Ethylene-Dependent and -Independent Processes
Associated with Floral Organ Abscission in Arabidopsis1
Sara E. Patterson* and Anthony B. Bleecker
Departments of Horticulture (S.E.P.) and Botany (A.B.B.), University of Wisconsin, Madison, Wisconsin 53706
Abscission is an important developmental process in the life cycle of the plant, regulating the detachment of organs from
the main body of the plant. This mechanism can be initiated in response to environmental cues such as disease or pathogen,
or it can be a programmed shedding of organs that no longer provide essential functions to the plant. We have identified
five novel dab (delayed floral organ abscission) mutants (dab1-1, dab2-1, dab3-1, dab3-2, and dab3-3) in Arabidopsis. These
mutants each display unique anatomical and physiological characteristics and are governed by three independent loci.
Scanning electron microscopy shows delayed development of the flattened fracture plane in some mutants and irregular
elongation in the cells of the fracture plane in other mutants. The anatomical observations are also supported by
breakstrength measurements that show high breakstrength associated with broken cells, moderate levels for the flattened
fracture plane, and low levels associated with the initial rounding of cells. In addition, observations on the expression
patterns in the abscission zone of cell wall hydrolytic enzymes, chitinase and cellulose, show altered patterns in the mutants.
Last, we have compared these mutants with the ethylene-insensitive mutants etr1-1 and ein2-1 to determine if ethylene is an
essential component of the abscission process and find that although ethylene can accelerate abscission under many
conditions, the perception of ethylene is not essential. The role of the dab genes and the ethylene response genes during the
abscission process is discussed.
Abscission, the developmental process regulating
detachment of organs from the main body of the
plant, can be regarded as valuable to the plant in
response to disease or pathogen challenge and the
shedding of organs that no longer provide essential
functions to the plant. Historically, ethylene treatment has been shown to result in early abscission and
increases in cell wall hydrolytic enzymes. In their
studies on Prunus serrulata senriko and Parthenocissus
quinquefolia, Jackson and Osborne (1970) concluded
that ethylene was not only responsible for accelerating abscission but an essential regulator of abscission. Alternatively, crops like Citrus sinensis appear
to have limited responses to ethylene treatment
(Lewis et al., 1968; Palmer et al., 1969). Although the
role of ethylene in hastening abscission has been
documented repeatedly in many plant species over
the last several decades, it has never been shown
definitively that ethylene perception in these plants
is essential for abscission (Addicott, 1982; Abeles et
al., 1992). Previously, we have illustrated that floral
organ abscission in Arabidopsis may be used as a
model system to study abscission (Bleecker and
Patterson, 1997). In this work, we will further elucidate the role of ethylene in floral organ abscission by
the identification and characterization of five novel
dab (delayed abscission) mutants (dab1-1, dab2-1,
1
This work was supported by the U.S. Department of Agriculture (grant no. 00 –35301–9085).
* Corresponding author; e-mail spatters@facstaff.wisc.edu; fax
608 –262– 4349.
Article, publication date, and citation information can be found
at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.028027.
194
dab3-1, dab3-2, and dab3-3), representing three independent loci, and the additional characterization of
two known ethylene response mutants (etr1-1 and
ein2-1).
In Arabidopsis, the cells of the floral organ abscission zones (filament, petal, and sepal) are characterized as small and densely cytoplasmic (Bleecker and
Patterson, 1997). The abscission zone is determined
by the presence of these cells and by being the region
of detachment and is generally characterized by a
band of cells four to six layers in depth. During
abscission, cells separate along the middle lamella,
and the organs are detached from the main body of
the plant (Esau, 1977). In Arabidopsis flowers, the
filaments are detached first, followed by the sepals,
and finally the petals. Concurrently or immediately
subsequent to detachment, the cells proximal to the
abscission zone remaining on the main body of the
plant begin to enlarge. In wild type, this progression
occurs within 2 to 3 d after anthesis, whereas in the
ethylene response mutant etr1-1, this sequence is significantly delayed, and cells are never fully enlarged
(Bleecker and Patterson, 1997; see also Patterson,
2001). If one observes the primary inflorescence, anthesis can be defined as position one and later positions as developmentally older flowers. In wild type,
the petals, sepals, and filaments are discarded between positions six and eight on the inflorescence,
whereas these organs are still turgid. We used this
knowledge to develop a massive screen for identification of novel delayed floral organ abscission
mutants by selecting for plants with inflorescences
retaining petals on flowers beyond position 10.
Plant Physiology, January 2004, Vol. 134, pp. 194–203, www.plantphysiol.org © 2004 American Society of Plant Biologists
Floral Organ Abscission in Arabidopsis
We have focused on the detachment of the petal
from the receptacle and have characterized wild type
and mutants by analyzing this region. Delayed abscission mutants dab1-1, dab2-1, dab3-1, dab3-2, and
dab3-3 display unique anatomical and physiological
characteristics. Each mutant is regulated by single
loci, and complementation tests show that dab3-1,
dab3-2, and dab3-3 are allelic. The responses of these
mutants to ethylene were analyzed by 0.001 ␮L L⫺1
ethylene treatment of dark-grown seedlings and
light-grown flowering plants. Anatomical characterization was generated using light microscopy and
scanning electron microscopy (SEM), and it provides
evidence for the changes or lack of changes in the
cells proximal to the abscission zone. In addition, the
SEM observations provide additional characterization of the fracture plane of the abscission zone. We
observed unique features of the fracture plane in the
delayed abscission mutants associated with the delay
in abscission. These anatomical observations are supported by breakstrength measurements in which the
force required to remove the petal is quantified
(Craker and Abeles, 1969). Breakstrength profiles of
wild type, ethylene response mutants, and dab mutants illustrate differences in the abscission program
of the plants. In addition, promoter-␤-glucuronidase
(GUS) constructs using the soybean (Glycine max)
chitinase (Broglie et al., 1989) and a bean (Phaseolus
vulgaris) abscission-related ␤-1,4-endoglucanase
(BAC; Tucker et al., 1988) were introduced to wild
type and all mutants. These observations provide a
foundation for developing a genetic model for the
genes regulating abscission and evidence for
ethylene-independent abscission.
RESULTS AND DISCUSSION
Identification and Selection of Delayed
Abscission Mutants
More than 32,000 T-DNA insertion lines were
screened for delayed floral organ abscission by selection of plants whose inflorescences had 10 or more
flowers and normal fertility (Fig. 1). We have designated these mutants as dab. These plants were selfed
and outcrossed to wild type (Ws) and selected for
delayed abscission for three or more generations to
eliminate any other mutations introduced by the
original Agrobacterium tumefaciens transformation.
