells or the opening of an intercellular
space.
Intercellular spaces
Intercellular spaces filled with air form the gas transport
system inside plants. Their presence in leaves, connecting
stomata with the sites of photosynthesis, dates back to some
of the earliest known land plants (Edwards, Kerp & Hass
1998). Intercellular channels in stems make root respiration
possible in anaerobic soils (Moog 1998). ‘Smart’ intercellular channels with variable resistance control gas exchange
in legume root nodules (Minchin 1997). Water-filled intercellular spaces provide pathways of low hydraulic resistance for apoplastic flow out from the xylem (Van der
Weele, Canny & McCully 1996).
These systems are well known. However, it is only
recently, with the introduction of new microscopy techniques and the microcasting method for visualizing interconnected intercellular spaces (Mauseth & Fujii 1994), that
they have been demonstrated to form complex and highly
patterned three-dimensional networks that could almost be
considered as a third vascular system in plants (Pyke, Marrison & Leech 1991; Prat et al. 1997). Individual intercellular spaces usually arise by limited separation of cells at the
corners (tricellular junctions) where the mechanical driving
force is clearly turgor. There are exceptions. Constriction
of growing maize mesophyll cells by bands of microfibrils
appears to pull cell walls apart in the vicinity of the bands
(Apostolakos, Galatis & Panteris 1991), a phenomenon
also observed in the formation of aerenchyma although in
that tissue the large intercellular spaces are formed by cell
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 977–989
984 M. C. Jarvis et al.
lysis rather than by schizogenous cell separation (Van der
Weele et al. 1996).
Where intercellular space formation is driven by turgor,
there must be a mechanism to permit separation only at
those points on the cell periphery at which an intercellular
space will form, and only to the degree required to give an
intercellular space of the appropriate size (Roland 1978;
Jeffree, Dale & Fry 1986; Kollöffel & Linssen 1984). Enzymic degradation of pectin is precisely targeted, beginning
in the primary wall of the cell that originated earliest in the
developmental sequence and soon spreading to the middle
lamella; see Fig. 1 for a schematic view of this process (Jeffree et al. 1986). As an intercellular space opens the turgorgenerated cell separation stress decreases (Jarvis 1998) but
this does not seem to be what limits the eventual size of the
intercellular space. Whatever its stage of development, it
always has reinforcing zones rich in linear galacturonan at
its corners (Roy et al. 1992), so as it expands these reinforcing zones must move outwards. Since the galacturonan that
binds the LM7 monoclonal antibody is restricted to the
reinforcing zones and to the region lining the intercellular
space (Willats et al. 2001), it is possible that this polymer is
continuously deposited just ahead of the point where the
cell walls diverge; the reinforcing zone later splits to allow
their divergence and its constituent polymers remain as a
residue on the inner face of the intercellular space.
The formation of a single intercellular space requires a
degree of co-ordination between the three cells involved,
with targeted extracellular metabolism at specific locations
on the surface of each cell. The formation of a continuous
line or network of intercellular spaces (Prat et al. 1997)
requires co-ordination of such activities among large numbers of cells. It is not clear how this is achieved. In simple
elongating tissues such as hypocotyls the intercellular
spaces are mainly longitudinal and it is necessary only that
these should extend as the cells extend. Leaves are more
complex and some form of positional signalling seems
likely.
Tucker 1998; reviewed in Hadfield & Bennett 1998) but
other enzymes such as b(1,4)-D-glucanases are also found
(e.g. Koehler et al. 1996; Gonzalez-Bosch, del Campillo &
Bennett 1997; Trainotti et al. 1998; Burns et al. 1998;
McManus et al. 1998; Lashbrook et al. 1998), and may be
connected with degradation of vascular tissue. Many of
these enzymes are isoforms specific to abscission zones.
Seed pods in the Cruciferae and other families open by
cell separation along a distinct dehiscence line to release
the seed. Premature dehiscence or pod shatter results in
considerable losses of yield in oilseed rape and legume
crops (Liljegren et al. 2000). In Arabidopsis and oilseed
rape, pod dehiscence is accompanied by expression of a
specific polygalacturonase gene between those cells that
separate (Petersen et al. 1996; Jenkins et al. 1996, 1999).
Cell separation is also involved in the development and
release of pollen. In the quartet mutants of Arabidopsis
(Rhee & Somerville 1998) microspores at the tetrad stage
fail to separate. This is associated with a defect in pectin
degradation.
The promoter of the polygalacturonase gene involved in
pod dehiscence in Arabidopsis, fused to a GUS gene, was
expressed in rape not only along the pod dehiscence line
but also at the point of attachment of the seeds and in
mature anthers (Jenkins et al. 1999). Sharing of genes for
cell separation between anthers and the dehiscence zone of
pods may explain why it has been so difficult to breed rape
varieties that are resistant to pod shatter, since male sterility is a likely consequence. In contrast to abscission of other
plant parts, these cell separation phenomena were not
inducible by ethylene (Jenkins et al. 1999). They share that
feature with seed shedding in the Gramineae (Sargent,
Osborne & Dunford 1984), another instance of a cell separation phenomenon whose inhibition was a key event in
crop domestication. It is possible that cell separation in the
release of pollen and seeds is under quite separate control
from other cell separation phenomena in plants.
Fruit ripening
Cell separation in abscission and the release of
pollen and seeds
Abscission and dehiscence have been discussed in recent
reviews (Roberts et al. 2000, 2002; Patterson 2001) and will
therefore be treated only briefly here. Abscission of leaves
and floral parts involves separation along the line between
two previously defined groups of cells, one group belonging
to the organ to be discarded and the other belonging to the
surviving part of the plant. Commonly at least one vascular
strand must be cleaved in addition to other tissues.
