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First published online June 15, 2006; 10.1104/pp.106.079277 Plant Physiology 141:1604-1616 (2006) © 2006 American Society of Plant Biologists Molecular Changes Occurring during Acquisition of Abscission Competence following Auxin Depletion in Mirabilis jalapa1,[W]Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel (S.M., V.H.); New Zealand Institute for Crop & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand (D.A.H.); and Department of Plant Sciences, University of California, Davis, California 95616 (J.-C.C., M.S.R.)
To understand how auxin regulates sensitivity of abscission zone (AZ) tissues to ethylene, we used a polymerase chain reaction-based subtractive approach to identify gene transcripts in Mirabilis jalapa AZs that changed in abundance during the time the zones became competent to abscise in response to exogenous ethylene. Transcript expression was then examined in leaf and stem AZs over the period they became ethylene competent following indole-3-acetic acid (IAA) depletion either by leaf deblading, treatment with the IAA transport inhibitor naphthylphthalamic acid, or cutting the stem above a node (decapitation). Transcripts down-regulated by deblading/decapitation included Mj-Aux/IAA1 and Mj-Aux/IAA2, encoding Aux/IAA proteins, and three other transcripts showing highest identity to a polygalacturonase inhibitor protein, a  -expansin, and a -tubulin. Application of IAA to the cut end of petioles or stumps inhibited abscission, and prevented the decline in the levels of transcripts in both AZs. Transcripts up-regulated in the AZ following deblading/decapitation or treatment with naphthylphthalamic acid were isolated from plants pretreated with 1-methylcyclopropene before deblading to help select against ethylene-induced genes. Some of the up-regulated transcripts showed identity to proteins associated with ethylene or stress responses, while others did not show homology to known sequences. Sucrose infiltration of stem stumps enhanced abscission following ethylene treatment and also enhanced the induction of some of the up-regulated genes. Our results demonstrate a correlation between acquisition of competence to respond to ethylene in both leaf and stem AZs, and decline in abundance of auxin regulatory gene transcripts.
The timing of developmental processes in plants, such as abscission, senescence, and ripening, is affected by increased tissue sensitivity to ethylene (Trewavas, 1986  ; Bleecker and Patterson, 1997; Zegzouti et al., 1999). The cause of this increased ethylene sensitivity is still not known, but it has been shown to be affected by other plant hormones. In abscission, the interplay between indole-3-acetic acid (IAA) and ethylene is important (Abeles and Rubinstein, 1964; Sexton, 1997; Taylor and Whitelaw, 2001). The generally accepted model is that a basipetal polar IAA flux through the abscission zone (AZ) prevents abscission by rendering the AZ insensitive to ethylene. If the source of IAA is removed, the AZ becomes sensitized to the presence of ethylene and abscission commences (Rubinstein and Leopold, 1963; Abeles and Rubinstein, 1964; Addicott, 1982; Sexton and Roberts, 1982). A classic experiment in support of the above model has been to deblade leaves (removing the IAA source), which leads to premature formation of an AZ and rapid petiole abscission in response to exogenous ethylene (Abeles and Rubinstein, 1964). The direction of the auxin flux is also important for determining whether abscission occurs (Morris, 1993; Roberts et al., 2002). Thus, unlike various auxin-mediated physiological processes that are a result of transient and local changes in auxin levels (Woodward and Bartel, 2005), prevention of abscission has been found to require a continuous and constant polar supply of auxin at the AZ (Taylor and Whitelaw, 2001). Auxin affects abscission not only by regulating sensitivity to ethylene, but also by affecting the intracellular transport of the hydrolytic enzyme polygalacturonase (PG) associated with cell wall degradation. A specific mechanism by which auxin delays or even prevents cell separation in silique dehiscence of oilseed rape (Brassica napus) has been reported. In this system the auxin analog 2-methyl-4-chlorophenoxyacetic acid retarded the secretion of PG to the cell wall, thereby inhibiting pod dehiscence (Dal Degan et al., 2001).