Surprisingly, only one of these mutations appears to
be linked to the original T-DNA insertion. Flowers
and siliques were identified by their position on the
inflorescence: A flower just opening at anthesis was
designated position one, and the later positions are
progressively older flowers. It is significant to note
that a single inflorescence provides flowers at all
stages of development. In wild-type Arabidopsis
(both Ws and Col), abscission of the floral organs
occurs between positions six and eight, and the average length of the silique at abscission is approxiPlant Physiol. Vol. 134, 2004
Figure 1. Floral organ abscission in wild-type Arabidopsis and selected delayed abscission mutants. 1A, i, Wild type (Columbia [Col]);
ii, etr1-1; iii, ein2-1. 1B, i, Wild type (Wassilewskija [Ws]); ii,
dab1-1; iii, dab2-1; iv, dab3-1; v, dab3-2; vi, dab3-3.
mately 7 mm (Table I). The dab mutants show a
significant delay in the timing of abscission as measured by position and length of the silique. In dab1-1,
petal abscission occurs between positions 15 and 17,
and siliques are approximately 14 mm in length. In
dab2-1, abscission is more variable and occurs between positions 12 and 16, and the silique is approximately 11 mm in length. In dab3-1, abscission of
petals typically occurs at position 10, and the average
silique length is 11.3 mm. In dab3-2 and dab3-3, abscission of the petals generally occurs between positions 10 and 12, and the average silique length is 12
mm. The ethylene-insensitive response mutants
etr1-1 and ein2-1 also displayed delayed floral abscission, and petals abscised at position 10 with an average silique length of almost 13 mm (Bleecker et al.,
1988; Bleecker and Patterson, 1997; Ecker, 1995). Although in wild type the sepals are green when the
petals abscise, in dab2-1, dab3-1, dab3-3, etr1-1, and
ein2-1, the sepals are yellow. Alternatively, in dab1-1
and dab3-2, the sepals are still green when the petals
abscise. Development of roots and seedling morphology in all of the dab mutants is comparable with wild
type. Similarly, all other aspects of plant growth with
the exception of floral organ abscission appear to be
normal.
195
Patterson and Bleecker
Table I. Characterization of abscission in wild-type and delayed abscission mutants
Linea
Average Position at Abscissionb
Silique Length at Abscissionc
Color of Silique
Condition of Petals
mm
dab1-1
dab2-1
dab3-1
dab3-2
dab3-3
ein2-1
etr1-1
Ws ecotype
Col ecotype
16.5 ⫾ 0.75
13.3 ⫾ 2.1
10.1 ⫾ 1.1
10.75 ⫾ 0.9
12.1 ⫾ 1.16
9.8 ⫾ 0.85
10.1 ⫾ 0.7
6.76 ⫾ 1.7
6.12 ⫾ 0.6
14 ⫾ 1.6
10.88 ⫾ 1.6
11.3 ⫾ 1.1
12 ⫾ 1.4
12 ⫾ 1.3
12.2 ⫾ 1.4
12.7 ⫾ 1.3
7 ⫾ 1.1
7 ⫾ 0.8
Green
Green
Green
Green
Green
Green
Green
Green
Green
silique,
silique,
silique,
silique,
silique,
silique,
silique,
silique,
silique,
green sepal
yellow sepal
yellow sepal
green sepal
yellow sepal
yellow sepal
yellow sepal
green sepal
green sepal
White, turgid
White, turgid
White, turgid
White, turgid
White, turgid
Senesced
Senesced
White, turgid
White, turgid
a
dab lines are WS ecotype. etr 1-1 and ein 2-1 are COL ecotype. Silique length and average position were determined for populations of 50
b
or more plants.
Average position at abscission was determined by counting the no. of flowers with attached petals on the primary
c
inflorescence. Position 0 represents the flower at anthesis.
The silique length at abscission was determined for each mutant.
Anatomical Analysis
To determine if there were discernable changes in
the cells of the petal abscission zone, we observed
both thin sections of the abscission zone (Fig. 2A) and
scanning electron micrographs (Fig. 3). Light microscopy showed that all lines developed a typical abscission zone for the filament, petal, and sepal (Fig.
2A). The abscission zones were characterized by
small, densely cytoplasmic cells two to six layers
deep. Separation and elongation of cells was delayed
in the mutants; however, general characteristics were
similar to wild type. Post abscission, the cells proximal to the abscinding organ were elongated (Fig. 2A).
SEM provided additional insights into the nature of
the abscission zone, and many distinct differences
were noted between wild type and the mutants (Fig.
3). For SEM, petal abscission was carefully observed
by removal of the petal at different stages of development (positions one–14) followed by immediate
fixation of tissues for microscopy. In most lines,
shortly after anthesis a smooth fracture plane was
observed within the revealed crater. The cells within
this crater, the abscission zone, then rounded up and
elongated as development progressed. The cells
within the fracture plane of mutants etr1-1, ein2-1,
and dab2-1 were initially broken rather than revealing
the flattened fracture plane. However, petal abscission zone cells did separate along the fracture plane
at later stages in development, thus paralleling the
processes observed in wild type. Generally speaking,
the cells passed quickly through the stage of development in which the flattened fracture plane was
observed. However, in the mutant dab1-1, this developmental stage was extended for a much longer duration. Rounding of the cells in the fracture plane was
delayed in all of the mutants including the ethylene
response mutants. Irregular rounding was observed
in dab2-1 at position nine as depicted in Figure 3.
These cells in the fracture plane often produced appendages, and the cells were more irregular in size,
with many cells being 2-fold larger than others. At
the final stages of normal abscission, cells became
196
fully elongated. This elongation of cells was observed
in all lines; however, the cells remaining proximal to
the abscission zone in dab2-1 were weaker and frequently collapsed with the critical drying steps of
sample preparation (see Fig. 3). This weakened cell
wall combined with irregular cell rounding is a
unique characteristic of dab2-1.
Genetic Analysis
Crosses were made between selected mutants and
wild type to determine if the delayed abscission phenotype was heritable and controlled by a single locus.
F1 plants of crosses between wild type and dab1-1,
dab3-1, dab3-2, or dab3-3 were normal in appearance,
whereas approximately 25% of the F2 progeny displayed delayed floral organ abscission. This allows
us to conclude that these phenotypes are controlled
by a single recessive locus (Table II). Alternatively,
the F1 progeny of dab2-1 backcrossed to wild type
were all delayed abscission, and approximately 70%
of F2 progeny displayed delayed abscission (Table II).
This is consistent with a dominant mutant allele.
