J. Embryol. exp. Morph. 83, Supplement, 179-195 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
179
Polarity, calcium and abscission: molecular bases
for developmental plasticity in plants
By A. J. TREWAVAS 1 , R. SEXTON 2 AND P. KELLY 1
1
Botany Department, University of Edinburgh, Mayfield Road, Edinburgh,
EH9 3JH, U.K.
2
Biology Department, University of Stirling, Stirling, U. K.
TABLE OF CONTENTS
Summary
Introduction
Polarity, electrical fields and calcium
Vascular tissue as an internal organizing polar axis
Are vascular materials freely available to all plant cells?
A basic polarity change must accompany root and shoot branching and regeneration . The role of excision
Molecular basis of polarity
Molcular effects of excision
Conclusion
Abscission as an example of plastic development
Many factors control abscission and plant development.
The regulation of abscission by growth substances
Molecular aspects of abscission
Phase changes in development
References
SUMMARY
This article considers novel aspects to plant development. It starts by outlining the concept
of plastic development. It then discusses polarity in plants arguing for the vascular tissue and
surrounding cells as a major internal polar axis. The requirement for a change in polarity in
this system when regeneration occurs is discussed and molecular mechanisms for specification
and changing of polarity are outlined. The process of abscission is then discussed as an example
of plastic development and it is concluded that many factors control abscission.
Molecular changes in abscission zone cellulase are summarized and the notion of two phases
in abscission are developed and generalized to other systems of development in plants.
INTRODUCTION
Plant and animal development is similar in that both commence their life cycles
as single fertilized cells. But marked differences occur in the way this cell develops
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A. J. TREWAVAS, R. SEXTON AND P. KELLY
into the adult. These differences originated as a result of early evolutionary
decisions concerning the basic life styles. Plants and animals separated in
evolutionary terms between one to two x 109 years ago when both were single
celled. One view is that the primordial eucaryote was a plant cell with chloroplasts
and a cell wall. Loss of these two organelles precipitated the animal/plant separation.
The primitive plant cell was therefore photosynthetic and thus autotrophic.
Since light was, theoretically, evenly distributed, there has never been strong
evolutionary pressure on plants to encourage the facility for movement.
Although motile algal cells are well known and mosses and ferns retain motile
sperms, genuinely motile cells have been lost altogether in angiosperms. Thus for
the great majority of plants the life cycle is conducted in their initial growth
position; plants are sessile organisms.
There is a price to be paid for remaining sessile. Predation, overgrowing and
a variable, often hostile environment have to be tolerated rather than avoided.
The strategies adopted by plants to deal with these problems were, firstly, to
lengthen the embryonic phase permanently so that development could continue
throughout the life cycle. This continued embryogenesis was permitted by the
evolution of an entirely novel tissue, the meristem. The capability for self
replication was built into the meristem so that growth and development could
take place at a large number of different points. Even if a number were
eliminated by predators, development could continue at others. Plants are
modular organisms; that is, they comprise repetitions of the same basic modular
structure. In the shoot the module is the leaf plus subtended bud or meristem,
and below ground repetitions of root meristems. Growth is accomplished by the
addition of more modules and continues throughout the life cycle (Harper &
Bell, 1979; Trewavas, 1983a).
The second strategy was the evolution of remarkable regenerative prowess.
Although many plants rely on dormant buds to recover from environmental or
predator damage others have evolved mechanisms for the reorganization of
shoot or root meristems from mature cells in many different tissues. Such
regenerative prowess is possible because of the very limited cell and tissue
specialization observed in all plants. Specialization and location of critical functions in one or two tissues, as observed in animals, makes the whole organism
very vulnerable to even limited predator damage. Thus plants have no central
controlling (nervous) system and as a consequence the various growing and nongrowing meristems enjoy a great degree of autonomy in their behaviour. There
is often an overlap in function in many of the main tissues (e.g. stems can be
photosynthetic, prop roots etc.). Many cells in plants can easily be reinitiated
into cell division to form callus and from these shoot and root meristems can be
regenerated. Organization in plants is a labile phenomenon.