Our knowledge of the biology of abscission has been increased greatly through anatomical, physiological, and biochemical studies (Sexton and Roberts, 1982
However, it has also been shown in Arabidopsis (Bleecker and Patterson, 1997
It seems that in the last years we are only beginning to get insight into regulatory control of abscission at the molecular level. For example, the tomato mutation jointless, which does not form pedicel AZs (Butler, 1936
We hypothesize that IAA regulates genes that control the sensitivity of the AZ tissue to ethylene. To examine this hypothesis we identified in this study transcripts that changed in abundance in the Mirabilis AZ as it became competent to respond to ethylene. We used the AZ tissue of Mirabilis jalapa for this purpose, as this plant has long been used as a model for abscission studies due to its easily identifiable and large AZs (Lloyd, 1916
Physiological Characterization of the M. jalapa Abscission Systems The petiole AZ of M. jalapa became sensitive to the abscission-inducing effects of ethylene when the leaf blades were removed (Fig. 1 ). The rate of petiole abscission was dependent upon the time between leaf deblading and ethylene exposure, with higher abscission rates obtained as this time period increased. The petioles of debladed leaves in the absence of exogenous ethylene eventually abscised 6 d after deblading, but their abscission rate was much slower than that of debladed petioles exposed to ethylene. Nondebladed petioles did not abscise over the time frame examined, even when exposed to ethylene (Fig. 1).
Naphthalene acetic acid (NAA) or IAA applied to the cut surface of the debladed plants reduced the number of petioles that abscised following ethylene exposure (Fig. 2 ). Both native (IAA) and synthetic (NAA) auxins were tested to guarantee a clear auxin effect. IAA was substantially more effective than NAA, resulting in close to complete inhibition of abscission. The ethylene action inhibitor 1-MCP applied prior to leaf deblading, completely prevented petiole abscission caused by exogenously applied ethylene (Fig. 2).
Cutting the stem above a node (decapitation) induces the formation of an AZ at the base of the cut internode that leads to abscission of the internodal stem section (stump) above the newly formed AZ (Beal and Whiting, 1945 ). In our experiments, the stumps abscised in response to 2 d of ethylene exposure (8–5 µL L–1), administered 4 (Fig. 3
), 6, or 9 (data not shown) d after they had been decapitated. The percentage of stump abscission increased with time reaching approximately 90% 5 d after the ethylene treatment. Ethylene did not cause stump abscission in intact plants. 1-MCP applied before decapitation or IAA applied after (in lanolin paste), effectively prevented stump abscission. By contrast, NAA applied to the cut surface either in a lanolin paste (Fig. 3) or by water infiltration (data not shown) was comparatively ineffective.
Suc was applied to the decapitated stumps to examine two possibilities: (1) whether elimination of Suc transport to Mirabilis roots and tuber via the node AZ might in part be responsible for the enhanced abscission, and (2) whether Suc has a possible signaling effect on abscission as a modulator of processes controlled by hormones (León and Sheen, 2003 ). Surprisingly, application of 2% Suc to the cut end of the decapitated stem stump increased the sensitivity of the AZ to ethylene, causing 100% of the stumps to abscise 1 d after ethylene treatment (data not shown).
We used a PCR-based subtractive procedure to isolate gene transcripts that declined in the petiole AZ of Mirabilis during the time the AZ became sensitive to respond to ethylene. This was notably 3 d before the debladed petioles without ethylene treatment would abscise (Fig. 1). To accomplish this we used RNA isolated from the petiole AZ of nondebladed leaves as tester RNA, and RNA isolated from the AZ of petioles that had been debladed for 3 d as driver RNA. The PCR-select procedure selected for those transcripts present in the tester RNA pool and not present in the driver RNA pool. Sequencing and homology searches of 140 insert-containing clones revealed that the deduced amino acid sequence of seven transcripts had significant similarities to a diverse array of auxin-related genes in the GenBank database (Table I ). SM-01 (AY137966) showed 54% identity to Arabidopsis auxin response factor 6 (ARF6 [At1g30330]), SM-32 (AY137965) displayed 62% identity to a Robinia pseudoacacia auxin-repressed protein (ARP) gene (AY009094), and clones Mj-Aux/IAA1 to 5 (AY137961–4 and DQ070872) showed 47% to 93% identity to various members of the Aux/IAA gene family.