After determining that delayed abscission was governed by a single locus in each of the mutants, the
lines were cleaned up by outcrossing to wild type
and selecting for delayed abscission for three generations. Crosses were then made between the mutant
lines to determine genetic interactions. In crosses
among dab3-1, dab3-2, and dab3-3, the mutations did
not complement each other; thus, these mutations
were determined to be allelic. The F1 progeny of
crosses between dab1-1 and dab3-1 or dab3-2 were all
wild type; thus, these mutations were considered
complementary, and dab1-1 was determined to be
independent of dab3. The F1 progeny of dab2-1
crossed with dab1 and dab3 were all delayed abscission as expected due to the dominant nature of
dab2-1. The appearance of wild type or normal abscission in approximately 40% of the analyzed F2
progeny indicate that dab1 and dab3 are not allelic to
dab2.
Plant Physiol. Vol. 134, 2004
Floral Organ Abscission in Arabidopsis
Figure 2. A, Longitudinal sections of the floral organ abscission zones. Left, Longitudinal section of wild-type inflorescence
at position three in which sepals, petals, and filaments are preparing for abscission. Right, Longitudinal section of wild-type
inflorescence at position seven showing the elongated cells of the remaining abscission layer proximal to the main body of
the plant. B, Longitudinal section showing chitinase-GUS expression in the abscission zone cells of the floral receptacle at
position four. Dark-field microscopy results in the GUS crystals appearing pink. C, BAC-GUS expression. Left, BAC-GUS
expression in the floral receptacle at different developmental positions in Ws wild-type Arabidopsis. Expression in the
inflorescence begins in anthers just before anthesis. Expression in the abscission zone begins with anthesis and is highest at
positions four to six and then begins to decrease. Top right, BAC-GUS expression in longitudinal sections of the receptacle
using dark-field microscopy. Note that the cells in the abscission zones appear pink. Bottom right, BAC-GUS expression in
the anther as viewed using dark-field microscopy. Note the cells at the suture (dehiscence zone) appear pink. D,
Chitinase-GUS expression in the floral receptacle at different developmental positions in wild-type Arabidopsis. i, Position
three; ii, position four; iii, position seven. Note the low level GUS expression at position four. At position seven, all organs
have abscised, and expression is very high. E, Chitinase-GUS expression in the floral receptacle at different developmental
positions in etr1-1. i, position three; ii, position four; iii, position seven. F, Chitinase-GUS expression in the floral receptacle
at different developmental positions in dab mutants at positions four, seven, and 13. i, dab1-1 position four; ii, dab1-1
position seven; iii, dab1-1 position 13; iv, dab2-1 position four; v, dab2-1 position seven; vi, dab2-1 position 13; vii, dab3-1
position four; viii, dab3-1 position seven; ix, dab3-1 position 13. Note that in dab1-1, highest GUS expression is represented
at position 13. In dab2-1, low-level GUS expression is just discernable at position seven, and high levels of expression in
the sepals, petals, and filaments are represented at position 13. In dab3-1, expression is not detectable in positions four or
seven, and high-level expression is represented at position 13.
Crosses made between the dab mutants and etr1-1
and ein2-1 confirmed the independent nature of the
dab mutants from the ethylene response mutants
(data not shown).
Ethylene Responses
All of the mutants were analyzed for ethylene responsiveness. All the dab mutants showed a characteristic shortened hypocotyl, thickened hypocotyls,
and more pronounced curvature of the hook. Mature
plants displayed yellowing of rosette leaves, yellowPlant Physiol. Vol. 134, 2004
ing of sepals, and withering of petals. Leaf senescence and abscission were also accelerated in response to ethylene treatment in all the dab mutants.
Alternatively, the ethylene-insensitive mutants
etr1-1, ein2-1, and ein2-5 were elongated and displayed no curvature of the hook, and the mature
plants showed no response to ethylene. The sensitivity of the dab mutants to ethylene at both the seedling
stage and as full-grown plants is an important observation in recognizing abscission can be regulated
independently of ethylene.
197
Patterson and Bleecker
Figure 3. Scanning electron micrographs of the
fracture plane of the petal abscission zone. Left
to right, Positions three, seven, nine, and 15.
The fracture plane is revealed after the petal
abscises or when the petal is forceably removed.
In wild type, the petals abscise at positions
seven or eight. Note that these cells are rounded
and fairly similar in size. These can be contrasted with flattened cells of several of the mutants at position seven (ein2-1, dab1-1, dab2-1,
and dab3-1) or the irregular cells of dab2-1 at
positions nine and 15. Bar ⫽ 10 ␮M size.
Breakstrength
Breakstrength provides a quantitative measure of
the force required to detach an organ from the main
body of the plant and has been used by many researchers in the field of abscission (Craker and Abeles, 1969; McKenzie and Lovell, 1992; Fernandez et
al., 2000). Breakstrength observations correlated with
the scanning electron micrographs at the different
positions. In the graph below (Fig. 4A), differences
between the breakstrength of Col wild type and the
198
ethylene response mutants etr1-1 and ein2-1 at each
position are illustrated. At position one, the petals
often tore; however, the small size of these petals
made it especially difficult to clamp resulting in damage of the petal or slipping of the clamp. A decrease
in breakstrength is observed in wild type after position three and becomes immeasurable after position
five or just before natural shedding of the floral
organs. In etr1-1 and ein2-1, breakstrength does not
begin to decrease until positions five or six and was
Plant Physiol. Vol. 134, 2004
Floral Organ Abscission in Arabidopsis
Table II. Genetic characterization of floral organ abscission mutants
Line
dab1-1
dab2-1
dab3-1
dab3-2
dab3-3
F1 Phenotypea
All
All
All
All
All
F2 Segregation Wild Type:
Delayed (Mutant)
␹2 Testb
107:41
41:129
71:23
88:32
37:11
0.576
0.071
0.014
0.177
0.111
wild type
mutant
wild type
wild type
wild type
a
F 1 progeny of cross between mutant and wild-type Ws.
Tested against expected 3:1 ratio for all lines except dab2-1, which
was tested against 1:3 ratio.
All ratios tested were found to be
acceptable with probabilities of 50% or greater.
b
measurable out to positions eight or nine. These measurements correlate well with anatomical observations of wild type, etr1-1, and ein2-1 and support the
idea that cell separation begins shortly after anthesis.
