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AOBPreview originally published online on October 25, 2007
Annals of Botany 2008 101(2):285-292; doi:10.1093/aob/mcm269
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© The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Further Examination of Abscission Zone Cells as Ethylene Target Cells in Higher Plants

Michael T. McManus*

Institute of Molecular Biosciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand

* For correspondence. E-mail M.T.McManus{at}massey.ac.nz

Received: 7 June 2007    Returned for revision: 19 August 2007    Accepted: 31 August 2007    Published electronically: 25 October 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

Background and Aims: Two aspects of the competence of abscission zone cells as a specific class of hormone target cell are examined. The first is the competence of these target cells to respond to a remote stele-generated signal, and whether ethylene acts in concert with this signal to initiate abscission of the primary leaf in Phaseolus vulgaris. The second is to extend the concept of dual control of abscission cell competence. Can the concept of developmental memory that is retained by abscission cell of Phaseolus vulgaris post-separation in terms of the inductive/repressive control of β-1,4-glucan endohydrolase (cellulase) activity exerted by ethylene/auxin be extended to the rachis abscission zone cells of Sambucus nigra?

Methods: Abscission assays were performed using the leaf petiole–pulvinus explants of P. vulgaris with the distal pulvinus stele removed. These (–stele) explants do not separate when treated with ethylene and require a stele-generated signal from the distal pulvinus for separation at the leaf petiole–pulvinis abscission zone. Using these explants, the role of ethylene was examined, using the ethylene action blocker, 1-methyl cyclopropene, as well as the significance of the tissue from which the stele signal originates. Further, leaf rachis abscission explants were excised from the compound leaves of S. nigra, and changes in the activity of cellulase in response to added ethylene and auxin post-separation was examined.

Key Results: The use of (–stele) explants has confirmed that ethylene, with the stele-generated signal, is essential for abscission. Neither ethylene alone nor the stelar signal alone is sufficient. Further, in addition to the leaf pulvinus distal to the abscission zone, mid-rib tissue that is excised from senescent or green mid-rib tissue can also generate a competent stelar signal. Experiments with rachis abscission explants of S. nigra have shown that auxin, when added to cells post-separation can retard cellulase activity, with activity re-established with subsequent ethylene treatment.

Conclusions: The triggers that initiate and regulate the separation process are complex with, in bean leaves at least, the generation of a signal (or signals) from remote tissues, in concert with ethylene, a requisite part of the process. Once evoked, abscission cells maintain a developmental memory such that the induction/repression mediated by ethylene/auxin that is observed prior to separation is also retained by the cells post-separation.

Key words: Phaseolus vulgaris, Sambucus nigra, abscission, auxin, β-1,4-glucan endohydrolase (cellulase), ethylene, target cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
The concept of target cells normally refers to the notion that a cell has a pre-determined competence to respond in a defined way to a specific hormonal signal. Only the specific hormone (or a chemically related analogue) can evoke that particular response in the cell (Osborne and McManus, 2005). The first substantive evidence of target cells in plants comes with the documentation of different target types in the cortical cells of plant tissues in terms of their growth responses to auxin and ethylene. This led to the identification of three types of cells with respect to their responses to these hormones (Osborne, 1977). In broad terms, type I cells elongate in response to auxin, while the addition of ethylene arrests this elongation growth and causes lateral expansion with the cell volume remaining essentially unchanged. Type II target cells are marked by expansive growth enhanced by ethylene, and not by auxin. The third type of target cortical cells, the type III cells, are found typically in the stems and petioles of many species confined to flooded or aquatic habitats, e.g. Ranunculas sceleratus or the water fern Regnellidium diphyllum. These plants possess cortical cells that will expand and extend with either auxin or ethylene (Osborne, 1977).