Third, to accommodate to a changing environment plants (ideologically)
learnt to adapt their development to cope. The environment in all its forms is
Molecular basis for developmental plasticity in plants
181
used by plants as signals to change development. As a consequence the size and
form of any individual plant can be extremely plastic. Of necessity plant cells are
sensitive to a much wider range of stimuli than any known animal sensory system.
These properties along with others have been considered a number of times
(Trewavas, 1979,1981,1982a, b) and are aptly described as plastic development.
This term is meant to contrast with the highly controlled, specialized and
canalized character of development clearly observable in animals. Plastic
development is the means whereby the sessile plant copes with predation and a
hostile environment. Animals solved these problems by specialization and movement, thus attempting to avoid predation altogether.
In this article we want to consider aspects of developmental plasticity. In
animal embryological development the production of many cell types each with
specific protein products leads naturally to theories of development which emphasize the unfolding of expression of specific genes. In plants with their very
limited number of cell types, their paucity of cell and tissue specialization and an
obvious lability in organization it is by no means obvious that the same molecular
mechanisms are paramount. For this reason we consider here aspects of plasticity, emphasizing polarity, and using leaf abscission as a typical example of plastic
development.
POLARITY, ELECTRICAL FIELDS AND CALCIUM
Vascular tissue as an internal organizing polar axis
Establishment of an axis or polarity is fundamental to plant development. The
major directions of plant growth are often considered to be gravitationally controlled and polarity is then discussed with reference only to the main shoot/root
axis which is already established in the dry seed. We think there is good reason
to suppose that the vascular tissue represents an internal organizing and polar
axis more basic than that of gravity. The evidence, admittedly limited at present,
which supports this view is as follows.
1. The direction of growth of the vascular tissue in both shoot and root is in
the main direction of growth of the organ. This is also the case for young leaves
(Esau, 1953) and evidence is available which shows that procambial strands
determine the new point of initiation of leaf primordia in the shoot apex itself
(Larson, 1975). Procambial strands like all vascular elements are believed to
be initiated by pre-existing vascular tissue (Esau, 1953).
2. The complexity of vascular tissue patterns can be dependent on shoot or
root size or vice versa (Trewavas, 1982b).
3. Jablonski & Skoog (1954) found that tobacco pith fragments underwent cell
division on simple media providing they already contained some vascular
tissue. In the absence of vascular tissue no cell proliferation occurred.
4. Lang (1965) has summarized many examples of homeogenetic induction in
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A. J. TREWAVAS, R. SEXTON AND P. KELLY
vascular tissue development. For example, a cut stem can reorganize its cambial ring by a progressive growing together of the two cut ends through newly
produced callus. Cambial cells can induce adjacent ones to become like themselves. There is evidence that cambial rings may have clockwise or anticlockwise polarity.
5. Sachs (1968) has carried out numerous experiments demonstrating how
developing vascular strands can repel one another suggesting each has the
capability of spatial recognition.
6. The generation or regeneration of both branch and adventitious roots commences in cells immediately adjacent to the vascular tissue.
7. The vascular tissue actively carries out a two-way transport in its own
direction. It acts as a sink for photosynthate in leaves. Both of these suggest
an established polarity within the system itself.
Are vascular materials freely available to all plant cells?
The vascular system transmits a mixture rich in photosynthate, minerals,
amino acids and growth substances (Pate, 1975; King, 1976). Are these freely
available to the stem and root cells through which they pass? All of the extensive
evidence available suggests that they are not. If materials are released at all there
is great selectivity about what is allowed out (Pate, 1975). Thus exogenously
applied nitrate taken up by roots induces branch root formation only in the
treated zone. Once in the vascular tissue it is not readily available to induce
branch root formation elsewhere (Drew, Saker & Ashley, 1973). The root and
shoot vascular tissue has 10- to 100-fold higher concentration of auxin than
surrounding cortical cells (Greenwood, Hillman, Shaw & Wilkins, 1973; Pengelly & Bandurski, 1981) and so on. Aphids with good reason penetrate the
vascular tissue and not the surrounding cortex. There is exchange between the
two components of the vascular system, the xylem and the phloem (Pate, 1975).
Since mature xylem is composed of dead cells there must, therefore, be additional permeability constraints to prevent non-specific leakage of vascular materials.