In all of the mutants (dab1-1, dab2-1, dab3-1, dab3-2,
and dab3-3), the breakstrength was determined for
each developmental position (Fig. 4B). These observations also correlate nicely with the scanning electron micrographs. Decreases in breakstrength for
mutants dab2-1, dab3-1, dab3-2, and dab3-3 were not
observed until positions eight or 10, whereas in Ws
wild type, these decreases were reflected in measurements as early as position four. In the mutant dab1-1,
changes in breakstrength were not observed until
position 10, and measurable breakstrength was maintained through position 14. It was not until position
14 that breakstrength began to noticeably decrease
and only at positions 15 and 16 that abscission occurred and breakstrength was unmeasurable.
was found to be developmentally regulated in the
abscission zones of sepal, petal, and the filament (Fig.
2, B and D–F; see also Bleecker and Patterson, 1997).
In wild type, chitinase-GUS expression begins to appear shortly after anthesis in correlation with the
rounding of the cells of the fracture plane and subsequent to the initial drop in breakstrength. Expression accumulates to maximal levels with the timing
of abscission and slowly decreases after abscission.
Note that if the duration of development for GUS
detection is shortened, then the pattern of expression
is limited to the abscission zone cells; however,
longer exposure is necessary for comparison of expression in other plant lines (Fig. 2B; see also
Bleecker and Patterson, 1997). In addition, expression
patterns changed both temporally and spatially depending upon the developmental stage of the tissues,
and it was observed that expression in the abscission
zone of the filament and sepal tissues occurred before
expression in the petal abscission zone. Expression of
chitinase-GUS in the mutant etr1-1 is delayed in timing and reduced in level, but spatial patterns of expression (tissue specificity) are the same as wild type
(Fig. 2E). Similarly, observations on the ethylene-
Promoter-GUS Expression Studies
We have used molecular markers for chitinase and
BAC (cellulase) to track the abscission process in
wild-type and mutant backgrounds. Previous studies
on ethylene regulation of the basic chitinase gene in
Arabidopsis (Chen and Bleecker, 1995; Bleecker and
Patterson, 1997) and the bean abscission cellulase in
Arabidopsis (Patterson, 1998) indicated that expression of these genes was abscission zone specific and
associated with organ loss. Although chitinase is
probably not involved in regulation of abscission,
many investigators have reported basic chitinase expression localized to abscission zones (del Campillo
and Lewis, 1992). Alternatively, the BACs have been
recognized for several decades as an important class
of cell wall hydrolases involved in regulating cell
separation (Horton and Osborne, 1967; Lewis and Varner, 1970; Sexton and Roberts, 1982). For these studies,
we used transgenic lines with the soybean chitinase
promoter driving GUS (Broglie et al., 1989; Chen and
Bleecker, 1995) and the bean abscission cellulase promoter (BAC) from bean (Koehler et al., 1996).
Expression of the chitinase-GUS reporter gene in
wild type and etr1-1 was observed, and expression
Plant Physiol. Vol. 134, 2004
Figure 4. Summary of breakstrength measurements of wild-type Arabidopsis and selected delayed abscission mutants. Petals were forcibly removed from the inflorescence at positions still retaining petals,
and the force to remove each petal was measured. A, Wild-type
(Col), etr1-1, and ein2-1 breakstrength profiles. B, Wild-type (Ws),
dab1-1, dab2-1, and dab3-1 breakstrength profiles (n ⬎ 10).
199
Patterson and Bleecker
insensitive line ein2-1 indicate that patterns of
chitinase-GUS expression are like etr1-1 because expression is specific to the abscission zone, delayed in
timing, and reduced (data not shown).
Analysis of the delayed abscission mutants dab1-1,
dab2-1, dab3-1, dab3-2, and dab3-3 containing the
chitinase-GUS transgene showed similar patterns of
GUS expression to wild type but much lower levels and
a delay in timing. This expression correlated with the
delay in abscission in each mutant (Fig. 2F) and changes
in the fracture plane as indicated by scanning electron
micrographs (Fig. 3). For example, in dab1-1, expression is not detectable until position nine and does not
begin to decrease until positions 16 or 17. This pattern
of expression correlates with the delay in timing of
petal abscission (position 16) and the observed flattened fracture plane and delayed rounding of cells in
the abscission zone (Figs. 2F and 3). In contrast, in
dab2-1, SEM observations show rounding of cells at
position eight, whereas GUS expression is initially
detectable at position seven. Expression begins to decrease at position 13 and is no longer detectable at
position 15. Last, in dab3-1 and dab3-3, chitinase-GUS
expression is not detectable until position nine and
does not decrease until position 15 (Fig. 2F). These
results indicate that in the mutants loosening of the
cell wall as evidenced by drop in breakstrength, and
the rounding of cells in the fracture planes correlates
with the expression of the chitinase-GUS marker.
The expression patterns of the BAC-GUS in wild
type and etr1-1 have many features similar to the
chitinase-GUS expression patterns. The highest levels
of BAC-GUS expression are observed in the abscission zone of sepals, petals, and filaments concurrent
with abscission. However, BAC-GUS is detectable at
earlier stages of development than CHIT-GUS because expression is visible shortly after anthesis (Fig.
2C). In addition, GUS expression is also detected in
dehiscent anthers (Fig. 2C) and the site of attachment
of the cauline leaf (data not shown). The levels of
expression of BAC-GUS compared with chitinaseGUS are significantly higher as indicated by both
histochemical and fluorometric assays (data not
shown). In the dab mutants, BAC-GUS expression
patterns do not differ significantly from chitinaseGUS patterns of expression, thus failing to distinguish differences in the regulation of the two enzymes within the floral organ abscission zones. We
present these data to indicate that in these lines,
regulation of chitinase and the bean BAC is similar
rather than distinguishable. Identification of mutants
with distinguishable patterns would be useful in establishing genetic pathways.
CONCLUSIONS
An examination of the ethylene-insensitive mutants in Arabidopsis has provided evidence that ethylene signaling may not be an essential component of
floral organ abscission in this system. All of the ex200
amined abscission processes listed above were also
observed in the mutants. The delay in the progression
of the abscission process, coupled with the reduction
in the magnitude with which some abscission related
processes occurred in the ethylene-insensitive mutants, are consistent with the concept that ethylene
acts as a modulator of abscission-related pathways.