The Type II cells that comprise the abscission zones in higher plants have been well characterized in terms of their responses to the hormones ethylene and auxin. Indeed, the time-course of abscission can be conveniently divided based upon the response of cells to these hormones such that the abscission process comprises two stages: a first stage denoted by the period in which added auxin can retard the abscission process while auxin added at the second stage can accelerate the process (Addicott, 1970). The repressive effect of added auxin prior to the addition of ethylene has been shown in a number of species including Citrus sinensis (Ratner et al., 1969; Jaffe and Goren, 1979) and P. vulgaris (Wright and Osborne, 1974). In cells of the rachis abscission zone of S. nigra, the application of auxin prior to treatment with ethylene also delays the abscission response, but Osborne and Sargent (1976) were also able to show that this repression can be overcome by a higher concentration of added ethylene. Together, these observations underline the target status of these cells in terms of their competence to respond to auxin and ethylene, and the specificity of this response. However, Thompson and Osborne (1994) revealed another essential component of abscission target cell competence in the leaf pulvinus abscission response of P. vulgaris. These authors have shown that some product of stelar degradation during ethylene-induced senescence of cells distal to zone cells is responsible for signalling an abscission sequence of events at the abscission zone, and that in the absence of the stelar-signal, ethylene alone is ineffective as the abscission inducer.

In the experiments performed by Thompson and Osborne (1994), the putative dual role of ethylene and the stelar signal in the regulation of the abscission process was not examined specifically. That is, while ethylene alone is not sufficient, is the stele signal alone sufficient to induce the abscission response at the primary abscission zone? The question of the role of ethylene in initiating or regulating the timing of abscission has been brought into sharp focus recently with studies using floral organ abscission and ethylene response mutants of the model plant species, Arabidopsis thaliana (Fernandez et al., 2000; Jinn et al., 2000; Butenko et al., 2003; Patterson and Bleecker, 2004). Together these studies suggest that ethylene, at least in floral organ shedding in Arabidopsis, is a modulator of the abscission process, but its perception may not be essential to initiate the process.

In this study, therefore, the role of ethylene and the regulation of the hormone with the stele-associated signal is first examined during pulvinus separation in abscission explants of P. vulgaris – is the stele signal alone sufficient to induce separation of the pulvinus? Then, this examination of the abscission cell as a target cell class for ethylene is further extended by looking at the dual auxin–ethylene control of cellulase activity. While the role of the hormone in the events up to cell separation is well established, auxin and ethylene can also exert similar repressive/inductive effects in cells post-separation (Osborne et al., 1985), i.e. the abscission target cells exhibit a developmental memory with respect to their response to these hormones. This has been shown for P. vulgaris and so it was of interest to see if the competence extends to abscission cells in other species, in this case Sambucus nigra.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Plant material and treatments
Seeds of P. vulgaris L. ‘Masterpiece’ (Asmer Seeds Ltd, Leicester, UK) were germinated in Levington's Universal Compost in a temperature-controlled glasshouse. The growing seedlings were maintained under 14-h-long days at a minimum temperature at 15 °C. To generate abscission explants, the first leaf pair, at the point of maximum leaf expansion (usually 12–15 d) were excised and used for experiments. From these primary leaves, 1·5-cm explants were excised to include the distal pulvinus, the distal abscission zone and the subtending petiole (McManus et al., 1998). To produce explants from which the stele from the distal pulvinus is removed [designated as (–stele) explants], the procedure as reported in Thompson and Osborne (1994) was used.

For some experiments, bean plants were maintained until the primary leaf developed visual signs (onset of chlorosis) of senescence (usually 30–40 d). As appropriate, specific tissues were excised from the primary leaf of either younger seedlings (at 12–15 d) or from older seedlings where the tissues of the primary leaf were displaying clear visual symptoms of senescence (at 30–40 d).

To generate ethylene-treated tissues, excised explants were supported on Perspex racks embedded in 2 % (w/v) agar, enclosed in humidified air-tight glass chambers and treated with ethylene gas to a concentration of 10 µL L–1. Explants were maintained in the dark at 23 °C, and the treated explants were aerated every 12 h. To generate ethylene-treated mid-rib tissues, 30- to 40-mm segments were excised, cut to preclude as much laminar tissue as possible, from the mid-rib of primary leaves of 12- to 15-d-old seedlings. These segments were supported on Perspex racks and treated with ethylene as described previously for the abscission explants.

