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Plant Physiol. (1998) 116: 891-899
Transdifferentiation of Mature Cortical Cells to Functional
Abscission Cells in Bean1
Michael T. McManus*,
D. Stuart Thompson,
Cledwyn Merriman,
Linden Lyne, and
Daphne J. Osborne
Department of Plant Biology and Biotechnology, Massey University,
Private Bag 11222, Palmerston North, New Zealand (M.T.M.); Department of Biological Sciences, University of Lancaster,
Bailrigg, Lancaster, United Kingdom (D.S.T.); Department of Plant
Sciences, University of Oxford, South Parks Road, Oxford OX1 3RA,
United Kingdom (C.M.); and Oxford Research Unit, The Open University,
Foxcombe Hall, Boars Hill, Oxford OX1 5HR, United Kingdom (L.L.,
D.J.O.)
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ABSTRACT |
Abscission explants of bean
(Phaseolus vulgaris L.) were treated with ethylene to
induce cell separation at the primary abscission zone. After several
days of further incubation of the remaining petiole in endogenously
produced ethylene, the distal two-thirds of the petiole became
senescent, and the remaining (proximal) portion stayed green.
Cell-to-cell separation (secondary abscission) takes place precisely at
the interface between the senescing yellow and the enlarging green
cells. The expression of the abscission-associated isoform of
 -1,4-glucanhydrolase, the activation of the Golgi apparatus, and
enhanced vesicle formation occurred only in the enlarging cortical
cells on the green side. These changes were indistinguishable from
those that occur in normal abscission cells and confirm the conversion
of the cortical cells to abscission-type cells. Secondary abscission
cells were also induced by applying auxin to the exposed primary
abscission surface after the pulvinus was shed, provided ethylene was
added. Then, the orientation of development of green and yellow tissue
was reversed; the distal tissue remained green and the proximal tissue
yellowed. Nevertheless, separation still occurred at the junction
between green and yellow cells and, again, it was one to two cell
layers of the green side that enlarged and separated from their
senescing neighbors. Evaluation of Feulgen-stained tissue establishes
that, although nuclear changes occur, the conversion of the cortical
cell to an abscission zone cell is a true transdifferentiation event,
occurring in the absence of cell division.
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INTRODUCTION |
We are of the view that the fundamental molecular mechanisms of
cell differentiation in plants and animals are intrinsically similar
but that, throughout the life of the organism, the majority of plant
cells retain a freedom of commitment with respect to the choice of
options for further cell determination (Osborne and McManus, 1986 ).
There is, however, good evidence that terminal commitment does occur in
certain restricted cell types in higher plants. Those forming the
aleurone layer in graminaceous seeds are one example, and, of
particular interest to us, the cells that comprise leaf abscission
zones are another. These cell types are confined to very precise
positions within the plant and display a high degree of functional
specialization with respect to hormonal cues. Once formed they do not
differentiate further. In certain plants (bean [Phaseolus
vulgaris] and Sambucus nigra), specific protein
determinants have been identified in leaf abscission cells that are
preferentially expressed compared with neighboring (nonabscission)
tissue (McManus and Osborne, 1990a, 1990b, 1991). The presence of such
determinants mark these cells as possessing a specific functional
competence with respect to their neighbors.
The question arises as to whether cells from higher plants that retain
a freedom of commitment can transdifferentiate to a different cell
type, i.e. convert into another distinct cell type without cell
division, or if there is always a requirement for prior cell division.
Evidence from studies with plant cells and tissues in culture suggests
that cells divide as part of a de-differentiation process (often to
form callus) before new cell types (usually arising from organized
apical regions) are formed (Schiavone and Racusen, 1990), although
transdifferentiation of parenchyma cells directly into tracheary
elements has been observed (Sugiyama and Komamine, 1990).
In the present study we provide evidence that cortical cells of mature
bean petioles have the flexibility to convert directly into
functionally competent and biochemically recognizable
ethylene-responsive abscission cells and exhibit a gene expression of
the new cell type. Further, this conversion occurs without the need for
cell division.