What these are can be deduced from studies on tropic bending.
The vascular bundles of coleoptiles are surrounded by a single ring of cells
containing the phototropic pigment (references in Trewavas, 1981); vascular
bundles in cocklebur, castor bean or corn are surrounded by a single ring of cells
containing the geotropic sensor, the starch grain (Salisbury, Sliwinski, Mueller
& Harris, 1982). Presumably the function of these is to release vascular
materials into growing cells in a controlled fashion thus initiating tropic bending.
But their main function must surely be that of restricting the flow of vascular
materials out of the vascular tissue. These 'bundle sheath' or pericycle cells in
stems and roots must therefore have a polarity for transporting materials in the
main direction of the vascular system and it may be suggested that the organizing
and polar properties of the vascular system may originate from these cells. It is
intriguing that the cells carrying out polar transport of auxin have recently been
Molecular basis for developmental plasticity in plants
183
reported to be located around the vascular tissue (Jacobs & Gilbert, 1983).
A basic polarity change must accompany root and shoot branching and regeneration. The role of excision
The breakage of dormancy in shoot buds or the phenomenon of root branching
must involve a fundamental change in polarity since the outgrowth is at right
angles to the prevailing polarity of the vascular system and their surrounding
cells. Before the growth of dormant lateral buds occurs their auxin content
increases, reflecting perhaps a polarity change in the cells surrounding the
vascular tissue and a leakage of auxin outwards (Van Overbeek, 1938). A similar
polarity change can be presumed to accompany the regeneration of adventitious
root formation and even the process of abscission. This latter event often involves some growth and cell division in a narrow band of cells situated at right
angles to the main vascular polarity (Sexton & Roberts, 1982).
How is this accomplished? When branch roots are induced they grow out into
the direction of the incoming stimulus and adventitious roots can occasionally be
induced to form on stems by simply keeping the outside very moist. The suggestion of this is that polarity may be reorientated by changing the direction of ion
fluxes or other electrical parameters through the critical cells. Dormant buds, in
intact plants, have with difficulty been induced to grow by very careful application of very high cytokinin levels to the bud itself (Guern & Usciati, 1972).
Cytokinin is known to act to mobilize materials inwards to treated cells. However
the easiest way to greatly stimulate the process of adventitious or branch root
formation, regeneration, bud outgrowth or abscission is simply to excize the
tissue. Thus Ballard & Wildman (1963) induced cell division in dormant buds by
excision and treatment with sucrose; Tran Thanh Van (1981) demonstrated the
regeneration of buds, roots, flowers or hairs in epidermal tissue providing it was
excised; excision of abscission zone tissue induces abscission in 2-3 days (Sexton
& Roberts, 1982) and so on.
Excision (or wounding) obviously has dramatic effects and it is pertinent to ask
what these effects are. 'Excision elicits a complex series of irreversible metabolic
changes beginning within one minute and unfolding over many hours' (MacNicol, 1976). These metabolic changes include changes in ion fluxes, initiation
of action potentials and enhanced production of ethylene, specific changes in
enzyme activity and protein synthesis and eventually cell division (Davies &
Schuster, 1981). Excision of growing root segments completely inhibits growth
within minutes and considerably slows that of shoots in 10-30 min (Hanson &
Trewavas, 1982). Davies & Schuster (1981) demonstrated that an excision
stimulus moves in both directions in wounded plants at a minimal speed of
3cm/min. The suggestion here must be that of a low-grade action potential
depolarizing cell membranes for very considerable distances from the wound.
How this results in changes in polarity will now be considered.
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A. J. TREWAVAS, R. SEXTON AND P. KELLY
Molecular basis of polarity
Studies on the developing Fucus zygote have clearly outlined a mechanism for
understanding the molecular basis of polarity. This initially apolar egg commences polarized growth and cell division within 24 h of fertilization. The initial
phase of polarity is instituted by an unequal distribution of cytoplasmic calcium
between the two ends of the zygote. Because calcium is a relatively immobile ion
in cytoplasm this leads to a net electrical difference between the two ends of the
egg, one (the future growing end) becoming more positive than the other. This
electrical difference institutes self-electrophoresis of internal charged cytoplasmic constituents (an idea first suggested in the 1930's) and accumulation of
charged membrane proteins mainly at the putative growing pole. A fuller
description of these events can be found in Trewavas (1982ft) and owes much to
the pioneering studies of Jaffe and collaborators.