RNA gel-blot analyses indicated that the IAA-related genes were expressed differently in the AZ in response to deblading. The expression of some of these genes, such as Mj-Aux/IAA4 (AY137964) and clone SM-32 (AY137965) was not affected by deblading or stem decapitation (data not shown). On the other hand, the expression of certain genes, such as Mj-Aux/IAA1 (AY137961) and Mj-Aux/IAA2 (AY137962) was substantially down-regulated within 1 d of deblading (Fig. 4 ) and within 6 to 9 d of stem decapitation (Fig. 5 ). The deblading-induced decline in Mj-Aux/IAA1 transcripts could be prevented by applying IAA, but not NAA, to the cut end of the debladed petiole (Fig. 4), whereas the reduction in Mj-Aux/IAA2 transcripts was unaffected by application of either auxin after deblading (Figs. 4 and 5). These transcripts were not specific to the AZ and were also detected in petioles of intact (control) leaves and adjacent stem tissues (Fig. 4). The expression of two of the Aux/IAA genes, Mj-Aux/IAA1 and Mj-Aux/IAA4, was also examined in a number of plant tissues and the data are shown in Supplemental Figure 1. Their expression patterns were very different. Mj-Aux/IAA1 was predominantly expressed in the petal-like calyx tissue during opening and senescence of the flowers, and was also expressed, but to a lesser extent, in young stems. Mj-Aux/IAA4, by contrast, was expressed in young leaf, stem, ovary, fruit, root, and tuber tissues, and in flowers only at the small bud stage.
The expression patterns of 12 additional clones from the subtracted cDNA library were examined by RNA gel-blot analysis (data not shown). Only three clones (MSR-27, MSR-127, and MSR-212) were found to be down-regulated in the petiole AZ following deblading (Fig. 4). MSR-27 (AY589714) showed 53% identity to the Actinidia deliciosa PG inhibitor protein (PGIP) gene (Z49063), MSR-127 was found to encode expansin gene ExpB2.1 (AY147412.1), and MSR-212 (AY589713) displayed 84% identity to the Arabidopsis
Isolation and Characterization of Gene Transcripts That Increased in the Petiole AZ of M. jalapa following Deblading/Decapitation An additional PCR-based subtractive library was constructed to isolate gene transcripts that were induced in the petiole AZ following deblading. This library was enriched with RNA species that were preferentially transcribed in the petiole AZ of 1-MCP-treated plants 3 d after deblading (tester RNA) and not present in the petiole AZ of nondebladed leaves (driver RNA). The tester RNA plants were pretreated with 1-MCP prior to deblading since this pretreatment eliminated abscission of the debladed petioles (Fig. 2). Therefore, 1-MCP pretreatment should help select for gene transcripts involved in competency of the AZ to respond to ethylene rather than for those involved in ethylene-mediated cell separation at the AZ. Using differential screening, we examined the effect of deblading on the expression of 94 clones selected from the subtractive library. The resulting dot-blot array is shown in Supplemental Figure 2. Of the 94 gene transcripts, 21 had a 95% probability of being deblading induced (according to the PCR-select cDNA subtraction kit user manual). These transcripts and the putative enzymes they encode are listed in Table III . Six transcripts (FS-18, FS-25, FS-39, FS-44, FS-55, and FS-87) were chosen for further study, and all were confirmed by RNA gel-blot analysis to be induced in the petiole AZ tissue within 3 d after deblading, whether the petioles were pretreated with 1-MCP (Fig. 6 , M/D3) or not (Fig. 6, D3). Of the six transcripts, FS-18 (AY589694), FS-44 (AY589704), and FS-55 (AY589707) did not show significant homology to any known sequence in the GenBank database, whereas FS-25 (AY589695) showed 40% identity to tomato ethylene-responsive transcript ER6 (AF096262.1). FS-39 (AY589698) displayed 83% identity to a Cicer arietinum cationic peroxidase (AJ271660.2), and FS-87 (AY589699) showed 66% identity to a Fragaria ananassa osmotin-like protein (AF532965.1). These transcripts differed in their levels of expression both before and after deblading (Fig. 6) or decapitation (Fig. 7 ). Some transcripts, such as FS-18, were not detected in the AZs of the nondebladed petioles (Fig. 6, lane C) or nondecapitated stems (Fig. 7, lane C), whereas others, such as FS-25, FS-39, and FS-87, were detected. FS-44, FS-55, and FS-87 were also expressed in the non-AZ region of the leaf petiole and in the stem. On the other hand, no detectable expression of FS-18, FS-39, and FS-25 transcripts was observed in these tissues (Fig. 6). The abundance of some of the transcripts (FS-25, FS-44, FS-55, and FS-87) increased considerably within 1 d after deblading, whereas for others (e.g. FS-39) it took 2 d before substantially higher expression levels were observed (Fig. 6). All the transcripts showed increased expression in the stem AZ at 6 d after decapitation, which was the shortest time period examined (Fig. 7). The expression of two of the transcripts (FS-18 and FS-87) was also elevated by exposure to 1-MCP in the AZ of petioles that were not debladed (Fig. 6, M/C). Application of Suc to the cut end of the stump hastened the formation of the stem AZ and its sensitivity to ethylene, resulting in accelerated abscission of the stem stumps. In addition, the Suc treatment enhanced the induction of some of the decapitation-induced transcripts (Fig. 7, FS-44 and FS-55). As was found for the deblading-repressed gene transcripts (Fig. 4), applying NAA to the cut petiole end (Fig. 6, N/D3) or to the cut stump end (Fig. 7, N/CT9) did not affect expression of the transcripts. However, application of IAA in lanolin paste to the cut end of the petiole was effective at inhibiting the deblading-induced increase in expression of FS-18 and FS-44 transcripts even 72 h after the treatment (Fig. 8 ).
The expression of the up-regulated gene transcripts, like that found for the down-regulated ones, was not specific to the AZ tissue. Expression of transcripts FS-44 and FS-25 in various tissues is shown in Supplemental Figure 3. The FS-44 transcript showed high expression in flowers at different stages of development, as well as in ovary, root, and tuber tissues. FS-25 showed an increased expression pattern during flower opening and senescence, in mature and senescent leaves, and in young and mature cotyledons, mature stems, and ovaries.
Leaf deblading or stem decapitation are very effective treatments for removing the auxin source, but they can also result in undesirable local and systemic wounding effects caused by the localized tissue damage (León et al., 2001
It is well known that organ abscission is controlled by the interplay between auxin and ethylene. While our knowledge of the biology of organ abscission and genes of hydrolytic enzymes involved in cell separation processes in the AZ has been increased greatly (Sexton and Roberts, 1982 ; Osborne, 1989; Sexton, 1997; Taylor and Whitelaw, 2001), there is still very little known about the control of abscission competence at the molecular level. This study aimed to identify mRNA transcripts in AZ tissues whose activated or suppressed expression enabled the AZ to gain competence to respond to ethylene. We hoped to accomplish this by isolating transcripts in the AZ whose expression changed in abundance as the AZ became sensitive to ethylene, but was not abscising. Such gene expression might be associated with increased competence to abscise. For this purpose we used the ethylene action inhibitor 1-MCP as a means for focusing our gene libraries on the acquisition of the abscission competence rather than on the ethylene-dependent abscission events.