Pertinent to our study is the organizational polarity of tissues that
is reiterated as plants differentiate and develop. The concept that
plant cells and mature tissues retain this inherent polarity (or
axiality) throughout their life span is widely accepted (Schnepf, 1986;
Warren Wilson and Warren Wilson, 1993), although the fundamental
mechanisms by which this polarity is established and maintained are not
understood. However, each cell is also the recipient of information
from each of its neighbors and will respond according to the dictates
of the signal and the flexibility of its own differentiation state. In
this paper we show that transdifferentiation of cortical cells to
abscission zone cells can be directed by a combination of auxin and
ethylene inputs, the response to which is not restricted by the fixed
axiality of the petiole tissue as a whole.
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MATERIALS AND METHODS |
Plants of bean (Phaseolus vulgaris var. Masterpiece)
(Asmer Seeds Ltd., Leicester, UK) were grown in a greenhouse in
Levington's Universal compost (Fisons, Suffolk, UK). The plants were
given additional illumination with 400-W mercury vapor lamps to
maintain long days (minimum 14 h), and the temperature was
maintained at a minimum of 15°C. The first leaf pair, harvested when
fully expanded (approximately 12-15 d), was used in experiments.
Secondary Abscission Assays
Explants, 1.5 cm long, were excised from the primary leaves to
include the distal pulvinus, the abscission zone, and part of the
petiole. These explants were supported on Perspex racks over 2% (w/v)
sterile agar in glass containers, and all manipulations were performed
in a sterile lamina flowhood. The explants were incubated in 10 µL
L 1 ethylene at 24°C in continuous cool-white
fluorescent light for 16 h, followed by 80 h in air, after
which time the senescent and abscinded pulvini were removed and the
remaining petioles were incubated in glass containers in which
endogenous levels of ethylene accumulated (up to 3 µL
L1). Once secondary zones formed (at d 8),
tissue from these explants was used (a) to determine
-1,4-glucanhydrolase activity and (b) for immune-recognition assays
(by ELISA), utilizing an antiserum raised against the bean abscission
zone cell-associated -1,4-glucanhydrolase isoenzyme (pI 9.5). From
the time of pulvinus separation (at d 4) until d 8, samples of petiole
tissue were taken daily for light and transmission electron microscopy
and for determinations of nDNA contents.
In separate experiments similar explants were incubated in ethylene (10 µL L1) for 16 h and then in air for a
further 48 h. At 64 h, the senescent and abscinded pulvini
were removed, and 1 µL of a range of concentrations from 10 µm to 1 mm of a sterile solution of IAA or
water was applied directly to the exposed surface of the primary
abscission zone at 12-h intervals for 24 h. During these
treatments and for a further 48 h, the explants were maintained at
24°C in continuous light either in air (with 0.25 m
mercuric perchlorate in 2.5 m perchloric acid included to
scavenge evolved ethylene) or in 10 µL L1
ethylene. Explants were scored daily for the frequency of secondary abscission zone formation, and for those in (10 µL
L1) ethylene, the distance from the newly
formed secondary abscission zone to the primary zone was measured.
Determination of -1,4-Glucanhydrolase Activity
The viscosity assay as described by Wright and Osborne (1974) was
used, with some minor modifications.
Sections approximately 1.0 mm thick were excised as appropriate from
explants after separation of the pulvinus at the primary zone (at d 4),
and after separation at the secondary zone (at d 8). Sections from 25 explants (approximately 120-150 mg fresh weight) were pooled and
homogenized in 3 mL of 0.02 m phosphate buffer, pH 6.1, containing 1.0 m NaCl (for maximum extraction of the pI
9.5 zone-specific isoenzyme; Lewis and Koehler, 1979) and 5 mm DTT, the resultant slurry was centrifuged at
10,000g for 5 min at 4°C, and the supernatant was cleared
by centrifugation at 25,000g for 30 min at 4°C. After
equilibration to 25°C, aliquots (250 µL) of the cleared
supernatant, diluted to a final volume of 1 mL in 0.05 m
phosphate buffer, pH 6.0, containing 5 mm DTT, were mixed
with 3 mL of 2% (w/v) carboxymethylcellulose (type 7HF, Hercules,
Inc., Wilmington, DE) in the same buffer, and the mixture was incubated
at 25°C. The viscosity of the mixture was recorded on mixing and,
thereafter, at timed intervals. Activity is expressed as enzyme units,
where 1 unit represents a percentage change in viscosity of 1 from the
time of mixing until 60 min per 1 mL of original extract.
