Plant Physiol. (1996) 11 1 : 81 3-820
Pedicel Breakstrength and Cellulase Gene Expression during Tomato Flower Abscission’ Elena de1 Campillo* and Alan B. Bennett Department of Plant Biology, University of Maryland, College Park, Maryland 20742 (E.d.C.); and Department of Vegetable Crops, University of California, Davis, California 9561 6 (A.B.B.)
Abscission is a process that allows plants to shed an organ from the parent body in a controlled fashion. Separation results from catabolic alterations in the middle lamellae and the primary cell wall in a few layers of specialized cells usually located at the base of the organ to be shed (Sexton and Roberts, 1982). The breakdown of cell-wall components in these specialized cells is so complete that after separation the cells of the scar surface look rounded, as if they were protoplasts (Sexton et al., 1989), and yet the destruction is gradual and spatially confined. These specialized cells have a unique ability to sense and respond to abscission signal(s)by initiating a gene expression program that causes separation (Sexton and Roberts, 1982). The enzymatic machinery engaged by the abscission program operates through severa1 classes of enzymes including endo-P-(1,4)-glucanases, commonly referred to as cellulases (Lewis and Varner, 1970; de1 Campillo et al., 1990),
and polygalacturonases (Taylor et al., 1990; Kalaitzis et al., 1995). In coordination with the abscission program, other programs with functions related to senescence and protection against pathogen invasion are also induced, which result in the accumulation of pathogenesis-related proteins in the Abzo and adjacent tissues (de1 Campillo and Lewis, 1992; Eyal et al., 1993). Ethylene and auxin have been implicated in the regulation of abscission of dicotyledonous plants (Sexton and Roberts, 1982). Ethylene is a potent accelerator of abscission, and IAA is a potent inhibitor of abscission (Abeles, 1968). Sexton et al. (1985) have also suggested that the complexity of the abscission process may require the interplay of other factors as well. In bean, the increase in mRNA and protein for the pI 9.5 cellulase isoenzyme was found to closely correlate with the initiation and progress of petiole abscission in debladed leaves exposed to ethylene (Lewis and Varner, 1970; Durbin et al., 1981; Tucker et al., 1988). Detailed studies of the pI 9.5 cellulase spatial distribution demonstrated that this enzyme begins to accumulate in the parenchymatous cells of the vascular bundles above the separation site and progresses outward through the cortical cells of the fracture plane (Sexton et al., 1980; de1 Campillo et al., 1990). Subsequently, Thompson and Osborne (1994) demonstrated that the vascular system is responsible for the production of a signal molecule essential for both cell separation and expression of the pI 9.5 cellulase in the cortical cells of the abscission site. A physical measure of the progress of abscission is the force necessary to separate the abscinding organ, the BKS, at the abscission site. For many systems, a force gauge has been adapted to quantitate abscission (Craker and Abeles, 1969). In all cases, BKS has been inversely correlated with the progress of abscission. Here we analyzed natural abscission of tomato (Lycopersicon esculentum Mill. cv Castlemart) flowers and used BKS measurements to establish possible steps through the process. Tomato flowers abscise naturally within a few days after flower opening if pollination fails and no fruit is set. If flowers are pollinated, they can also abscise if they are excised from the plant and exposed to ethylene (Jensen and Valdovinos, 1967).
This work was supported by U.S. Department of Agriculture grant no. 94-02996 to E.d.C. * Corresponding author; e-mail
[email protected]; fax 1-301-314-9082.
Abbreviations: AB, abscising; Abzo, abscission zone; BKS, breakstrength; FAZ, flower abscission zone; INTM, intermediate; NA, nonabscising; RPA, RNase protection assay; RT-PCR, reversetranscription PCR; ST, strong flower.