Results from our initial physiological characterization of the abscission process in M. jalapa indicated that petiole abscission of debladed leaves and internodal stump abscission of decapitated shoots were mediated through ethylene, as pretreatment of the plants with 1-MCP prevented shedding of these organs following deblading/decapitation (Figs. 2 and 3). We showed that this abscission was not solely the result of increased ethylene produced by the detachment, because exogenously applied ethylene did not cause petiole (Fig. 1) or stump (Fig. 3) abscission in intact plants. This suggests that deblading or decapitation caused abscission by increasing the competence of the petiole or stump AZs, respectively, to respond to ethylene. Previous research indicates that the increased competence of AZ tissues to respond to ethylene is due to the reduced auxin flux across the AZ, that occurs either during aging or by removing the auxin source (i.e. the leaf blade or the stem above a node; Beal and Whiting, 1945
Further confirmation for auxin regulation of the process was obtained by application of the IAA transport inhibitor NPA, which caused leaf abscission in response to ethylene similar to the deblading-induced petiole abscission (Fig. 9A). This clearly shows that reducing IAA flux across the AZ without wounding leads to increased competence of the AZ to respond to ethylene. The gradual leaf abscission following the NPA treatment compared to the fast petiole abscission following deblading (Fig. 9A) may be attributed to the relative effectiveness of these two treatments in IAA elimination, as in the presence of NPA the IAA source is still present in the tissue. The NPA results ruled out any local and systemic wounding effects (León et al., 2001
We applied Suc to the cut end of the decapitated stem to test whether Suc flow to the Mirabilis roots and tuber via the node AZ might also have a role in inhibiting abscission. Surprisingly, we found that rather than inhibiting abscission, the Suc treatment increased the rate at which abscission occurred. Why this is so is unclear, but it may be related to the ability of Suc to affect hormone signaling (León and Sheen, 2003
The time interval obtained between the deblading and actual petiole abscission in response to ethylene (Fig. 1) indicates that there was a clear separation in time between the tissue's ability to respond to ethylene and the occurrence of the hydrolytic processes necessary for separation of cells in the AZ. This suggested that we could isolate RNA from tissue that was undergoing a change in sensitivity to ethylene, but likely not yet undergoing changes associated with cell separation at the AZ. We hypothesized that the increased ethylene sensitivity of the AZ resulting from deblading would be due to changes in gene expression resulting from the altered auxin flux across the AZ. Decreased gene expression would likely occur for genes whose expression was normally maintained by the auxin flux across the AZ, whereas increased gene expression would likely occur for genes that were normally repressed by this auxin flux.
We made two subtractive libraries to identify these gene transcripts. In the first we selected for mRNA transcripts in the AZ tissue that were down-regulated within 3 d of deblading, during which the AZ had already gained competence to abscise in response to ethylene. We speculated that these would be the genes whose expression would normally be maintained by the auxin flux. Interestingly, we found that five of the mRNA transcripts we identified in this screen showed homology to members of the Aux/IAA gene family (Table I). This gene family contains members that are rapidly induced within 5 to 60 min of IAA application (Abel and Theologis, 1996
RNA gel-blot analysis confirmed that the expression of two of the transcripts that showed homology to members of the Aux/IAA gene family (Mj-Aux/IAA1 and 2) was repressed by deblading or decapitation. This repression was inhibited by applying IAA to the cut end of the petiole (Fig. 4) or stump (Fig. 5), which also prevented abscission (Figs. 2 and 3). A similar correlation between the effectiveness of auxin in delaying abscission and induction of Aux/IAA gene expression was reported recently in Cestrum elegans (Abebie et al., 2005b