ELISA
Tissue extracts identical to those prepared for assay of
-1,4-glucanhydrolase activity were diluted in 50 mm
sodium carbonate, pH 9.6, and coated onto 96-well
Micro-ELISA plates (Dynatech Laboratories, Billinghurst, Sussex, UK). A
rabbit polyclonal antibody raised against the pI 9.5 isoenzyme of
-1,4-glucanhydrolase purified from bean abscission tissue
(kindly supplied by Dr. Sexton, Department of Biological Sciences,
Stirling University, UK) was used as the primary antibody, and
peroxidase-conjugated goat anti-rabbit IgG (Nordic Immunological
Laboratories, Maidenhead, Berks, UK) was used as the secondary
antibody. The peroxidase-bound peroxidase was quantified by the
addition of 0.05% (w/v) o-phenylenediamine and 0.03% (v/v)
H2O2 in 0.02 m
sodium acetate, pH 5.0, and the developed color was measured at 495 nm
using a Micro-ELISA MR 580 plate reader (Dynatech Laboratories).
Scanning Electron Microscopy
Scanning electron microscopy was performed using a cryo-system
developed by EM Technology (Hexland, Ltd., Oxford, UK), attached to a
scanning electron microscope (model 505, Philips). Abscission explants
with partial or complete cell separation at the secondary abscission zone were examined.
Light and Transmission Electron Microscopy
Explant tissue was fixed for 4 h in 3% (w/v)
paraformaldehyde in 0.1 m sodium phosphate buffer, pH 7.0. After washing in buffer, the tissues were postfixed for 1 h in 1%
(w/v) osmium tetroxide, washed, and then held in 1% (w/v) uranyl
acetate for 18 h. The fixed tissue was dehydrated through an
ethanol series and embedded in TAAB resin (TAAB Laboratory Equipment,
Ltd., Reading, Berkshire, UK). For light microscopy, 2-µm sections
were cut and stained in 1% (w/v) basic fuchsin or 1% (w/v) toluidine
blue, and the sections were examined and photographed using an Axiophot
microscope (Zeiss). For electron microscopy, thin sections were mounted
on grids, stained with uranyl acetate followed by lead citrate, and examined at 100 kV using a Jeol 200 Ex electron microscope.
Quantification of nDNA
Sections of explant tissue from the different developmental stages
of secondary zone formation were fixed overnight in ethanol:acetic acid
(3:1, v/v). The nDNA was then hydrolyzed for 30 min in 5 n
HCl at room temperature, and the tissue was rinsed in distilled water
and stained with Feulgen reagent (BDH Chemicals, Poole, Dorset, UK) for
1 h in darkness at 25°C. After rinsing three times in a solution
of 0.5% (w/v) KH2SO3 in 50 mm HCl and then once in water, the stained sections were
softened in 45% (v/v) acetic acid for 10 min, rinsed with water,
transferred to slides, and then squashed and mounted for viewing and
quantification of nDNA in a M85 scanning microdensitometer (Vickers
Instruments, York, UK). At least 200 nuclei were measured for each
sample.
To obtain relative values for the diameter of the different nuclei, the
slides were assessed using a marked graticule within the eyepiece of a
binocular microscope at a magnification of ×200.
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RESULTS |
Conditions Leading to Secondary Abscission Zone Formation
To determine whether a secondary abscission zone could be induced
as a transdifferentiation event, rather than the result of new cell
divisions, we set up an explant system from the primary leaves of bean,
in which we had previously ascertained that secondary abscission zones
would form (Osborne and McManus, 1986).
The explants were induced to abscind at the primary zone by a brief
treatment with ethylene, the shed pulvini were removed, and the
remaining petioles were maintained in closed containers in which
endogenously produced levels of ethylene accumulated (up to 3 µL
L1). After 7 to 8 d, distinct yellow:green
junctions appeared in the petiole with a discrete zone of cell-cell
separation between the distal yellow and proximal green portions (Fig.