To conduct abscission assays, (–stele) explants were supported on Perspex racks embedded in 2 % (w/v) agar, enclosed in humidified air-tight glass chambers and treated with ethylene gas to a concentration of 10 µL L–1. Explants were maintained in the dark at 23 °C, and the treated explants were aerated every 12 h. For treatment with the ethylene-action blocker, 1-methyl cyclopropene (1-MCP), the explants were again enclosed in humidified glass air-tight containers, the 1-MCP was released by the addition, through a suba-seal port, of warm (60 °C) distilled water and, again, the treated explants were maintained in the dark at 23 °C, with aeration every 12 h. To expose the (–stele) abscission explants to excised tissues that had been ethylene-treated, as appropriate, approx. 3 mm x 3 mm segments were held in place using 1 µL droplets of cooled, molten 1 % (w/v) agarose, and the explants enclosed in humidified air-tight glass containers, and treated with ethylene or 1-MCP, as described previously.

Compound leaves of Sambucus nigra L. were collected from local sites around Oxford, UK. In the shortest time possible, 2·5-cm rachis abscission explants were excised from the leaves as described in Osborne and Sargent (1976). The explants were excised to include both the rachis and the leaflet abscission zone, enclosed in air-tight glass dishes with the physiological basal end of the explant placed in 2 % (w/v) agar to a depth of approx. 5 mm to hold the explants in place. For the ethylene treatment, explants were maintained in the sealed containers in which endogenously evolved ethylene accumulated (typically to a concentration of 1–3 µL L–1, as determined by gas chromatography). At appropriate time intervals, the explants were treated with IAA (1 mM) or water by placing 2-µL droplets onto the cut rachis and leaflet petiole surfaces, or, after separation of the rachis and leaflet base, directly to the exposed cells.

Tissue extraction and β-1,4-glucanhydrolase enzyme assay
For extraction, tissue was homogenized in 50 mM sodium phosphate buffer, pH 6·0, containing 100 mM NaCl at a standard ratio of 3 mL extraction buffer : 1·0 g fresh weight of tissue. After sufficient homogenization, the slurry was centrifuged at 10 000 g for 5 min, the supernatant removed and either used directly to treat the exposed abscission zone of the (–stele) explants, or used for protein or enzyme measurements or for SDS–PAGE and western analysis. Cellulase (β-1,4-glucan endohydrolase) activity in the supernatant was measured in excised tissues of P. vulgaris or S. nigra as described in McManus et al. (1998). Cellulase activity is expressed as arbitrary units (AU) where 1 unit is a reduction in viscosity of 1·0 %, per minute, per millilitre of extract.

SDS–PAGE and western analysis
SDS–PAGE and subsequent western analysis was carried out using established procedures. Extracts for separation were prepared as described previously for the β-1,4-glucan endohydrolase assay. Depending on the tissue, 1–4 µg of total protein was separated through a 12 % polyacrylamide gel, and antibody recognition by antibodies raised against the pI 9·5 isoform of β-1,4-glucan endohydrolase was determined using the Supersignal West Pico Chemiluminescent system (Pierce Biotechnology).

Antibodies were raised to the pI 9·5 isoform in New Zealand rabbits through immunization of a recombinant protein using established procedures. To synthesize the recombinant protein, the pBAC1 gene (kindly gifted by Dr Mark Tucker, USDA Beltsville, MD, USA) was directionally cloned into the pPROEX-1 vector (Life Technologies) and then transformed into Escherichia coli. Induction and purification of the recombinant protein was as described previously (Hunter et al., 1999).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Ethylene as a signal to regulate cell-to-cell separation in the leaf pulvinus abscission zone of bean
For the initial assay, (–stele) explants were either treated with extracts of a separated abscission zone or the senescent pulvinus after treatment with ethylene for 48 h, or a segment of similarly treated senescent pulvinus was placed in direct contact with the exposed petiole–pulvinus abscission zone. Cellulase activity assays confirmed that both extracts contained enzyme activity (data not shown). The explants were enclosed in air-tight humidified glass dishes and aerated at 12-h intervals. Scoring for pulvinus separation was undertaken daily but by 4 d, no separation had been observed for any of the (–stele) explants (Table 1). After 4 d, the explants were treated with ethylene (10 µL L–1) and scored for separation of the pulvinus at 1 d and 2 d post-ethylene treatment. After 2 d in ethylene (6 d in total), all of the explants where the abscission zone was in contact with a segment of the senescent pulvinus had separated. In contrast, no separation was observed for explants treated with extracts from 48-h-treated abscission zone or pulvinus tissue.