The regulation by IAA is important in our study, as the cells in the debladed petiole tissue are potentially influenced by numerous signals (e.g. wound and dehydration) resulting from the excision that may not be related to the AZ formation. The restoration of their expression by IAA application provides evidence that it is the removal of the IAA source rather than the stress of excision that causes their altered expression. The expression of Mj-Aux/IAA1 was also examined in other plant tissues, and it was found not to be specific to the AZ. Rather, it was shown to accumulate in the non-AZ portion of the petiole, the stem, and in high amounts in the petal-like calyx (Supplemental Fig. 1), in agreement with the findings of Gookin et al. (2003)
Other mRNA transcripts that were down-regulated by deblading showed homology to a PGIP,
Our study showed that the
Our second subtractive library enabled us to isolate gene transcripts that were induced in the AZ by either deblading or NPA treatment during the time the zone became competent to respond to ethylene, but before abscission of the plant organs. We pretreated the plants with 1-MCP to select for genes that increased in expression independently of ethylene. We identified gene transcripts in our differential screen that showed homology to a variety of mRNAs that encode proteins shown to have roles in defense (terpene-synthase-like protein and thaumatin-like protein), stress (ER6 protein and osmotin-like protein), abscission (peroxidase), cellular signaling (FKBP12-like protein, cap-binding protein, and phosphorylase-domain protein), microtubule function (kinesin-like protein), and others whose functions are still unknown (Table III). We used RNA gel-blot or real-time PCR analyses to confirm the deblading or NPA induction, respectively, of a number of the transcripts (Figs. 6 and 9A; Supplemental Fig. 4), including those encoding a putative thaumatin-like protein, peroxidase, and three unknown transcripts, FS-18, FS-44, and FS-55. The roles of these identified proteins in abscission are currently not clear. Among the unknown transcripts, FS-18 was strongly up-regulated in the petiole AZ, apparently independently of ethylene, although the finding that this transcript was induced in nondebladed tissue by 1-MCP (Fig. 6) suggests that it might be actively repressed by endogenous ethylene. The induction of FS-18 was also found to be repressed by IAA in the leaf AZ (Fig. 8), strongly indicating its regulation by IAA.
The addition of Suc to the cut end of the decapitated internode further increased the expression levels of some of the decapitation up-regulated transcripts (Fig. 7). This suggests that the regulation of these decapitation-induced transcripts via carbohydrate signaling is probably additional to their up-regulation by auxin depletion. Suc application also accelerated the abscission of the cut internode (data not shown), confirming previous findings showing that Suc induced abscission of internodal explants of Impatiens sultani (Warren-Wilson et al., 1986
In our study we were able to isolate gene transcripts that increase or decrease in the petiole AZ during the time it developed competency to respond to ethylene by petiole abscission. We tested some of these transcripts for their expression in leaf and stem AZs, and for their regulation by abscission-modulating factors such as 1-MCP, IAA, NPA, and Suc. The similarity in the expression patterns of transcripts, isolated only from the leaf AZ, in both leaf and stem AZs following deblading/decapitation and abscission-modifying treatments, points to a general abscission mechanism. This suggests that auxin induces or represses in both AZ tissues the same type of regulatory gene transcripts, which retain the AZ insensitive to ethylene. It is possible that the change in abundance of part of these transcripts leads to changes that affect the ability of the tissue to respond to ethylene. It should be noted that the expression of the majority of the transcripts we studied was found to be unaffected by the 1-MCP pretreatment, suggesting that if they are involved in abscission they are not regulated by ethylene but by IAA. It is possible that this IAA regulation of AZ sensitivity to ethylene may operate by affecting components downstream to ETR1 or ERS1 receptors in the ethylene signal transduction pathway (Zhou et al., 1996 Many of the transcripts we isolated encoded proteins of unknown functions and merit further examination. It will be particularly interesting to examine the effect on abscission of the modulated expression of Aux/IAA and other genes that we identified in our study in M. jalapa. However, despite the obvious anatomical advantages that the Mirabilis system provides in studying genes in the AZ, we are aware that other common model systems, such as tomato or Brassica, will likely be more useful for further research and functional analysis. Accordingly, experiments with genes isolated from tomato AZs and their analysis by tomato microarrays are currently under way.