1A). In these petioles the orientation of
the senescent and nonsenescent tissue with respect to the separating
cells was the same as that observed in vivo when a senescent pulvinus
is shed at the primary abscission zone (i.e. a distal yellow
pulvinus-proximal green petiole; Fig. 1B).
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| Figure 1.
A, Cell-cell separation at secondary abscission
zones that form between the distal yellow and proximal green tissue in
petioles maintained in conditions in which endogenously produced auxin and ethylene accumulate. B, The naturally occurring leaf
pulvinus:petiole abscission zone. C, Petioles treated with 1 mm IAA in the absence of ethylene. D, Cell-cell separation
at the secondary abscission zone induced to form in petiole tissue by
the application of 1 mm IAA in the presence of ethylene.
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Evidence That Separation of Induced Yellow-Green Junctions Is a
True Abscission Event
Microscopic examination of many yellow-green junctions (example
shown in Fig. 2) revealed that, in common
with separation at the primary zone, only cells of the green side of
the induced zone enlarge to form turgid and rounded cells (Fig. 2, side
B). Those of the adjacent yellow and senescing petiole eventually become collapsed and flaccid (Fig. 2, side A), and the epidermal cells
on either the green or yellow side do not enlarge.
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| Figure 2.
Light micrograph of a separation between the
distal yellow (A) and proximal green (B) tissue at a secondary
abscission zone induced by endogenously produced auxin and ethylene
(see Fig. 1A) (magnification ×47).
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An induction of -1,4-glucanhydrolase activity is concomitant with
the initiation of separation in the primary leaf pulvinus-petiole abscission zone (Horton and Osborne, 1967; Wright and Osborne, 1974),
and is attributable to an abscission zone-associated pI 9.5 isoenzyme
(Sexton et al., 1980; Durbin et al., 1981). We show that
-1,4-glucanhydrolase activity is also induced in the secondary abscission zone at the site of the yellow-green junction by d 8 (Table I). The isoenzyme has been
identified immunologically as the abscission-associated pI 9.5 isoform,
with a high level of antibody binding to both extracts of primary and
secondary zone tissue. No pI 9.5 enzyme induction and negligible
antibody binding were observed to petiole tissues at a distance from
the secondary abscission zone (Table I).
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Table I.
Conversion of cortical cells to abscission cells
directed by endogenous hormone levels
Activity of -1,4-glucanhydrolase and immune recognition by the pI
9.5 isoenzyme antibody in extracts from tissues of the primary
abscission zone at d 0 and 4 and tissues from the secondary abscission
zone at cell-cell separation at d 8. NA, Not assayed.
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Ultrastructurally, the enlarging cells at primary abscission zones
undergo cytoplasmic activation, including dilation of dictyosomes and
enhanced vesicle formation. Such activity is not observed in other
petiole cells at sites remote from the zone (Osborne et al., 1985).
Ultrastructural examination of secondary zones generated in the present
study show that cells at the proximal (green) side of the zone
exhibited the highly dilated dictyosomes with many associated vesicles
(Fig. 3A), similar to those observed in
the cells at the proximal (green) side of primary zones. Cytoplasmic activation in these secondary zones was restricted to those green cells
that enlarge and separate; it did not occur in other green cells (Fig.
3B) or in the yellow, nonseparating parts of the petiole (not shown).
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| Figure 3.
Transmission electron micrographs of the secondary
abscission zone that forms between the distal yellow and proximal green tissue in petioles maintained in conditions in which endogenously produced auxin and ethylene accumulate (see Fig. 1A). A, Electron micrograph of a separating cell from the proximal (green) side of the
newly formed zone. B, Electron micrograph of a nonseparating cell from
the proximal (green) side of the newly formed zone, 12 rows of cells
distant from the separating cells (magnification ×16,000).