Normally this unequal cytoplasmic calcium distribution is instituted by a light
gradient across the zygote but gradients in ions, osmotic potential, pH, temperature, dinitrophenol, various organic substances and even mechanical distortion are equally effective. Many growing plant (and in some cases animal)
systems have now been shown to drive polarized electrical currents through
themselves (Jaffe & Nuccitelli, 1977).
It is the surface membrane proteins/enzymes which are critical to our
discussion here. Most of these have a net negative charge at neutral pH's (Barber, 1982) and thus would accumulate at the positive pole of the polarized egg.
Once these are concentrated they can aggregate into membrane patches with a
likely consequent modification of function. These patches exhibit threshold
characteristics in their formation and the aggregation can be modified by variations in local electrostatic forces (Gershon, 1978; Barber, 1982). Patches have
been observed to form at the putative growing pole of Fucus (Trewavas, 1982ft).
Once formed these patches can be stabilized in at least three ways. First, a
number of membrane proteins are, on their cytoplasmic side, attached to actin,
myosin and tubulin. Once a patch is formed these proteins act as nucleation
centres for growing microtubules and microfilaments in the cytoplasm stabilizing
the patch itself (Trewavas, 1982ft). Second, membrane proteins which are concerned with ion fluxes may undergo self-stabilization as they aggregate and
modify the local electrostatic environment (Barber, 1982). Third, yet other
membrane patches will be stabilized by the prevailing electrical fields.
The net effect of patch formation is to provide a distinctive biochemical,
enzymatic and molecular character to certain areas of the surface membrane in
cells and the immediately adjacent cytoplasm. In other words the cell has
established a polarity.
Much work in the earlier part of this century established that plants contain
internal electrical fields between all tissues, and parts of tissues. The size and
direction of these fields vary with development (Rosene & Lund, 1953). This
Molecular basis for developmental plasticity in plants
185
presumably reflects the summation of individual cell asymmetries in ion fluxes
which are maintained even in the mature parts of a plant. Recent work, by way
of support, has shown potential differences of several millivolts between the top
and bottom of individual shoot cells (Etherton & Depolph, 1972). The fact that
a potential difference can be observed at all, between, for example, the top and
bottom of a coleoptile suggests a certain uniformity in the polarity patterns of the
individual cells comprising that tissue.
Rosene & Lund (1953) point to a number of ways in which the electrical field
can be altered. Laying growing stems horizontally leads to the lower, more
rapidly, growing side becoming positive with respect to the upper. But a major
way of altering the pre-existing electrical field is by wounding and excision.
Molecular effects of excision
From the previous discussion it should be apparent that excision disrupts the
pre-existing cell polarity. We can now enquire how this happens and suggest
some consequences.
Membrane patches self-stabilized by ion fluxes and pre-existing electrical
fields will obviously disaggregate as the source of ions and the electrical field is
disrupted by excision. The lowering of membrane potential may then (as in many
animal tissues) activate voltage-regulated calcium channels leading to the lowgrade action potential previously described as resulting from wounding and to an
increased inward flux of calcium ions. The evidence that this partly involves
altered calcium fluxes may be found in Trewavas (19826). As a consequence
cytoplasmic calcium will increase progressively and persistently through tissues.
Three separate events may result.
1. High levels of cytoplasmic calcium are known to disrupt microtubules.
Membrane patches stabilized by these, of necessity, disaggregate. Calciumactivated protein kinases have been detected in plant membranes
(Hetherington & Trewavas, 1982). Phosphorylation (i.e. addition of negative
charge) of membrane proteins will increase the electrostatic repulsion between individual membrane proteins (Barber, 1982) lengthening the effects of
depolarization. As a result many pre-existing membrane patches and thus cell
polarity will be completely disrupted. The previous enzymatic activities of the
surface membrane will diminish and changes in cytoplasmic composition commence leading to alterations in chromatin structure, protein synthesis and
gene expression. All these are known to result from elevated cytoplasmic
calcium levels (Dunham & Walton, 1982; Trewavas, 19836).