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  		TABLE 1. Abscission assays with (–stele) explants of P. vulgaris

 
The results displayed in Table 1 suggested that a signal or signals that emanate from the whole senescent pulvinus tissue was necessary for separation at the abscission zone of the (–stele) explants to occur and essentially confirm the earlier observations of Thompson and Osborne (1994). Separation occurred after ethylene treatment, but since there were no explants maintained in an atmosphere without ethylene (to act as a suitable control), it is not clear whether ethylene is also essential for the abscission process. Therefore, in further abscission assays, an identical set of explants was maintained in the ethylene action blocker, 1-MCP (at 1 µL L–1). Further, to test for the role of the stele in the separation process, pulvinus, abscission zone and pulvinus cortex tissue that had been treated with ethylene for 48 h were placed in contact with the exposed abscission zone of the (–stele) explants. The abscission zone tissue and the pulvinus contain stele tissue, while the cortex tissue has been excised from the pulvinus to avoid stele tissue. In these assays (Table 2A), separation in response to ethylene treatment after 3 d was observed in the ethylene-treated and stele-containing tissue (Eth-treated PV; Eth-treat AZ), but no evidence of separation was evident in the 1-MCP-treated explants in contact with ethylene-treated and stele-containing tissue (Eth-treated PV; Eth-treat AZ). Also, no separation was observed in the ethylene-treated cortex tissue that was placed in contact with explants and either treated with ethylene or 1-MCP (Table 2A). Assays of cellulase activity revealed that the ethylene-treated pulvinus and abscission zone tissue contained activity, while no activity was present in ethylene-treated pulvinus cortex tissue that was devoid of stele (Table 2B).


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  		TABLE 2. Abscission assays with (–stele) explants of P. vulgaris

 
The results displayed in Table 2 suggest that ethylene and the presence of stele tissue is necessary for separation of the distal pulvinus in the (–stele) explants. To confirm this observation and to examine whether only the stele tissue from the distal pulvinus is necessary for separation, pulvinus tissue or leaf mid-rib tissue, excised from a senescent primary leaf, was placed in contact with the exposed abscission zone of the (–stele) explant and were either treated with ethylene (10 µL L–1) or with 1-MCP (1 µL L–1) (Table 3). After 2 d, none of the explants had separated but by 5 d all of the ethylene-treated explants that were in contact with the senescent pulvinus and most of those that were in contact with the senescent midrib tissue had also separated. None of the explants that were treated with 1-MCP had separated after 5 d (Table 3A). To detect the occurrence of cellulase activity in the segments used, enzyme activity was assayed in extracts of the pulvinus and midrib tissue excised from the senescent primary leaf, and for comparative purposes, extracts of pulvinus tissue treated with ethylene for 48 h (Table 3B). The cellulase assays showed that both the mid-rib and pulvinus tissue excised from the senescent primary leaf contained enzyme activity with more present in the extract from senescent pulvinus tissue. However, this activity was considerably less than that observed in the extracts of the ethylene-treated pulvinus tissue (Table 3B).


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  		TABLE 3. Abscission assays with (–stele) explants of P. vulgaris