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Evidence That Secondary Zone Cell Formation Results from
Cortical Cell Transdifferentiation
Light Microscopy
From the time of pulvinus shedding at the primary zone until
separation of cells at the secondary zone, photographs of each petiole
stage are shown (Fig. 4A). Samples of
petiole tissue from each of these stages were excised and fixed for
light microscopy, and a series of sections from the developing zones is
shown in Figure 4B. At no stage during secondary zone development was
any evidence for cell division observed (as defined by the deposition of cell plates) in either the cortex or in epidermal cells. Many samples of tissue have been examined in this way, and under all of the
conditions for secondary zone formation that have been employed, none
has shown evidence of any division or cell-plate formation in cells
prior to, or during, yellow-green junction formation or during
subsequent cell separation.
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| Figure 4.
A, Photographs of the developing stages (a-e) of
distal (yellow) and proximal (green) junctions in petioles of abscinded
explants. To induce secondary zone formation, abscinced explants were
treated as described in Figure 1A. B, Photomicrographs of sections of cortex cut from the five stages of secondary zone development described
in A (magnification ×100). a, Primary abscission just completed,
petiole wholly green (d 0); b, slight loss of green color (d 2); c,
beginning of formation of a yellow-green junction (d 4); d,
yellow-green junction well defined (d 6); and e, cell separation at the
yellow-green junction (d 8).
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Determination of nDNA Content
Samples of petiole tissue, similar to those used for light
microscopy, were fixed and stained with Feulgen reagent for
determinations of DNA content by microdensitometry. Throughout the
period of the development of yellow-green junctions, no changes were
found in terms of total DNA content of the nuclei in any part of the petiole. The relative DNA values remained similar to those in the
petioles at the time of pulvinus shedding at the primary abscission zone (data not shown). Considerable changes were observed, however, in
the size of the nuclei and in the distribution of chromatin and
chromatin granules within the nuclei of the yellowing, green, and separating cells (Fig. 5).
Nuclei in cortical tissue at the time of pulvinus shedding are shown in
Figure 5A, with a mean relative diameter of 2.36 ± 0.57 units. As
senescence proceeded in the yellow portion of the petiole, nuclei
contracted in size (mean relative diameter: 2.03 ± 0.53 units)
and chromatin became condensed (Fig. 5B). All cortical nuclei showed
some slight enlargement (mean relative diameter: 3.89 ± 0.50 units) in those portions of the petiole that remained green (Fig. 5C),
but in cells that enlarged and separated at the yellow-green junctions,
nuclear volume greatly increased (mean relative diameter: 8.0 ± 1.45 units) and chromatin became highly dispersed (Fig. 5D).
There was, however, no evidence of DNA replication or mitotic activity in cells at or neighboring the secondary zone, or in yellowing or green
tissue of the petiole. These results, together with observations made
with the light microscope, show that formation of secondary zone cells
is a true transdifferentiation event.
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| Figure 5.
A, Feulgen-stained nuclei of cortical cells from
petiole segments from which pulvini has just abscinded. B, Senescent
(yellow) tissue at the time of secondary zone formation. C, Cells of
green petiole tissue remote from the secondary zone. D, Cells of green tissue separating at the junction with yellow tissue at the secondary zone (magnification ×150).
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Induction of Secondary Abscission Zones by Auxin and Ethylene
To explore the role of hormones in inducing these
transdifferentiation events, we have manipulated those known to be
involved in abscission control, i.e. ethylene and auxin, and recorded
the frequency and position of secondary zone formation. To accomplish this, auxin was applied to the exposed petiole abscission cells at the
primary abscission zone (after shedding of the senescent pulvinus), and
ethylene was supplied in the ambient air. The position of secondary
zone formation was then determined by the concentration of auxin
applied and the presence or absence of ethylene (Table II). If ethylene was excluded,
no new abscission zones were formed either in the presence or absence
of auxin (Table II; Fig. 1C). When ethylene was present, the frequency
of new zone differentiation was determined by the added auxin (Table II), with the location of the cortical-to-zone cell change dependent upon the concentration of IAA applied (Table
III): the higher the concentration, the
greater the distance between the primary zone and the
induced secondary zone.
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Table II.
Frequency of conversion of cortical cells to
secondary abscission zone cells following applications of IAA and
ethylene
IAA (1 mm) or H2O was applied directly to the
exposed cells of the separated primary abscission zone at 12-h
intervals over 24 h. During treatment, and for a further 48 h, explants were incubated in ethylene (10 µL L1) or
air (with MP) and scored every 24 h for cell-cell separation at
the green-yellow junction. MP, Mercuric perchlorate.