However, it is clear that ethylene is not necessary for
the activation of these processes because floral organ
abscission does occur in etr1-1 and ein2-1. This conclusion is strengthened by the fact that the dab mutants show a greater delay in floral organ abscission
than the ethylene-insensitive mutants, yet the dab
mutants show no insensitivity to other ethylene response pathways in the plant. In contrast, there are
also many previous studies that have emphasized a
central role for ethylene in the regulation of abscission (Abeles and Rubenstein, 1964; Jackson and Osborne, 1970; Sexton and Roberts, 1982). These studies
indicate a role for ethylene in the cell separation
process (Abeles and Rubenstein, 1964), the expansion
of cells proximal to the fracture plane (Wright and
Osborne, 1974), and the induction of hydrolytic enzymes in the abscission zone (Boller et al., 1983;
Taylor et al., 1990). More recently, it has been demonstrated in tomato (Lycopersicon esculentum) that
fruit abscission could effectively be blocked by the
use of the ethylene agonist MCP or a mutation in
NR-1 (Never-ripe) (Lanahan et al., 1994; Wilkinson et
al., 1997; Giovannoni, 2001). This is distinct from the
jointless tomato, which lacks development of the
pedicel abscission zone (Szymkowiak and Irish,
1999). In the cut flower industry, ethylene-induced
abscission has been recognized as a serious problem
for decades, and the use of silver nitrate has become
an essential component of marketing. As a result, in
many of these studies, it is the role of ethylene in
stimulating or accelerating abscission that has been
addressed (Osborne, 1989; Roberts et al., 2000).
Differences in gene regulation in ethylene signaling
among plants are not at all unprecedented. For example, CTR1 is constitutively expressed in Arabidopsis
(Kieber et al., 1993), whereas LeCTR1 is developmentally regulated during fruit ripening in tomato (Giovannoni et al., 1999). Studies with citrus and Sambucus
nigra indicate that in specific tissues abscission is an
ethylene-independent process (Lewis et al., 1968; Osborne and Sargent, 1976). Osborne and McManus
(1984) found that many members of the Gramineae
were not responsive to ethylene and that organs often
senesced before abscission. More recently, van Doorn
and Stead have conducted comprehensive studies
looking at floral organ abscission and responses to
ethylene. They observed that in most dicots, ethylene
levels are elevated in response to pollination and abscission subsequently results. However, they also
found that many monocots do not show similar ethylene responses (van Doorn and Stead, 1997; van
Doorn, 2002).
Plant Physiol. Vol. 134, 2004
Floral Organ Abscission in Arabidopsis
The above considerations may be reconciled if one
imagines that many regulatory pathways may impinge on the core pathways that actually drive the
abscission process. The outcome of any one set of
experiments may be dictated by which regulatory
inputs are rate limiting under the specific physiological conditions employed. A particularly relevant example was provided by the analysis of transgenic
Arabidopsis that ectopically express AGL15, an
embryo-associated MADS box transcription factor
(Fernandez et al., 2000; Fang and Fernandez, 2002).
Ectopic expression of AGL15 in flowers results in
substantially greater delay in floral organ abscission
than that observed with the ethylene-insensitive mutants (Fernandez et al., 2000). Yet, application of ethylene to these plants resulted in a rapid induction of
organ abscission. Thus, the pathway regulated by ethylene and the pathway regulated by ectopic AGL15
expression appear to be parallel and, to some degree,
independent. Thus, the specific genetic and physiological conditions dictate which of these pathways appears to be most important in regulating abscission.
The concept of multiple pathways can be extended
to the biochemical and physiological activities that
define the abscission process per se. The shedding of
an organ may not be defined by a single process or
even a linear sequence of processes. Attempts to
single out individual hydrolytic enzymes as rate limiting for cell separation have met with limited success
(Gonzalez-Carranza et al., 1998; del Campillo, 1999).
Transgenic plants containing RNA antisense to ␤-1,4glucanases and polygalacturonases targeted to the
abscission zone have failed to specifically regulate
abscission in several species (Lashbrook et al., 1998;
Sander et al., 2001; Taylor and Whitelaw, 2001; Roberts et al., 2002). Even if scientists have guessed correctly which kinds of cell wall activities drive cell
separation, the likelihood of functional redundancy
is great, given that the candidate cell wall enzymes
are represented by large gene families in plants
(Gonzalez-Carranza et al., 1998; Patterson, 2001; Roberts et al., 2002). Thus, different regulatory pathways
regulating different suites of genes may lead to the
same outcome—cell separation.
Despite these caveats, the identification of mutations affecting floral organ abscission will ultimately
provide a means of distinguishing the different components of the abscission process in Arabidopsis. The
characterization of the dab mutants and others such
as ida (Butenko et al., 2003), nevershed (Liljegren et al.,
2002), and EXPANSIN10 (Cho and Cosgrove, 2000)
are important stepping stones in unraveling these
mysteries of organ abscission.
MATERIALS AND METHODS
Plant Material
Three ecotypes of Arabidopsis were used for this research: Col, Bensheim
(ROO2A), and Ws. ein2-1 and ein2-5 were kindly provided by Joe Ecker
Plant Physiol. Vol. 134, 2004
(University of California, San Diego). Mutant lines were all in the Ws
ecotype unless otherwise noted.
Plant Growth
Seed was surface sterilized with 10% (v/v) commercial bleach (0.5%
[w/v] sodium hypochlorite NaOCl) and 0.05% (w/v) surfactant for 15 to 20
min, followed by three rinses in sterilized deionized water, and then plated
on petri plates containing one-half-strength Murashige and Skoog basal
media (Murashige and Skoog, 1962) with 8 g L⫺1 agar (pH 5.6–5.7), no Suc,
and no hormones. Plates were wrapped with stericrepe (3M Micropore
Tape, 3M, Minneapolis) to prevent drying but to permit good gas exchange.
The plates were placed in the dark for 3 to 7 d at 4°C to guarantee even
germination. Plates were transferred to 22°C with continuous light and
grown for 5 to 7 d before transplanting. Seedlings were transplanted to soil
(1:2 [v/v] Perlite [Midwest Perlite, Appleton, WI]:Jiffy Mix [Jiffy Products of
America, Batavia, Illinois]) and placed in an environmental growth chamber
(Enconair Ecological Chambers, Inc., Winnipeg, MN, Canada) with 16-h
days and 8-h nights at 22°C to 23°C. Light intensity was approximately 150
␮E m⫺2 s⫺1 and provided by a mix of incandescent and cool fluorescent
light. The pots were covered with saran wrap and black shade cloth for the
first 3 to 5 d to minimize transplant shock. A wicking system using strips of
Vattex (Hummerts, St. Louis) was developed to allow for constant bottom
watering with 10% (v/v) Hoagland solution (Hoagland and Arnon, 1938).
All seeds were harvested from single plants with siliques that were brown
and dry unless otherwise specified. All seeds were desiccated for a minimum of 2 weeks before replanting.