 
The results displayed in Table 3 suggest that both ethylene and senescent stele tissue is required for separation of the pulvinus in (–stele) explants. However, to examine whether aged tissue is essential for the separation process, further abscission assays were performed in which the exposed abscission zone of the (–stele) explants was placed in contact with leaf mid-rib tissue excised from either green or senescent primary leaves, and either treated with ethylene (10 µL L–1) or 1-MCP (1 µL L–1) (Table 4A). In these experiments, after 3 d, the pulvinus had separated from all of the (–stele) explants that were ethylene-treated and in contact with mid-rib tissue excised from green leaves, while 80 % of the ethylene-treated (–stele) explants with the abscission zone in contact with mid-rib tissue excised from senescent primary leaves also displayed separation of the pulvinus. In contrast, no separation was observed in any of the 1-MCP treated (–stele) explants (Table 4A). To investigate the presence of cellulase activity, extracts from freshly excised mid-rib tissue from green primary leaves and senescent primary leaves were assayed. In these assays, only some activity was determined in the mid-rib tissue excised from senescent primary leaves while no detectable activity, under the conditions of the assay used, could be determined in the extracts made from mid-rib tissue freshly excised from green leaves (Table 4B). However, treatment of the mid-rib tissues with ethylene (for 48 h) was sufficient to generate an increase in cellulase activity, and it is reasonable to assume that cellulase activity is induced in the green mid-rib tissue when it is contact with the exposed abscission zone of the (–stele) explants and then ethylene-treated.


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  		TABLE 4. Abscission assays with (–stele) explants of P. vulgaris

 
To confirm that the cellulase activity determined in the ethylene-treated green mid-rib tissue is immunologically related to the activity determined in the ethylene-treated pulvinus and abscission zone tissue, western analysis was performed using antibodies raised against the abscission-associated pI 9·5 isoform. In the extracts tested, a protein of identical molecular mass (51 kDa) was recognized in the separated pulvinus, abscission zone and mid-rib extracts, which was the expected mass of the pI 9·5 isoform (Tucker et al., 1988) (Fig. 1). Although not strictly quantitative, western analysis also confirmed the relative proportions of cellulase activity detected in each tissue with a more intense signal observed for the ethylene-treated pulvinus and abscission extracts, tissues with a higher level of cellulase activity (see Table 2B) when compared with the ethylene-treated mid-rib (see Table 4).


Figure 1
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  		FIG. 1. Recognition, using western analysis, of the pI 9·5 isoform of β-1,4-glucan endohydrolase in extracts of ethylene-treated pulvinus (Pv; 1 µg total protein loaded), ethylene-treated abscission zone (Az; 1 µg total protein loaded) and ethylene-treated green leaf mid-rib (Md; 4 µg total protein loaded) after separation using SDS–PAGE. Antibodies were raised against a recombinant pI 9·5 isoform after expression of pBAC1 in E. coli. Antibody recognition was determined using the Supersignal West Pico Chemiluminescent system.

 
Regulation of cellulase activity in the leaf abscission zones of S. nigra by auxin and ethylene
To extend the investigations into the nature of the abscission zone as a target cell, the regulation of cellulase activity, post-separation, by the hormones auxin and ethylene was examined. To do this, abscission explants of S. nigra were chosen as the experimental material. It is known, using abscission explants of both P. vulgaris and S. nigra, that if a sufficient concentration of auxin is added to the abscission zone prior to ethylene treatment abscission will not occur (Wright and Osborne, 1974; Osborne and Sargent, 1976). Further, Osborne et al. (1985) were able to show in abscission explants of P. vulgaris that the addition of auxin to the exposed abscission zone after separation of the distal pulvinus again repressed any further increase in cellulose activity. However, if ethylene is added again, then cellulase activity resumes. The purpose of the experiments described here, therefore, was to determine whether this post-separation auxin repression–ethylene induction can be extended to other plant species, and so abscission explants of S. nigra were examined.

In this assay, explants were incubated in enclosed glass dishes where endogenously evolved ethylene accumulated for a 24-h period (data not shown), by which time a significant level of cellulase activity could be measured and evidence for some cell-to-cell separation is evident (Fig. 2). At this (24 h) point, the explants were either treated with IAA or H2O by application to the exposed rachis and leaf petiole surfaces of the explants, and these were then maintained, as before, in an enclosed atmosphere in which endogenous ethylene accumulated. After a further 24 h, there was no further induction of activity in the IAA-treated explants, whereas the level of enzyme activity measured in the H2O-treated explants had further increased. At this (48 h) point, the IAA-treated explants were either retreated with IAA or with H2O, while the H2O-treated explants were retreated with H2O. At this stage, the treatments were now directly applied to the exposed cells. The explants were returned to the enclosed atmosphere for a further 24-h period, after which time cellulase activity was again measured. Here, no increase in cellulase activity was observed in the IAA-treated explants that were re-treated with IAA, while an increase in activity was measured in those IAA-treated explants that were re-treated with H2O. Interestingly, a slight decrease in the measured activity of explants that had been treated with H2O only was also observed (Fig. 2).