Plant Systems and Treatments
Experiments were performed with Mirabilis jalapa plants. The plants were grown in pots filled with a mix of one-third fir (Abies spp.) bark, one-third sand, and one-third peat moss under standard greenhouse conditions. Seedlings were trimmed at the fifth node to initiate side shoots, and flower buds were constantly removed. Both leaf and stem AZ systems of M. jalapa were studied. IAA flow from the leaf blade or stem internode to the corresponding AZs was eliminated by either leaf deblading or stem decapitation, respectively. Leaf deblading was performed by cutting the petiole at 1.5 cm from the AZ with a sharp razor blade. Lanolin paste was applied on the cut surface to prevent water stress and as a control for the auxin treatments (see below). Stem decapitation was performed by cutting the stem 5 cm above the stem node AZ. The cut surface was treated similarly to that of the petioles. To avoid wounding, IAA flow from the leaf blade was also eliminated by application of the auxin transport inhibitor NPA. An aliquot of 0.025 g of NPA (Chem Service) was dissolved in 0.5 mL DMSO and mixed with 5 g of warmed (50°C) lanolin paste to a final concentration of 0.5% (w/w). The lanolin paste containing the NPA was applied by spreading it as a ring around the leaf petiole, in the section close to the leaf blade. The same amount of lanolin paste with DMSO was similarly applied as control. To renew the auxin supply following deblading or decapitation, IAA (10–3 M) or NAA (2x10–2 M) was immediately applied in lanolin paste to the cut surface of the remaining tissues. Control plants had lanolin alone applied to the cut surface. Both native (IAA) and synthetic (NAA) auxins were used to guarantee a clear auxin effect. The ethylene action inhibitor 1-MCP (0.2 µL L–1 for 24 h at 20°C) was applied 1 d before leaf deblading or stem decapitation, to block any direct effects of ethylene on gene expression (Sisler and Serek, 1997
Total RNA was isolated from the stored tissue as outlined by Hunter et al. (2002)
Transcription levels were quantified by means of the real-time PCR method, using Sybr-green amplification kits (Absolute QPCR SYBR Green mix) and the Rotor-Gene 3000 sequence detection system (Corbett Research) according to the manufacturer's instructions. cDNA was prepared by using Moloney murine leukemia virus reverse transcriptase and random primers (Promega). Ribosomal 18S RNA served to normalize expression. The primers used (detailed in Table IV ) were designed with the Primer Express software (Applied Biosystems).The amplification efficiency for each primer pair was determined by serial dilutions of the cDNA (1:5, 1:25, 1:125, 1:625, and 1:3,125 for the target genes and 1:104, 1:105, 1:106, 1:107, and 1:108 for the ribosomal 18S). The reaction mixture contained 2.5 µL of 1:10 diluted cDNA (or 1:104 diluted cDNA for Ribosomal 18S amplification), 5 µL of Absolute QPCR SYBR Green mix, primers at a final concentration of 2.5 µM, and water to a final volume of 10 µL. PCR conditions were 95°C for 15 min (activation of Thermo-start DNA polimerase), 40 cycles at 95°C for 1 s, at 62°C for 15 s, and at 72°C for 20 s. After the amplification steps, the melting curve was determined for each primer pair to verify the presence of only one specific product. The reactions were performed in triplicate using the same cDNA and the results were averaged. Two different experiments were carried out. The comparative quantitation values were calculated with the analysis software supplied with the detection system. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AY137961-4, AY137965-6, and AY589693-714.
The authors are thankful to Dr. Sonia Philosoph-Hadas from the Volcani Center for her valuable comments and critical reading of the manuscript. Received February 14, 2006; returned for revision June 2, 2006; accepted June 5, 2006.
1 This work was supported by the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel (440/05). 
2 These authors contributed equally to the paper. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Shimon Meir (shimonm{at}volcani.agri.gov.il).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.079277. * Corresponding author; e-mail shimonm{at}volcani.agri.gov.il; fax 972–3–9683878.
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ABSTRACT
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, 3 d debladed then ethylene; 
, 2 d debladed then ethylene; 
, 1 d debladed then ethylene; 
, debladed then ethylene immediately; 
, 3 d debladed without ethylene; 
, nondebladed + ethylene. Accumulated percentage of petiole abscission is presented, and data represent means ± SE of three replicates of eight petioles per treatment.
-tubulins, make up the microtubule arrays of the cytoskeleton that determine cellular shape in response to a variety of internal and external cues (Breviario and Nick, 2000