Identification of Delayed Abscission Mutants
The Arabidopsis Biological Resource Center (University of Ohio, Columbus) T-DNA insertion lines were screened by selecting delayed abscission
mutants after gently brushing inflorescences of all of the plants to facilitate
removal of unattached floral organs. Individual plants in which petals
abscised after position 10 were selected and further characterized.
Genetic Analysis of Mutants
Crosses were performed by manually emasculating the female recipient
before anthesis and just before the petals emerged above the sepals. Pollen
from the male donor parent was transferred to the female parent by rubbing
a recently shed flower on the female stigma. This was done immediately
after emasculation and then again the next morning (18–24 h later). Each
cross was treated independently and labeled as such. F1 plants were allowed
to self-pollinate and seed collected as specified for all plants. Simple genetic
analysis was determined by ␹2 tests. Selected mutants were backcrossed to
the wild-type parent to determine if the delay in abscission was a single
gene trait and if this trait was recessive or dominant. Multiple crosses were
performed in both directions to give greater confidence to the cross and to
rule out any possible maternal effects. Populations of approximately 100 F2
plants were screened to determine whether a trait was a single gene and
whether it was recessive or dominant. After determining whether each
mutant was a single dominant or recessive trait, mutants were intercrossed
to determine if genes were complementary.
Ethylene Responses
Ethylene responsiveness was determined by treating germinating seedlings with 10 ppm ethylene for 3 d in the dark as described by Chen and
Bleecker (1995) and gassing mature plants with 10 ppm ethylene for 48 h in
16-h days and 8-h nights. Ethylene-responsive seedlings showed a characteristic shortened hypocotyl, thickened hypocotyls, and more pronounced
curvature of the hook. Mature plants displayed yellowing of rosette leaves,
yellowing of sepals, and withering of petals. The ethylene-insensitive mutants etr1-1, ein2-1, and ein2-5 were elongated and displayed no curvature of
the hook.
201
Patterson and Bleecker
Light Microscopy and Image Processing
Fresh tissues were fixed in 4% (v/v) gluteraldehyde (Sigma, St Louis) or
4% (v/v) gluteraldehyde and 1% (w/v) paraformaldehyde (Sigma) in a 0.05
m potassium phosphate buffer (pH 7.4), rinsed four times in buffer, and then
dehydrated in a graded ethanol series. Tissues were then embedded in
medium-grade LR White resin (Ted Pella, Inc., Redding, CA). One- to 2-␮m
sections were made on an MT-2 ultramicrotome (Sorvall, Inc., Norwalk, CT),
and sections were stained with 0.05% (w/v) Toluidine Blue O. Sections were
mounted on glass slides and coverslips annealed with Cytoseal 60 (Thomas
Scientific, Riverdale, NJ). All observations were done on a BX50 microscope
(Olympus Optical Company, Tokyo) using bright-field, phase contrast, or
florescent microscopy. Objectives used included PlanApo 10⫻, 20⫻, 40⫻,
and 60⫻ objectives. For observation of precipitate in the transgenic lines
with GUS activity, tissues were left unstained and observed by dark-field
microscopy. Photographic images were recorded on Ektachrome 160T color
slide film and Kodacolor print film ISO100 and 200 (Eastman Kodak, Rochester, NY), which were subsequently scanned with a Kodak RFS 2035
Professional film scanner. Images were assembled for figures using Adobe
Photoshop 4.0 software (Adobe, Inc., Mountain View, CA).
search Service, Beltsville, MD) and is the same as the construct used to
analyze abscission in the embryo MADS domain AGL-15 (Fernandez et al.,
2000). This construct containing the BAC promoter and GUS gene was
inserted into the pBIN19 transformation vector (Bevan, 1984) and introduced into Arabidopsis using Agrobacterium tumefaciens strain GV3101 and
vacuum infiltration techniques of Bechtold et al. (1993). Arabidopsis plants
were grown in short-day conditions (10-h-light/14-h-dark cycle) to encourage vigorous rosette development. At 4 weeks, when small inflorescence
shoots developed, these inflorescences were removed, and plants were
allowed to recover for 5 to 7 d. After recovery, plants were vacuum infiltrated with A. tumefaciens GV3101 containing the bean abscission cellulase
(BAC)-GUS construct pBAC:GUS (Koehler et al., 1996). Plants were grown
out for two generations (M2) before analysis. The number of T-DNA inserts
was determined by Southern and segregation of kanamycin resistance. A
line with a single insert was selected and used for further studies. The
pBAC:GUS was introduced into etr1-1 and the delayed abscission mutants
dab1-1, dab2-1, dab3-1, dab3-2, and dab3-3 in the same way as the pCHIT:GUS
construct by backcrossing mutant and transgenic lines as described above.
Histochemical GUS Assays
SEM
Individual flowers from each stage of abscission were fixed in 4% (v/v)
gluteraldehyde in a 0.05 m potassium phosphate buffer (pH 7.4), rinsed four
times in buffer, and then dehydrated in a graded ethanol series. Samples
were critical point dried in liquid CO2 at the SEM facility (Entomology
Department, University of Wisconsin, Madison) and stored desiccated for
future use. Samples were mounted on steel plates covered with double-stick
tape and sputter coated with gold palladium. Samples were viewed at 10K
accelerating voltage on a Hitachi S-570 Scanning Electron Microscope (Hitachi, Ltd., Tokyo). Images were recorded on Polaroid 55P-N film (Polaroid
Corporation, Cambridge MA) or digitally captured using a Gatan digital
image capture system and digital micrograph 2.5 software (Gatan Inc.,
Pleasanton, CA). Fifteen or more flowers were observed at each position of
floral organ abscission, and these samples were collected from plants grown
on two or more replications.
Breakstrength Measurements
Breakstrength measurements were conducted on inflorescences containing 15 or more open flowers and before floral meristem arrest. Flower
position was designated relative to the initial flower at anthesis with the
whites of petals just showing. Flowers in positions two to four were progressively older and further down the inflorescence. Breakstrength measurements were conducted on a stress transducer developed by Edgar P.
Spalding and Anthony B. Bleecker (Botany Department, University of Wisconsin, Madison) and described by Patterson (1998). Petals were clamped
individually at each position, and the electrical resistance required to remove the petal was measured by a Fort 10 force transducer (World Precision
Instruments, Inc., Sarasota, FL) and a voltmeter (Radio Shack, Fort Worth,
TX). The voltage (range 0–10 V) was converted to gram equivalents (0–10 g),
and petal breakstrength was plotted as gram equivalents required to remove
the petal from the receptacle. Each breakstrength determination represents
10 or more flowers unless otherwise noted.