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Table III.
Effect of applied IAA concentration on the
positional formation of secondary zones
IAA at the appropriate concentration or H2O was applied
directly to the exposed cells of the separated primary abscission zone
at 12-h intervals over 24 h. During treatment and for a further 48 h explants were incubated in ethylene (10 µL
L1) to induce cell separation at the secondary zone, and
the distance along the petiole from the site of this induced zone to
the primary zone was then measured.
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In contrast to the explants described in the previous section, the
tissue below the auxin application and extending as far as the induced
zone remained green and nonsenescent, but below the induced zone the
petiole was senescent. Again, separation always occurred at a distal
green and proximal yellow junction of cells (Fig. 1D). In the secondary
zones induced by these treatments, therefore, the orientation of
senescent and nonsenescent tissue was completely reversed compared with
that of separation events normally occurring at a primary zone (compare
Fig. 1D with Fig. 1A). The performance of these secondary zones,
however, does not differ either in the induction of
-1,4-glucanhydrolase activity on the green side of the
junction, with pI 9.5 immunological recognition (data not shown), or,
in the restriction of cell enlargement, to only those cells of the
green side of the junction (Fig. 6, side
B). Again, no enlargement was observed in any of the epidermal cells
(Fig. 6).
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| Figure 6.
Scanning electron micrograph of the secondary
abscission zone that forms between the proximal yellow (A) and distal
green (B) tissue of the petiole after incubation with applied auxin in
the presence of ethylene (see Fig. 1D) (magnification ×170).
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DISCUSSION |
This study has explored the conversion of cortical cells of bean
petioles into another distinct cell type, the abscission zone cell. The
conversion has been shown to be directed by ethylene and auxin, and to
take place in the absence of cell division, thus fulfilling the
definition of transdifferentiation.
The differentiation of abscission zones in abnormal positions on stems,
petioles, and branches can occur in vivo in response to tissue injury
or infection (Addicott, 1982). For example, in the "shot-hole"
response to fungal infection of Prunus amygdalus leaves, a
disc of infected tissue from the leaf blade is shed from the
surrounding palisade and mesophyll area by the formation of an
encircling ring of separating cells (Samuel, 1927). When excised from
the whole plant, certain parts of petioles and stems will also form
secondary abscission zones in vitro (Addicott, 1982). Usually, the
formation of these new zones is preceded by cell division, as in
GA-induced shedding of cotton stems (Bornmann et al., 1968) and the
auxin- and ethylene-regulated abscission in bean shoots (Webster and
Leopold, 1972). However, the question as to whether cell division is a
necessary prerequisite for such events has not been addressed directly.
The evidence we present here shows that in excised bean petioles a
conversion of one cell type (the cortical cell) to another (the
abscission zone cell) can take place without the advent of cell
division. From examination using light microscopy of many sections
through the developing region of the bean secondary abscission zone, we
have found no evidence either for cell division or for the deposition
of new cell plates. Using microdensitometry we found no increases in
nDNA contents that might indicate an activation of cells to the S-phase
or to the G2M phase. We cannot, however, say
whether specific regions of DNA are replicated or amplified. It is
possible that some DNA endoreduplication may occur, but these values
would then fall within the range of variation of the DNA determinations
by microdensitometry. The doubling of DNA content that accompanies the
differentiation of an abscission cell at the base of the fruit in
Ecballium elatarium (Wong and Osborne, 1978) does not occur
as part of secondary zone formation in bean.
One of the few well-characterized examples of transdifferentiation in
plants is the conversion of parenchyma cells into tracheary elements
(for a review, see Sugiyama and Komamine, 1990). There, the use of
specific inhibitors showed that formation of the mitotic spindle or DNA
replication is not required for transdifferentiation, but
"repair-type" synthesis of nDNA was a necessary prerequisite. An
unscheduled DNA repair synthesis may accompany our bean cortical cell
transdifferentiation, but we can exclude S-phase DNA synthesis. However, changes in the nuclei of the differently responding tissues clearly do take place. Those of the senescing tissue become
progressively smaller and pycnotic, whereas cells of the newly forming
abscission zone enlarge, and just prior to and at separation their
nuclei exhibit the dispersed chromatin of highly active cells. These secondary zone cells, therefore, give every indication of an induced and increased genomic activity.