It appears that the formation of a functional abscission
zone is specified by signals from the innermost meristem
layer to the outer layers (Szymkowiak & Irish 1999). Once
an abscission zone is established in vegetative plant tissues,
the cascade of gene expression characteristic of the abscission process is triggered by ethylene and retarded by auxin
(Mao et al. 2000; reviewed by Taylor & Whitelaw 2001). The
gene products include polygalacturonases (e.g. Hong &
Most fruits soften on ripening, but they do so in different
ways (Redgwell et al. 1997). It is common, but not universal, for the cell wall to be degraded and swell. Pectin degradation is particularly conspicuous in those species where
swelling of the the cell walls occurs (Redgwell et al. 1997),
but enzymes with degradative activity on a wide range of
polysaccharides have been implicated in different species
and the role of the polygalacturonases in swelling and
weakening the cell wall is not wholly clear (Hadfield &
Bennett 1998; Brummell & Harpster 2001).
The presence of active polygalacturonases in ripening
fruit suggests that cell separation might be expected, and in
some species this is indeed observed. Examples are apples
(Roy, Jauneau & Vian 1994b; Roy et al. 1995; Siddiqui &
Bangerth 1996); cherries (Batisse et al. 1996); plums (Taylor
et al. 1993); kiwifruit (Hallett, MacRae & Wegrzyn 1992)
and tomato (Roy et al. 1992, 1994a). Cell separation
requires dissolution of the middle lamella, but this does not
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 977–989
Intercellular adhesion and cell separation in plants 985
occur evenly: calcium-rich zones at tricellular junctions or
the corners of intercellular spaces remained intact after the
rest of the middle lamella had been degraded (Roy et al.
1994a, b, 1995), and the regions around plasmodesmata
were also resistant to degradation (Hallett et al. 1992).
While cell separation is not the principal mechanism of fruit
softening in these species, the relative extents of cell separation and wall breakdown modulate the eventual texture
in ways that can be important for quality. In apples, high
levels of cell separation lead to undesirably mealy texture
and to loss of flavour because if cells separate instead of
rupturing, the flavour components within them are not
released. In processing tomatoes, facile cell separation is
necessary for quality in puree.
The question then is the relative extent of pectin degradation in the middle lamella and the primary cell wall,
together with the degree to which pectins matter in maintaining the structural integrity of the primary cell wall. Pectin degradation in ripening tomato occurs in irregular
blocks of the cell wall, some of which appear to encompass
the middle lamella (Steele, McCann & Roberts 1997). Pogson et al. (1992) found polygalacturonase in both cell wall
and middle lamella, but pectin methylesterase was
restricted to the cell wall. Since only de-esterified pectins
are degraded by polygalacturonase, the de-esterification
step might in principle be limiting although the pectins of
the middle lamella in tomato never have a high degree of
esterification (Roy et al. 1994a). It is evident that mechanisms exist for targeting polygalacturonases and possibly
other pectin-degrading enzymes to specific zones in the
extracellular matrix, but the details are not yet clear. A
better understanding of this phenomenon could provide
the basis for a rational approach to quality improvement in
tomatoes and other fruit.
Cooking of vegetables
The thermally induced b-elimination reaction, which occurs
during the cooking of vegetables, is a relatively specific
method of depolymerizing pectic galacturonans esterified
on the galacturonoyl carboxyl group with no known effect
on other covalent bonds within the plant cell wall. During
cooking it is commonly accompanied by chelation of divalent cations by organic acids released from within the cell,
but even then it is not normally sufficient to induce cell
separation after the cells are dead and the mechanical stress
induced by turgor pressure is no longer present. However
in vegetables that contain starch, like peas, beans and potatoes, starch swelling pressure can substitute for turgor pressure and induce visible cell separation (Jarvis, MacKenzie
& Duncan 1992; Binner et al. 2000). Starch swelling pressure is a necessary condition for cell separation in cooked
vegetables, but not a sufficient one. Starch swelling pressures in cooked potatoes are comparable with turgor pressures in raw potatoes, where cell separation does not occur
(Jarvis et al. 1992). Degradation of the pectic polymers
involved in cell adhesion is therefore also necessary (Parker
et al. 2001), and the degree to which it occurs can be mod-
ulated by raw material characteristics and by the details of
cooking conditions.
With processed potatoes and some other vegetables it is
common to introduce a precooking step at a temperature
below 100 ∞C, during which pectin methyl esterases are
activated and the degree of methyl-esterification of pectins
is reduced. This diminishes the susceptibility of the pectic
galacturonans to b-eliminative degradation and, if sufficient
uncomplexed calcium is available, provides new sites for
ionic aggregation of pectic chains. It has also been suggested (Hou & Chang 1997) that certain pectin methyl
esterases may generate intermolecular ester linkages by a
trans-esterification reaction, thus contributing to covalent
network formation; however, the nature of any such new,
non-methyl esters requires clarification before this can be
confirmed.
The ‘mealy’ textures that result from a high degree of cell
separation are an important quality factor in potatoes,
sweet potatoes and legumes – good or bad depending on
the preference of specific consumer groups. Cell separation
also has a major influence on the suitability of potatoes and
peas for processing and because it can be manipulated
through both raw material quality and process variables,
there is an opportunity for a better scientific understanding
of the process to make a major contribution to technological development.
CONCLUSION
Intercellular adhesion is central to the way in which plants
grow and take shape, and controlled separation of specific
cells is an essential feature of plant development. Mutants
in which cell adhesion or separation is abnormal are promising tools for the dissection of mechanisms in plant morphogenesis, and are also likely to be useful in improving the
quality of raw materials derived from plants.
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Received 7 August 2002; received in revised form 17 January 2002;
accepted for publication 24 January 2002
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 977–989