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First published online December 30, 2003; 10.1104/pp.103.028027 Plant Physiology 134:194-203 (2004) © 2004 American Society of Plant Biologists Ethylene-Dependent and -Independent Processes Associated with Floral Organ Abscission in Arabidopsis1Departments 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, 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
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 µL 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
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 approximately 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
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 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.
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. 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).
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, yellowing 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.
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
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.
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
Expression of the chitinase-GUS reporter gene in wild type and etr1-1 was observed, and expression 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 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 chitinase-GUS 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 chitinase-GUS 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.
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 examined 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
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
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
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
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 (University of California, San Diego). Mutant lines were all in the Ws ecotype unless otherwise noted.
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
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.
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
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
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-µm 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 10x, 20x, 40x, and 60x 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).
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 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
The chitinase promoter:GUS construct was introduced into Arabidopsis ecotype ROO2A by Grace Chen (Chen and Bleecker, 1995
The bean (Phaseolus vulgaris) BAC genomic DNA was originally obtained from Dr. Mark Tucker (U.S. Department of Agriculture/Agricultural Research 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
Lines containing GUS were stained according to Koltunow et al. (1990
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.
Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.028027.
1 This work was supported by the U.S. Department of Agriculture (grant no. 00–35301–9085). * Corresponding author; e-mail spatters{at}facstaff.wisc.edu; fax 608–262–4349.
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ABSTRACT
RESULTS AND DISCUSSION
-glucuronidase (GUS) constructs using the soybean (Glycine max) chitinase (Broglie et al., 1989
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. 