In common with transdifferentiated animal cells (Okada, 1983; Kineman
et al., 1992; Patapoutian et al., 1995), we also observed a change in
gene expression with the production of a marker protein (diagnostic for
abscission cells) that accompanied the conversion of cortical cells to
abscission cells. Upon separation at the primary zone, the
immunologically distinct and abscission-associated pI 9.5 isoform of
-1,4-glucanhydrolase is newly expressed but only in abscission cells
and their close neighbors and not in other cortical cells of the
petiole. However, when these cortical cells are converted to abscission
cells, they too express the specific pI 9.5 isoform of
-1,4-glucanhydrolase. Again, expression of the enzyme is confined to
only the secondary abscission zone and is not detected in the other
cells of the petiole. This biochemical and immunological evidence,
taken together with the absence of cell division, confirms that this
cortical-to-abscission cell conversion is a true transdifferentiation
event.
In demonstrating the flexibility of cortical cells to
transdifferentiate to committed abscission cells, we have shown that the conversion is not dependent on the orientation in which the green
and senescent tissue develops in the petiole. If the
transdifferentiation of cortical cells occurred only when the distal
tissue became senescent and the proximal tissue remained green (as in
separation of naturally occurring abscission zones), then one could
conclude that a directional signaling or a polarity of perception or
response operates. However, our experiments show the contrary. If the
distal tissue is retained green and the proximal tissue becomes
senescent, it is still the green cells at the junction with the
senescing tissue that transdifferentiate. In other words, it is not
critical to achieving the transdifferentiation response whether the
distal or proximal tissue becomes senescent. Rather, the specific
requirement appears to be the generation of a juxtaposition of green
and senescing cells, irrespective of their orientation within the
overall morphological axiality of the petiole. This apparent absence of
directional perception and response to particular signals from their
neighbor cells does not conflict with the concept that all cortical
cells possess an established basipetal polarity (or axiality) in common with other cells in the plant body (Sachs, 1991; Warren Wilson and
Warren Wilson, 1993).
At present the chemical nature of the signals that are transmitted
between the adjacent senescent and nonsenescent petiole tissues has not
been characterized, although we have established that ethylene provides
one of the requirements for transdifferentiation and that the auxin
concentration can provide positional information.
In analysis of extracts of bean explants (data not presented here), we
have shown that applied 14C-IAA is conjugated in
the petiole tissue (probably to IAA-aspartate) within 6 h. This
could, as Hangarter and Good (1981) suggest, then be the source for a
slow release of free IAA. In our IAA-treated petioles, in which the
apical region remains green, a release of free IAA could be responsible
for maintaining a region of tissue proximal to the point of IAA
application in a green and nonsenescent condition, and, as we have
observed, the concentration of IAA applied determines the length of
tissue that is retained green. Warren Wilson et al. (1986) have
described a mathematical model for Impatiens sultanii that
accommodates such positional differentiation of abscission zones
regulated by the concentration of auxin applied.
In this study we conclude that certain mature cells of higher plants
retain a flexibility for direct transdifferentiation to functionally
specialized and committed cell types. In the example we describe,
cortical cells of the bean leaf petiole are seen to be sufficiently
uncommitted to be converted into fully competent, terminally
differentiated abscission zone cells. Further, we propose that certain
cells, such as cortical cells, which retain differentiation flexibility, can undergo positional transdifferentiation independently of the distal-proximal axiality of the tissue.
| |
FOOTNOTES |
1
This work was supported in part by a New Zealand
Ministry of Research, Science, and Technology Marsden Fund grant (no.
MAU 509) to M.T.M.
*
Corresponding author; e-mail M.T.McManus{at}Massey.ac.nz; fax
64-6-350-5694.
Received July 3, 1997;
accepted October 28, 1997.
| |
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Abstract
Introduction