2. High levels of cytoplasmic calcium interrupt intercellular communication
(Loewenstein, 1979) and thus isolate cells. Cells thus become embryo-like,
capable therefore of regenerating roots, shoots etc.
3. High levels of cytoplasmic calcium initiate the cell cycle if the cells are
preprogrammed to do this, as in dormant bud or pericycle cells (Trewavas,
19826).
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A. J. TREWAVAS, R. SEXTON AND P. KELLY
Conclusion
The important conclusion to be deduced from this discussion is that cell surface
organization in plants may be labile because it is stabilized electrically by the
overall tissue structure itself. When that tisue structure is disrupted the controls
on organization are likewise disrupted and regeneration, or changes in polarity
can then be instituted. This means that the emphasis in investigating cell
development in plants must be much more directed towards understanding membrane structure and organization than it is currently. Although genomic changes
are obviously of relevance they seem to represent the minimum compatible with
the multicellular state and are obviously not so advanced or irreversible as in
animal development.
The disruption of polarity in cells adjacent to the vascular system should lead
to a leaking out of vascular materials into cells which previously were isolated
from it, with the obvious consequences on cell division, growth etc.
ABSCISSION AS AN EXAMPLE OF PLASTIC DEVELOPMENT
Abscission is the means whereby a plant can eliminate leaves, fruits, seeds,
branches and almost any part of its structure. It enables the mature plant to
adjust its structure to the prevailing circumstances. Most abscission occurs
because of enzymatic weakening of cells in a small zone located at the base of a
petiole or pedicel although mechanical means play a role in certain circumstances.
On a whole plant basis what is the mechanism that determines which tissues
are abscized? There is general agreement that competition between the parts of
a plant is a major one (Addicott, 1982). Trees, for example, adjust the amount
of growing fruit to the photosynthate they produce, or the number of leaves to
the amount of water available, and then abscize the excess. Competition is a form
of communication, and is termed negative control. What one organ removes
from the vascular system is not available to others and changing the competitive
ability of some can lead to qualitative alterations in what is left to circulate in the
vascular system.
Many factors control abscission and plant development
One of the most unusual aspects of plant development is the wide variety of
factors that specifically modify it. Table 1 compares the factors that can be used
to specify polarity in Fucus, to promote abscission or to specifically modify
regeneration in epidermal explants or callus. They are compared to those that
induce parthenogenesis in sea urchin eggs, a process known to result from cell
surface changes particularly increased influx of calcium ions.
The remarkable feature of Table 1 is the great similarity between these lists.
Light gradient or u.v. light
gradient
Inorganic ions
Dinitrophenol gradient
u.v. light
Osmotic pressure (water
availability) mechanical
constraints
Light quantity and quality
Inorganic ions
Fatty acids
Numerous hydrophobic
substances, steroids
phenolamines
Auxin, kinin, ethylene
Sucrose
pH ammonia
Osmotic potential (water
availability) excision (mechanical
or osmotic shock)
Light quantity and quality
Inorganic ions
Numerous hydrophobic
substances hydrazines, petroleum
fractions
3
•§
00
3
S3-
'cit
References. Trewavas (1982b). Addicott (1969,1982). Tran Thanh Van (1981).