Six cellulase genes were isolated from total RNA of the ethylenetreated tomato (Lycopersicon esculentum Mill.) flower abscission zone by reverse-transcription polymerase chain reaction using degenerate primers to conserved amino acid sequences from known plant cellulases. Four of the gene fragments are homologous to fruit pericarp cellulases. l h e other two are nove1 cellulase genes, referred to as Ce15 and Ce16. Breakstrength and cellulase gene expression were then analyzed in naturally abscising flowers and flower explants. I n both naturally abscising flowers and flower explants induced to abscise in air or ethylene, both new cellulase mRNAs were correlated with flower shedding. Whereas the Ce15 mRNA increased in later stages of abscission, the Ce16 mRNA was present i n nonabscising flowers and then decreased i n the final stage of abscission. A third cellulase, Cell, increased during the final stage of abscission in flower explants and yet did not increase during shedding i n planta, although it was detectable at low levels in all abscission stages. Cell and Ce15 mRNA decreased 99% when indole-3-acetic acid was added during ethylene treatment, consistent with low levels of abscission (3%). I n contrast, Ce16 mRNA increased slightly when indole-3-acetic acid was added. These results suggest that abscission is a multistep process involving both activated and repressed cellulase genes and that the relative importance of each cellulase in the process depends on the physiological conditions under which abscission takes place.
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The FAZ in tomato is found in the middle of the pedicel and is demarcated by a circumferential groove. The abscission cells are located in the plane of the groove and it comprises different types of cells. A11 cells across the pedicel except those of the vascular vessels, which must be broken mechanically, take part in separation (Kendall, 1918). The breakdown of the cell walls during separation is enzymatic, and two types of enzyme activities, those of endo-P-1,4-glucanase and polygalacturonase, have been shown to increase during abscission of tomato flower explants exposed to ethylene (Tucker et al., 1984). Furthermore, from tomato flower explants treated with ethylene, two tomato cellulase genes, Cell and Ce12 (Lashbrook et al., 1994), and one polygalacturonase gene, TAPG (Kalaitzis et al., 1995), have been isolated and shown to be regulated in coordination with flower shedding. However, these studies also indicated a significant basal level of expression of these genes in nonabscising tissue. This is contrary to what has been observed in other abscission systems, such as bean, in which the pI 9.5 cellulase is de novo synthesized (Lewis and Varner, 1970; Durbin et al., 1981; Tucker et al., 1988). A possible explanation for the expression of these RNA in nonabscising flowers is that the method used to measure abscission did not correctly distinguish nonabscising flowers from those that are committed to abscise and are already in the initial stages of weakening. In this work we developed a quantitative physical measurement to analyze abscission of tomato flowers. We used BKS distributions to define empirically stages of naturally abscising flowers. The method was then used to characterize abscission of flowers in planta and to address the function of cellulase genes during abscission. MATERIALS A N D METHODS Flower Preparation
Twenty to 30 tomato (Lycopersicon esculentum Mil1 cv Castlemart) inflorescences bearing two to three flowers were collected from plants growing in the field at Davis, CA. Within a single tomato inflorescence there are flowers of different developmental stages with the youngest located at the distal end. Harvested flower clusters were washed extensively with running water to remove dust and insects from the surface of the tissue. To avoid secondary effects derived from possible communication between flowers, each pedicel was separated from the cluster and inserted from the proximal end into a moistened foam pad or placed in water. Flower explants were incubated in ethylene as previously described (de1 Campillo et al., 1988). To avoid significant bacterial contamination, incubation of flowers in water for more than 48 h was avoided. BKS BKS of tomato flowers was measured within 2 to 3 h after collection from the field using a force gauge modified by addition of a chuck at the end of the spring scale. The proximal side of each flower pedicel was inserted into a piece of Tygon tubing, set into the chuck, and slightly tightened. The force was applied from the distal side, and