Figure 2
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  		FIG. 2. Cellulase activity in extracts of the leaf rachis abscission zone of S. nigra. Explants were maintained in a sealed atmosphere in which endogenous ethylene accumulated and at 24 h were treated with either 2·0-µL droplets of H2O and enzyme activity measured 24 h later (at 48 h; {blacktriangleup} — {blacktriangleup}), or with IAA (arrowed) and enzyme activity measured 24 h later (at 48 h; {blacktriangleup} — •). At 48 h, the H2O-treated explants were again treated with H2O and cellulase activity measured 24 h later (at 72 h; {blacktriangleup} — {blacktriangleup}) and the IAA-treated explants were either treated again with IAA (arrowed) and cellulase activity measured 24 h later (at 72 h; • — •), or with H2O and cellulase activity measured 24 h later (at 72 h; • — {blacksquare}). Values are means ± s.e., n = 5.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
The positionally differentiated and functionally specialized cells that comprise the abscission zones of plants represent good examples of hormone target cells in higher plants (Osborne and McManus, 2005). In many species, the abscission zone represents an anatomically distinct region of cells strategically positioned at the physiological base of the organ to be shed (Sexton and Roberts, 1982; Osborne, 1989; Roberts et al., 2000). This region of cells, which is distinctive in the mature plant body of most plant species where it has been examined can, for example in P. vulgaris, differentiate early in development (Osborne, 1989). To accompany this positional differentiation, specific protein determinants have been identified in both S. nigra (McManus and Osborne, 1990a, 1991) and in P. vulgaris (McManus and Osborne, 1990b) that marks these cells as differentiated from their neighbours. As well, in flower buds of Ecballium elaterium, the competence of the ovary-pedicel abscission zone is marked by the attainment of an 8C endoreduplicated state in the cells of the separation zone (Wong and Osborne, 1978). More recently, the characterization of these determinants has been extended with the identification of a plasma membrane-localized serine/threonine receptor-like kinase, HAESA, that is localized in the competent floral organ abscission zone as well as at the base of pedicels and of leaf petioles of A. thaliana (Jinn et al., 2000). The role of the kinase appears to maintain abscission cell competence in the floral organs of Arabidopsis since down-regulation of protein accumulation delays abscission (Jinn et al., 2000).

Further, characterization of the inflorescence deficient in abscission (ida) mutant of Arabidopsis has led to the identification of the secreted 77-residue IDA peptide (Butenko et al., 2003). Using IDAp::GUS transgenics, expression of the IDA gene was shown to be specific to the floral organ abscission zone (Butenko et al., 2003), although overexpression of the IDA imparts the competence to abscise (without added ethylene) to vestigial abscission zones at the bases of the pedicels, inflorescence branches and cauline leaves (Stenvik et al., 2006). The ida mutants have a normally differentiated floral organ abscission zone suggesting that the peptide, in common with HAESA, does not play a role in the differentiation of the abscission zone, but accumulation of the peptide acts as a marker for cellular competence (a functional role for these peptides in the separation process has yet to be shown).

The identification of cellular constituents that may impart competence to abscise has also invited studies on the role of ethylene in the initiation of the abscission process. Experiments with the ethylene-insensitive mutants etr1-1 and ein-2-1 have shown that these plants abscise normally, albeit in a delayed time course when compared with the wild type (Patterson and Bleecker, 2004). Further, the more recently characterized delayed floral organ abscission (dab) mutants also display a delayed abscission response when compared with the ethylene-insensitive mutants, but show normal responses when treated with ethylene (Patterson and Bleecker, 2004). Indeed, observations of the ida mutant of arabidopsis (Butenko et al., 2003), or of transgenic plants with expression of the HAESA gene down-regulated (Jinn et al., 2000) where floral organ abscission is either inhibited or delayed, has shown that ethylene perception is not affected in any of these genetic lines. Evidence from such studies suggests, therefore, that at least in the regulation of floral organ tissues in Arabidopsis, ethylene does not induce the process per se, but instead, is a modulator of the process.