Generation of Transgenic Arabidopsis
The chitinase promoter:GUS construct was introduced into Arabidopsis
ecotype ROO2A by Grace Chen (Chen and Bleecker, 1995) using cotyledon
and root transformation techniques (Valvekens et al., 1988). Transformed
progeny were backcrossed to wild type (Col or Ws ecotypes) for five
generations to minimize the Bensheim (ROO2A) background that had been
used initially because it was more amenable to transformation. The gene
was introduced to the etr1-1, dab1-3, dab2-1, dab3-1, dab3-2, and dab3-3
mutant backgrounds by backcrossing with subsequent selection for the
delayed abscission phenotype and GUS expression. GUS expression was
easily identified because expression is constitutive in vascular tissues of
seedlings whether wild type or mutant.
The bean (Phaseolus vulgaris) BAC genomic DNA was originally obtained
from Dr. Mark Tucker (U.S. Department of Agriculture/Agricultural Re-
202
Lines containing GUS were stained according to Koltunow et al. (1990),
with minor modifications as specified by Fernandez et al. (2000). Samples
were quickly fixed in 0.3% (v/v) glutaraldehyde (Sigma) in 50 mm potassium phosphate buffer (pH 7.2) and then incubated with 0.5 mg mL⫺1
5-bromo-4-chloro-3-indolyl-␤-Dglucuronic acid (X-gluc; Research Organics,
Cleveland), 0.825 mg mL⫺1 ferriccyanide, 1.0 mg mL⫺1 ferrocyanide, and
0.05% (v/v) betamercaptoethanol for 4 to 12 h at 37°C. After several washes
with 50 mm potassium phosphate buffer (pH 7.2), the tissues were fixed at
4°C overnight with 4% (v/v) gluteraldehyde in the same buffer. Samples
were dehydrated by using a graded ethanol series to 100% (v/v) ethanol
and then embedded in medium-grade LR White (Ted Pella, Inc.). Semithin
sections (2 ␮m thick) were cut with glass knives on a Sorvall MT-2 ultramicrotome and heat fixed to glass slides. Slides were covered with #1
coverslips and sealed with Cytoseal 60 (Thomas Scientific) and viewed
unstained using dark-field microscopy. For general anatomical analyses of
GUS expression, tissues were immediately incubated with 0.5 mg mL⫺1
X-gluc (Research Organics), 0.825 mg mL⫺1 ferriccyanide, 1.0 mg mL⫺1
ferrocyanide, 0.05% (v/v) TritonX 100, and 0.05% (v/v) betamercaptoethanol in 50 mm potassium phosphate buffer (pH 7.2) for 4 to 12 h at 37°C. After
X-gluc incubation, tissues were rinsed several times in 70% (v/v) ethanol
and stored at room temperature.
ACKNOWLEDGMENTS
We would like to thank members of the Patterson and Bleecker labs for
constructive criticism and reading of the manuscript, especially Ayala Most.
A special thanks to Edgar Spalding for development of the stress transducer
(Love-me-not meter) to measure breakstrength on Arabidopsis petals.
Thanks also to Heidi Barnhill and Phillip Oshel for assistance with SEM and
to Dr. Mark Tucker for the BAC construct. We would also like to thank
Claudia Lipke for photography of plant samples and assistance in microscope imaging.
Received June 3, 2003; returned for revision July 21, 2003; accepted October
1, 2003.
LITERATURE CITED
Abeles FB, Morgan PW, Saltveit ME (1992) Ethylene in Plant Biology, Ed 2.
Academic Press, San Diego
Abeles FB, Rubenstein B (1964) Regulation of ethylene evolution and leaf
abscission by auxin. Plant Physiol 39:963–69
Addicott FT (1982) Abscission. University of California Press, Berkeley
Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium gene transfer
by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris Life
Sci 316: 1194–1199
Bevan MW (1984) Binary Agrobacterium vectors for plant transformation.
Nucleic Acids Res 12: 8711–8721
Plant Physiol. Vol. 134, 2004
Floral Organ Abscission in Arabidopsis
Bleecker AB, Estelle MA, Somerville C, Kende H (1988) Insensitivity to
ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 241: 1086–1089
Bleecker AB, Patterson SE (1997) Last exit: senescence, abscission, and
meristem arrest in Arabidopsis. Plant Cell 9: 1169–1179
Boller T, Gehri A, Manch F, Vogeli V (1983) Chitinase in bean leaves:
induction by ethylene, purification, properties, and possible function.
Planta 157: 22
Broglie KE, Biddle P, Cressman R, Broglie R (1989) Functional analysis of
DNA sequences responsible for ethylene regulation of a bean chitinase
gene in transgenic tobacco. Plant Cell 1: 599–607
Butenko MA, Patterson SE, Grini PE, Stenvik G-E, Amundsen SJ, Mandal
A, Aalen RB (2003) IDA controls floral organ abscission in Arabidopsis
and identifies a novel family of putative ligands in plants. Plant Cell 15:
2296–2307
Chen GQ, Bleecker AB (1995) Analysis of ethylene signal-transduction
kinetics associated with seedling growth response and chitinase induction in wild-type and mutant Arabidopsis. Plant Physiol 108: 597–607
Cho H-T, Cosgrove DJ (2000) Altered expression of expansin modulates leaf
growth and pedicel abscission in Arabidopsis thaliana. Proc Natl Acad Sci
USA 97: 9783–9788
Craker LE, Abeles FB (1969) Abscission: quantitative measurement with a
recording abscissor. Plant Physiol 8: 1139–1143
del Campillo E (1999) Multiple endo-1,4-beta-d-glucanase (cellulase) genes
in Arabidopsis. Curr Top Dev Biol 46: 39–63
del Campillo E, Lewis LN (1992) Identification and kinetics of accumulation
of proteins induced by ethylene in bean abscission zones. Plant Physiol
98: 955–961
Ecker JR (1995) The ethylene signal-transduction pathway in plants. Science
268: 667–675
Esau K (1977) Cell wall. In Anatomy of Seed Plants. John Wiley and Sons,
New York, pp 43–59
Fang S, Fernandez DE (2002) Effect of regulated overexpression of the
MADS domain factor AGL15 on flower senescence and fruit maturation.