Plant Physiol. Vol. 11 1, 1996
BKS is the weight in grams indicated on the spring scale at the time of separation. Only open flowers were selected from each flower cluster for BKS determination, which was measured throughout the population for a11 experiments. For in planta experiments, RNA was isolated from Abzos sorted according to BKS categories, but for explant experiments a11 Abzos were combined prior to RNA isolation to have enough material to analyze mRNA levels. Therefore, mRNA levels in explant experiments do not correspond to each range of BKS category but indicate the average level of mRNA in the population. Enzyme Extraction and Cellulase Activity
Abscission cells were removed from the flower pedicel by cutting approximately 1mm on each side of the Abzo. Tissue was frozen in liquid nitrogen, weighed frozen, reduced to powder with a mortar and pestle, and homogenized in ice-cold extraction buffer (20 mM Tris, 0.5 M NaC1, pH 8.1), at a ratio of 4 mL per g of tissue. After 20 min of incubation on ice, the homogenate was centrifuged for 30 min at 12,000 rpm in a microfuge. The supernatant was decanted and used for enzyme assays and protein determination. Cellulase activity was determined viscometrically using carboxymethyl cellulose as substrate. Assays were performed by mixing 50 p L of enzyme extract with 200 pL of 1.3%carboxymethyl cellulose in 20 mM Tris buffer (pH 8.1), 0.1 M NaC1. Incubation was at room temperature (22-23°C). Activity is expressed as relative units as described by Durbin and Lewis (1988). Protein determinations were made by the method of Schaffner and Weissmann (1973) using BSA as the standar d. RT-PCR and Cloning of PCR Products
Cellulase genes expressed in abscission of tomato flowers were isolated from total RNA of FAZ treated with ethylene by RT-PCR using degenerate primers to conserved amino acid sequences from known plant cellulases. For PCR, the upstream primer, referred to as cell f, was GGN-TAY-TAY-GAY-GCN-GGN-GA, which is a degenerate sequence with 512 possible combinations. The downstream primer, referred to as cell r, was GC-NSC-CCANAR-NAR-YTC-RTC, which is also a degenerate sequence with 2048 possible combinations. Two complete PCR runs of 30 cycles each were carried out: in the first PCR run, the template was denatured at 94°C (1min), annealed at 50°C (1.5 min), and extended at 74°C (2 min). Under these conditions, the PCR reaction generated at least four bands. The expected 500-bp band was excised from the agarose gel, purified, and used as a template for the second run of PCR, for which the template was denatured at 94°C (1 min), annealed at 63°C (1.5 min), and extended at 74°C (2 min). A single band of 500 bp was obtained. The resulting PCR products were then concentrated, cloned into the vector pCR I1 (Invitrogen, San Diego, CA), and used to transform Escherichia cozi XL1-Blue (Stratagene) with a 5 p L / L ligation reaction. Plasmids derived from clones containing 500-bp
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inserts were analyzed by sequencing and cross-hybridization with each other and with fruit-derived tomato cellulase genes. RPA
Accumulation of each cellulase mRNA was analyzed with antisense RNA transcripts of high specific activity. Transcripts were synthesized in the presence of [32P]ATP using the bacteriophage promoters present in the PCR I1 vector. The labeled RNA was gel purified to eliminate the presence of shorter transcripts using a 0.75-mmthick, 8 M urea, 5% acrylamide gel. After electrophoresis the gel was covered with polyvinyl film and visualized by autoradiography. The developed film was aligned with the gel and used to localize and excise the area of the gel that contained the labeled transcript of appropriate size. After elution from the gel, the labeled probe was mixed with 5 Fg of the sample RNA, keeping approximately 4-fold molar excess of the labeled probe over the target RNA and co-precipitated with ethanol. The molar concentration of the target RNA was calculated, assuming that the target mRNA was 2.0 kb long and that mRNA makes u p 3% of the total cellular RNA. After precipitation, the mixture was resuspended in hybridization buffer (SOo/, deionized formamide, 100 mM sodium citrate, pH 6.4, 300 mM sodium acetate, pH 6.4, l mM EDTA) and incubated at 42°C for 12 to 16 h to promote hybridization of complementary transcripts. After hybridization, the mixture was treated with RNase to degrade single-stranded unhybridized probe. The protected, labeled fragments were separated using a 0.75-mm-thick, 8 M urea, 4% acrylamide gel. Gels were dried and scanned by a phosphorimager analyzer BAS1000 (Fuji, Tokyo, Japan).