However, consideration of the critical cues that may induce or regulate abscission can no longer be solely confined to the abscission zone itself. Indeed, the question of initiating signals, other than ethylene, has already been postulated previously in the abscission literature. The concept of a ‘senescence factor’ which accelerates the separation process was proposed by Osborne and colleagues some years ago, but the identity of the compound has not been confirmed (Osborne et al., 1972). More recently, the concept of initiating signals remote from the abscission zone has been shown for the proximal leaf abscission zone of the bean, P. vulgaris (Thompson and Osborne, 1994). In earlier studies, the accumulation of significant levels of mRNA coding for the abscission-associated cellulase (the pI 9·5 β-1,4-glucan endohydrolase isoform) was observed in the stele tissue in the distal pulvinus (Tucker et al., 1991). However, in elegant experiments involving micro-dissections, Thompson and Osborne (1994) were able to show that if the vascular tissue is removed surgically from the distal pulvinus of an abscission explant, ethylene is no longer effective in inducing abscission nor the abscission-associated pI 9·5 cellulase isoform. Replacement of the vascular tissue restores the full abscission response, but if any phloroglucinol-positive staining vascular tissue is retained in the explant for 24 h, abscission is initiated and separation is normal (Thompson and Osborne, 1994). The identity of the stelar product is not yet known but its activity is clearly essential for the separation of the distal leaf pulvinus.

More significantly, perhaps, these experiments challenged the view of the leaf abscission zone of bean as a self-contained hormone target cell class. Clearly, a signal (or signals) arising from distal tissue was important for the initiation of the cell-to-cell separation process. However, prior to the current study, it was not known whether the signal that arises from the induced stele could, alone and without ethylene, induce cell-to-cell separation at the abscission zone and subsequent abscission of the pulvinus. The results from the experiments performed in this study suggest that the stele-signal requires ethylene for the functional evocation of cell-to-cell separation and pulvinus separation in the (–stele) explants. If pulvinus tissue, excised from full explants that have been treated with ethylene for 36 h (i.e. induced pulvinus), is placed in contact with the abscission zone and treated with ethylene, then separation of the distal pulvinus on the (–stele) explant is observed. However, if the ethylene blocker 1-MCP is added, then no separation is observed, suggesting that ethylene, in concert with the stele-generated signal from the induced pulvinus, is essential (Table 2). However, the stele tissue does not necessarily need to be localized in the distal pulvinus, but can be generated from both green and senescent leaf mid-rib tissue (although in planta, this tissue is also located distal to the abscission zone) (Tables 3 and 4). This study has shown that ethylene is essential for the generation of the signal from stele tissue (although it has not been shown that ethylene induces the generation of the signal). Likewise, it has been shown that the treatment of the pulvinus and isolated mid-rib tissue will increase cellulase activity in these tissues, although it is not known whether ethylene induces the transcription of the cellulase gene(s) (Table 4). Thompson and Osborne (1994), using immunological recognition, were able to show that the cellulase activity induced in the distal pulvinus was the abscission-associated pI 9·5 β-1,4-glucan endohydrolase. In this study it has been shown that the mid-rib-associated cellulase is also immunologically related (and of the identical molecular mass) to the pI 9·5 isoform (as the gene product of pBAC1; Tucker et al., 1988). However, it is not known whether cellulase activity is essential for the generation of the stele signal. If cellulase is added directly to the (–stele) explants, no separation was observed (Table 1), although it may be that the activity of the protein is not preserved in such treatments. Further experiments are required in which the activity of cellulase is repressed in planta via some anti-sense or RNAi procedures.