pH gradient
Calcium gradient and calcium
ionophore
Temperature
Temperature
Auxin, ethylene
Sucrose
pH change ammonia
Decline in calcium
Factors specifically altering
regeneration
Factors promoting abscission
£•
a
basis f(
mtal pi
Sucrose
pH change ammonia
Calcium and calcium ionophore
Numerous hydrophobic
substances, e.g. ethanol, ether,
acetone
Uncoupling agents
Fatty acids
Auxin gradient
Temperature gradient
Osmotic potential gradient
mechanical alteration of cell
shape
Temperature shock
Osmotic and mechanical shock
Inorganic ions
Factors used for polarity
specification in Fucus
Factors inducing parthenogenesis
in sea urchin eggs
Table 1. A comparison of the factors inducing parthenogenesis in sea urchin eggs, specifying polarity in Fucus zygotes,
promoting abscission or specifically manipulating regeneration
olecu
188
A. J. TREWAVAS, R. SEXTON AND P. KELLY
This suggests a common mode of action and the likely identity of the receptive
components as being the cell surface. The fact that membrane patches may have
their stability altered by changes in lipid viscosity (modified by hydrophobic
substances, inorganic ions and temperature) or by ionic strength, pH changes,
other hydrophobic substances such as poly amines, anaesthetics, by various
shock treatments, metabolic poisons, membrane potentials, local (electrical) ion
currents and calcium ionophores (Edidin, 1981; Barber, 1982) probably goes far
to explaining these observations. Critical to this are the internal effects of
cytoplasmic calcium on the disaggregation of microtubules, filaments etc which
destabilize patches.
That abscission should be sensitive to so many different factors is what one
would expect from a process which must constantly change with the environment
that is, from a plastically developing organism. However, response to a wide
variety of signals is not limited to abscission and callus regeneration; similar lists
in scope and variety have been published for plant cell division, seed dormancy,
adventitious root formation, bud dormancy, flowering, cambial growth,
stomatal aperture and most other aspects of plant development which have been
examined (Trewavas, 1982a, b, 1983a, b, 1984). Clearly it would be unrealistic to
argue that such processes are controlled by one or two factors.
The regulation of abscission by growth substances
The lists in Table 1 include the growth substances ethylene and auxin. Calling
growth substances 'hormones' has led to the unfortunate assumption that growth
substances are the only critical controlling factor in abscission and indeed plant
development. To what extent is this assumption true for abscission?
Ethylene has often been quoted as the abscission regulator or inducer. However in a well-constructed series of experiments Beyer (1975) showed that treating
only the abscission zone of intact leaves with ethylene did not lead to abscission.
Only when the leaf itself commenced senescence could exogenously applied
ethylene accelerate the process in the zone. Ethylene will accelerate abscission
in cells which are predisposed to do so (Sexton, Lewis, Trewavas & Kelly, 1984).
The normal experimental means of producing that predisposition or making
them ethylene sensitive is by excision. In general it seems that the factors which
induce leaf abscission, i.e. deep shade, lack of water, defoliants, induce a permanent dysfunction in the leaf itself and it is this that initiates the subsequent
process in the abscission zone (Addicott, 1982). In excision-induced abscission,
ethylene seems to accumulate only after the zone has abscized. On the other
hand sensitivity to ethylene shows a progressive increase during the preabscission period.
It has been argued that a reduction in the level of auxin transported in a polar
direction from leaf to abscission zone cells might be the means of conveying the
leaf dysfunction to the abscission zone. Beyer (1975), like many others, found
he could counteract the abscission-accelerating effects of treating whole leaves
Molecular basis for developmental plasticity in plants
189
and zones with ethylene by auxin treatment. He calculated that under his conditions abscission occurred when the auxin content of abscission zone cells had
reduced some seven-fold. Others have reported a two- to five-fold reduction in
auxin levels correlated with abscission (references in Addicott, 1982; Sexton et
al. 1984). However the abscission-inhibiting concentrations of exogenous auxin
required are very high (10~3 M) and Beyer's data clearly shows a dose-response
curve which suggests reductions of three orders of magnitude would be necessary
for abscission to occur. This makes a seven-fold change relatively insignificant.
Most auxin transport is now known to occur in the vascular tissue (Goldsmith,
1977) and from the known enzymology of auxin synthesis it is difficult to see how
a variety of factors (Table 1) could modify the synthesis of auxin (Sheldrake,
1973). The abscission-inhibiting effects of auxin can be mimicked by fusicoccin
(Sexton & Roberts, 1982). Further complications with the notion that abscission
is regulated by one or two substances are considered in depth in Sexton et al.
(1984) and for other plant development systems in Trewavas (1981, 1982a).
The data in Table 1 are directly relevant to assessing the role of growth substances in abscission. These substances are often talked of as though they are
controllers in a rheostat type of fashion. However they are only one of a variety
of inputs, many others of which are environmental, and therefore, for a plant,
internally uncontrollable variables. As these vary, the responsiveness of the
system will vary. It is difficult, if not impossible, to see how a rheostat type of
control could operate, or even evolve, under such circumstances. The response
could never be guaranteed.