Plant Physiol 130: 78–89
Fernandez DE, Heck GR, Perry SE, Patterson SE, Bleecker AB, Fang S-C
(2000) The embryo MADS domain factor AGL15 acts post-embryonically:
inhibition of perianth senescence and abscission via constitutive expression. Plant Cell 12: 183–197
Giovannoni JJ, Yen H, Shelton B, Miller S, Vrebalov J, Kannan P, Tieman
D, Hackett R, Grierson D, Klee H (1999) Genetic mapping of ripening
and ethylene-related loci in tomato. Theor Appl Genet 98: 1005–1013
Giovannoni JJ (2001) Molecular regulation of fruit ripening. Annu Rev
Plant Physiol Plant Mol Biol 52: 725–749
Gonzalez-Carranza ZH, Lozoya-Gloria E, Roberts JA (1998) Recent developments in abscission: shedding light on the shedding process. Trends
Plant Sci 3: 10–14
Hoagland DR, Arnon DI (1938) The water-culture method for growing
plants without soil. Calif Agric Exp Stn Circ 347:
Horton RF, Osborne DJ (1967) Senescence, abscission and cellulase activity
in Phaseolus vulgaris. Nature 214: 1086–1088
Jackson MB, Osborne DJ (1970) Ethylene, the natural regulator of leaf
abscission. Nature 225: 1019–1022
Kieber J, Rothenberg M, Roman G, Feldmann K, Ecker J (1993) CTR1, a
negative regulator of the ethylene response pathway in Arabidopsis,
encodes a member of the Raf family of protein kinases. Cell 72: 427–441
Koehler SM, Matters GL, Nath P, Kemmerer EC, Tucker ML (1996) The
gene promoter for a bean abscission cellulase is ethylene-induced in
transgenic tomato and shows high sequence conservation with a soybean
abscission cellulase. Plant Mol Biol 31: 595–606
Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990)
Different temporal and spatial gene expression patterns occur during
anther development. Plant Cell 2: 1201–1224
Lanahan MB, Yen H-C, Giovannoni JJ, Klee HJ (1994) The Never Ripe
mutation blocks ethylene perception in tomato. Plant Cell 6: 521–530
Lashbrook CC, Giovannoni J, Hall BD, Fischer RL, Bennett AB (1998)
Transgenic analysis of tomato endo-B-1,4-glucanase gene function: role of
cel1 in floral abscission. Plant J 13: 303–310
Lewis LN, Palmer RL, Hield HZ (1968) Interactions of auxins, abscission
accelerators, and ethylene in the abscission of Citrus fruit. In F Wightman,
Plant Physiol. Vol. 134, 2004
G Setterfield, eds, Biochemistry and Physiology of Plant Growth Substances Runge Press, Ottawa pp 1303–1313
Lewis LN, Varner JE (1970) Synthesis of cellulase during abscission of
Phaseolus vulgaris leaf explants. Plant Physiol 46: 194–199
Liljegren SJ, Yanofsky MF, Ecker JR (2002) NEVERSHED controls floral
abscission in Arabidopsis. In L Romero, C Gotor, JM Pardo, MA Botella,
eds, XIII International Conference on Arabidopsis Research, Palacio de
Exposiciones y Congresos, Sevilla, Spain. University of Seville, Spain.
Abstract no. 5-39
McKenzie RJ, Lovell PH (1992) Perianth abscission in Montbretia (Crocosmia ⫻ crocosmiiflora). Ann Bot 69: 199–207
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497
Osborne DJ (1989) Abscission. Curr Rev Plant Sci 8: 103–129
Osborne DJ, McManus MT (1984) Abscission and recognition of zone
specific target cells. In Y Fuchs, E Chalutz, eds, Ethylene: Biochemical,
Physiological, and Applied Aspects. Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, The Netherlands, pp 221–230
Osborne DJ, Sargent JA (1976) The positional differentiation of abscission
zones during the development of leaves of Sambucus nigra and the
response of the cells to auxin and ethylene. Planta 132: 197–204
Palmer RL, Hield HZ, Lewis LN (1969) Citrus leaf and fruit abscission. In
AC Hulme, ed, Proceedings of the First International Citrus Symposium,
University of California, Riverside, CA. Academic Press, NY, pp 1135–
1143
Patterson SE (1998) Characterization of delayed floral organ abscission and
cell separation in Arabidosis thaliana L. Heynh. PhD thesis. University of
Wisconsin, Madison
Patterson SE (2001) Cutting loose: abscission and dehiscence in Arabidopsis. Plant Physiol 126: 494–500
Roberts JA, Elliot KA, Gonzalez-Carranza ZH (2002) Abscission, dehiscence, and other cell separation processes. Annu Rev Plant Biol 53:
131–158
Roberts JA, Whitelaw CA, Gonzalez-Carranza ZH, McManus M (2000)
Cell separation processes in plants: models, mechanisms and manipulation. Ann Bot 86: 223–235
Sander L, Child R, Ulvskov P, Albrechtsen M, Borkhardt B (2001) Analysis
of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica
napus) and Arabidopsis thaliana: evidence for roles in cell separation in
dehiscence and abscission zones, and in stylar tissues during pollen tube
growth. Plant Mol Biol 46: 469–479
Sexton R, Roberts JA (1982) Cell biology of abscission. Annu Rev Plant
Physiol 33: 133–162
Szymkowiak EJ, Irish EE (1999) Interactions between jointless and wildtype tomato tissues during development of the pedicel abscission zone
and the inflorescence meristem. Plant Cell 11: 159–175
Taylor EJ, Whitelaw CA (2001) Signals in abscission. New Physiol 151:
323–339
Taylor JE, Tucker GA, Lasslett Y, Smith CJS, Arnold CM, Watson CF,
Schuch W, Grierson D, Roberts JA (1990) Polygalacturonase expression
during abscission of normal and transgenic tomato plants. Planta 183:
133–138
Tucker ML, Sexton R, del Campillo E, Lewis LN (1988) Characterization of
a cDNA clone and regulation of gene expression by ethylene and auxin.
Plant Physiol 88: 1257–1262
Valvekens D, Van Montagu M, Van Lijsebettens M (1988) Agrobacterium
tumefaciens-mediated transformation of Arabidopsis thaliana root explants
using kanamycin selection. Proc Natl Acad Sci USA 85: 5536–5540
van Doorn WG (2002) Effect of ethylene on flower abscission: a survey. Ann
Bot 89: 689–693
van Doorn WG, Stead AD (1997) Abscission of flowers and floral parts. J
Exp Bot 48: 821–837
Wilkinson JQ, Lanahan MB, Clark DG, Bleecker AB, Chang C, Meyerowitz EM, Klee HJ (1997) A dominant mutant receptor from Arabidopsis
confers ethylene insensitivity in heterologous plants. Nat Biotechnol 15:
444–447
Wright M, Osborne DJ (1974) Abscission in Phaseolus vulgaris: the positional
differentiation and ethylene induced expansion growth of specialised
cells. Planta 120: 163–170
203