The experiments described in the first part of this study have shown that ethylene in association with some stele-generated signal is essential for cell-to-cell separation and the abscission of the distal pulvinus in bean leaf petioles. Whether ethylene is the primary inducer of the process is less certain, but the presence of the hormone is obligatory. When examining events at the abscission zone, it has been shown previously that the continuous presence of ethylene is required to maintain the transcription of the gene encoding the pI 9·5 isoform (Tucker et al., 1988). Using a similar leaf abscission explant system from P. vulgaris, Tucker et al. (1988) showed that if ethylene is withdrawn after 24 h of treatment of the explants (after the pI 9·5 β-1,4-glucan endohydrolase mRNA can be detected at the zone), and replaced with the ethylene action inhibitor, 2,5-norbornadiene, expression of the pI 9·5 β-1,4-glucanhydrolase mRNA at the zone is repressed. However, it has also been shown previously, using the same petiole–distal pulvinus explant system described in the first part of this study, that the addition of auxin, after the induction of cellulase and separation of the distal pulvinus, can repress further induction of cellulase activity (Osborne et al., 1985). Critically, after this repression by auxin, further treatment of the explants with ethylene can induce an increase in cellulase activity – in other words, auxin and ethylene continue to repress/induce the enzyme activity even after cell separation. This suggests that an integral component of the attainment of a specific target cell status by abscission zone cells is to display this developmental memory in which auxin and ethylene can regulate cellular competence post-separation. However, thus far, this functional specialization post-separation has only been shown for the target cells that comprise the leaf abscission zones of P. vulgaris (Osborne et al., 1985).

As the second part of this study, therefore, it was of interest to determine if the cells comprising leaf abscission cells of other species also display this induction/repression of the target cell competence post-separation, and so dual hormonal regulation in the rachis abscission zone cells of S. nigra was examined. In common with bean leaf abscission, it had also been shown previously that the addition of auxin to rachis abscission zone explants prior to ethylene treatment prevented separation of the rachis, a repression that can be overcome with the addition of a higher concentration of ethylene (Osborne and Sargent, 1976). In the experiments performed in the present study, the addition of auxin, after the induction of cellulase activity (at 24 h) and when cell-to-cell separation is first apparent, did repress the subsequent increase in cellulase activity over the 24- to 48-h period in non-IAA-treated explants (Fig. 2). As cell-to-cell separation has been induced in these explants, the retardation of cellulase by IAA is distinct from the retardation observed in phase I of the abscission process and prior to cell-to-cell separation. Over the next 24-h period (48–72 h), activity did increase in the IAA-treated explants if the hormone treatment was not maintained (Fig. 2). However, to confirm that the repression of activity is in response to IAA treatment, some explants were treated again with auxin at 48 h, and these explants did not show any increase in cellulase activity over the 48- to 72-h period (Fig. 2). This cycle of induction/repression in terms of measurable cellulase activity confirms, therefore, the control mediated by ethylene and auxin post-separation observed in the bean leaf abscission zone (Osborne et al., 1985), and it will be interesting to determine if this functionality extends to yet other species.

The induction of cellulase by ethylene and subsequent repression by auxin is also reminiscent of another dual hormone regulation of enzyme activity, that is the control of the secretion of {alpha}-amylase by the aleurone cells surrounding the endosperm in graminaceous seeds by gibberellic acid (the inducer) and abscisic acid (the repressor) (Bethke et al., 1997). Indeed, it has been shown that the control of the target status of the cells comprising the aleurone cell is quite exquisite as a recruitment model has been shown to operate for their response to gibberellin (GA) (Ritchie et al., 1999). Here, as the concentration of GA that irrigates the aleurone layer increases (as biosynthesis of the hormone and secretion from the embryonic axis increases) then more cells are ‘triggered’ to secrete {alpha}-amylase. Further, these cells with differential sensitivity to GA also display precise positional differentiation such that those that respond to lower concentrations of the inducer (GA) are located in closer proximity to the embryo.

It is not known whether abscission cells also display such differential sensitivity to the signals that initiate or regulate the separation process. Such comparisons may be quite elucidatory. However, it is clear that the triggers that initiate and regulate the separation process are complex with, in bean leaves at least, the generation of a signal (or signals) from remote tissues being an essential part of the process. Once evoked, the cells maintain a developmental memory such that the induction/repression mediated by ethylene/auxin that is observed prior to separation is also retained by the cells post-separation.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
The provision of a Travel Fellowship from the Royal Society of London, UK, is acknowledged. The technical assistance of Marissa Roldan is also acknowledged.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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