Ethylene and auxin are essential inputs into the process of abscission. However all essential inputs can be made limiting and a graded dose response easily
produced. One should not confuse the observations made on carefully manipulated and controlled experimental material where ethylene (or auxin) can be
made limiting, and thus apparently controlling, with the realities of natural
abscission. Since it is a dysfunction of leaf metabolism that initiates abscission we
suggest that it is the transit of many factors, sugars, ions, growth substances etc
through the abscission zone which are the critical event. When the transit rates
decline below a certain level, abscission may proceed. Like many other aspects
of plant development, it is a change in the balance of many factors, not just one
or two, which regulate the process.
MOLECULAR ASPECTS OF ABSCISSION
Fig. 1 summarizes some important molecular aspects of abscission. Details of
other accompanying metabolic changes may be found in Sexton & Roberts
(1982). It is assumed that the process of excision initiates a set of metabolic
changes which lead to abscission. The known events are placed at their
approximate starting times and the time values are only a rough guide because
of biological variation in individual abscission zones.
EMB 83S
190
A. J. TREWAVAS, R. SEXTON AND P. KELLY
Phase 1
Phase 2
phase 3
Abscission
1 .
!
Excision
Chitinase mRNA Chitinase
accumulates.
translated
Cellulase mRNA
accumulates.
Ethylene
accumulates
Cellulase translated
Senescence
Wall breakdown
Abscission not inhibited
by auxin auxin insensitive
Abscission inhibited by
auxin auxin sensitive
Wound physiology.
Cork formation
Abscission accelerated
by ethylene
1
I
Rough E.R. Dyctyosomes
accumulate
[
48
14 20
hours
Fig. 1. Summary of some important metabolic changes during abscission of excised
abscission zone tissue. Note that the accumulation of chitinase mRNA occurs before
cellulase mRNA.
There are two known enzymatic changes of interest amongst many unknowns.
Wall breakdown has been correlated with the appearance of an isozyme of
cellulase. This enzyme has been purified and antibodies to it used to identify and
quantify cellulase mRNA levels by in vitro translation (Kelly et al. 1984).
Cellulase mRNA commences accumulation 6 h after excision and is a constant
proportion of the total by-20 h. Translation (and indeed continued transcription)
seem to be ethylene dependent and commence about 20-24 h after excision.
There is no increase in endogenous synthesis of ethylene at this time and the
onset of translation presumably reflects a change in sensitivity to ethylene
although its nature is not understood. If excized tissue is treated with auxin
during the first 12-18 h (called phase 1) then subsequent wall breakdown is
severely inhibited. In addition auxin inhibits the transcription and translation of
cellulase mRNA in this phase-1 period but is no longer able to do so if this phase
has been completed in its absence. The accumulation of chitinase mRNA and
chitinase is believed to have anti-fungal functions.
Phase changes in development
The metabolic events of abscission are divisible into at least two phases. The
first phase lasts some 12-18 h and is recognized by its sensitivity to auxin. If
exogenous auxin is applied during phase 1 then the time for the tissue to reach
phase 2 is either very greatly increased or it is never reached. If phase 1 is
Molecular basis for developmental plasticity in plants
191
accomplished in the absence of auxin then phase 2 is reached when auxin is no
longer an effective inhibitor. In phase 2 the tissue is, however, very ethylene
sensitive. If endogenous ethylene is removed during phase 2 then the progress
of abscission is inhibited. If, on the other hand, abscission zone tissue is treated
with ethylene during phase 1 and then the ethylene removed during phase 2, the
cellulase mRNA which has been accumulated is degraded and continued cell
wall weakening stops. Cellulase mRNA has a short half life. The rate of accumulation of cellulase mRNA in phase 1 is itself ethylene dependent. Thus there are
at least two characterizable phases in abscission which presumably reflect different metabolic states of the tissue.
There are a number of stages of plant development where a similar change of
phase can be recognized and these have been listed in Table 2. Such changes in
phase are often referred to as commitment. However, bearing in mind the reversibility and lability of much of plant cell development we are unhappy with the
use of that term here. Obviously in excized abscission zones there are metabolic
changes going on during phase 1 coupled with some timing device which eventually lead to phase 2. But commitment to phase 2 may not necessarily lead to its
expression. It too is dependent on the right balance of factors in the cellular
environment including ethylene. Ordinarily, in intact plants, the environmental
circumstances may initiate expression of the appropriate phases immediately
they appear, but, because of developmental lability, changes in sensitivity or the
term 'competence' more accurately describe what is going on.
What do these metabolic changes involve? As Table 2 shows most phase l's
occupy a period of around 16 h. From the previous discussion it can be suggested
that this may involve disaggregation of membrane patches and breakdown of
membrane proteins and calcium influx leading to changes in gene expression. It
is to be expected that the specific genomic changes will in part be determined by.
the previous chromatin structure but new membrane proteins and other cell
enzymes will appear. Membrane patches will reform but their positioning,
degree of aggregation and anchorage will be subject to the particular local external environment in terms of pH, ion fluxes, calcium, temperature etc in which
the cell finds itself. A new electrical field, a new polarity, is subsequently
produced, self-stabilizing the structure by a process of feedback. Characteristically, Rosene & Lund (1953) found the electrical symmetry of tissues to change
during development.
The reported ability of auxin to change H + ion fluxes across membranes may
explain why it figures prominently in Table 2. There has been much confusion
about this and the role of other growth substances. Thus it is often argued that
auxin acts to initiate changes in development. But from the discussion here
(Table 2) it is apparent that it is excision or dehydration/imbibition which are the
main initiators of developmental change; auxin acts passively like any other
factor in the cellular environment, able to influence development only when the
cell has acquired the sensitivity to allow it to act. Thus in cell division in excized
Kelly et al. (1984)
Trewavas (1982a)
Jaffe(1969)
Setterfield (1963)
Melanson & Trewavas (1982)
Imbibition
Anthocyanin synthesis
6.
7.
8.
9.
Auxin
Gibberellin
Abscisic acid
Auxin
Not known
Insensitive to auxin
Insensitive to giberellin
Reversibility of polarity
specification
Insensitivity to
phytochrome
PFR
Auxin
Mohr(1982)
Smart & Trewavas (1983)
Blakeley, Rodaway, Hollen & Croker
Fabijan, Yeung, Mukherjee & Reid
27
8-16
12-16
Ethylene
Auxin causes expansion
Auxin
Auxin sensitivity
Auxin causes division
Expansion insensitive to
auxin
Insensitive to auxin
Sensitive to ABA
Insensitivity to auxin
Factor needed for
expression of phase 2
How is phase 1
recognized
6
3
2
7
2
9
8
4
4,5
1
Reference
w
>
to
13
>
Z
D
m
X
C/3
TON
References 1.
2.
3.
4.
5.
Fertilization
14-20
Development
Fucus zygote polarity
20-24
Imbibition
Development
16
Excision
Adventitious root
regeneration
Aleurone amylase
Turion-formation
Cell expansion coleoptile,
hypocotyl
24
(maximum)
Excision
Branch root formation
12-18
12-16
16-20
Excision
Excision
Excision
Abscission zone cellulase
Cell division
Cell expansion
Phase 1 length
(hours)
Initiating event
Developmental event
Table 2. Phase changes recognisable in plant development
'AVA;
CELLY
Molecular basis for developmental plasticity in plants
193
tissue auxin can only act on cells which are predisposed to divide. It is excision
that initiates the predisposition; auxin is one of the factors that helps the process
along (Hanson & Trewavas, 1982). We would suggest that auxin may only
contribute significantly to developmental change when the pre-existing polarity
is disrupted and membrane patches have to reform. It is interesting that fusicoccin, a fungal toxin which also promotes H + ion flux, mimics the abscissioninhibiting and growth-promoting properties of auxin. Again, like auxin, it only
initiates these changes in excized tissue (Hanson & Trewavas, 1982).
The research on abscission zone cellulase described here was supported by the A.R.C. and
theS.E.R.C.
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