whole and indicates that the Abzo is at least as strong as any other part of the pedicel. In the abscinding group, the flowers broke at the abscission site, indicating that the Abzo had started to weaken. Within the abscinding group three different categories could be distinguished: (a) ST, in these the abscission site is the weakest part of the pedicel; however, a strong force (>200 g) is required to obtain separation; (b) INTM, separation at the Abzo occurred by applying a small force (50-200 g); and (c) AB, flowers separated readily but the force was too small to be measured. Since late-season field-grown plants do not set a high proportion of fruit (Rick and Dempsey, 1969), a significant proportion of abscinding flowers was expected. In fact, only 35% of the flowers in the population were strongly attached and broke outside the Abzo (NA). The rest of the flowers separated at the abscission site. Of those, 20% separated at the abscission site, but they were strongly attached (ST). About 26% separated after applying a small force (INTM), and the rest, about 20%, were AB, i.e. they were weakly attached. Despite efforts to select flowers at similar developmental stages, it is apparent from the analysis of BKS of tomato flowers growing in the field that this is not a synchronized event. Each collection was a mixture of flowers, some of which showed no sign of BKS decline and others were weak or were ready to abscise. It was impossible to distinguish visually which flowers were strongly attached and which flowers were weak during the collection. This inherent variability in the system was addressed by quantitatively monitoring abscission in the starting material and in a11 experimental treatments and then assigning individual flowers to BKS categories. Functional Significance of BKS Categories
RESULTS
BKS Distribution in Planta
To establish the typical distribution for BKS of tomato flowers growing in the field under late summer conditions at Davis, CA, six independent collections of approximately 50 flowers were made in September and October, 1993. Figure 1 shows the average BKS distribution over a11 collections of flowers growing in the field. Initially, flowers were arranged into two main groups: nonabscinding and abscinding. In the nonabscinding group, the pedicel broke outside the Abzo with a mean force of about 250 g. This force established the overall strength of the pedicel as a
To establish the functional significance of the BKS categories determined empirically, we analyzed BKS during the progress of abscission to see whether there were transitions between stages. We compared the progress of abscission in flower explants exposed for 48 h to air or ethylene after discarding flowers that at time O had a BKS of less than 50 g. Therefore, the BKS distribution at time O of the flowers used for the experiment consisted of three categories: 42% NA, 43% INTM (BKS 50-200 g), and 15% ST. The explants were then incubated in air or 13 p L / L ethylene for 48 h, and at the end of the treatment the distribution of BKS was determined (Fig. 2). After 48 h of treatment in air, the distribution of BKS was quite different
20
o
Strong
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Non-Abscising
3
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ò?
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50 100150200 200 250 300 350 400 50 100 150 200 250 300 350 400 450 500
Breakstrength (9)
Figure 1. Distribution of BKS in flowers growing in the field during late summer at Davis, CA. Average distribution over six independent collections of approximately 50 flowers.
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INTM (50-200 g), ST (200400 g), and NA BKS were larger compared to the respective proportions found for ethylene treatment alone. Compared to time 0, the IAA treatment showed no major changes in the proportion of INTM Abzos but showed a larger proportion of ST Abzos and a reduction in the proportion of NA Abzos. This suggested that IAA also inhibits the transfer of ST categories to a weaker stage and may slow down but not completely inhibit the transition of NA to weaker categories.
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3 o 40 u. 30 $? 20
Comparison of Cellulase Activity in Abscising Flowers in Planta and Flower Explants
10 O O
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Breakstrengt h (g) Figure 2. Distribution of BKS in tomato flower pedicels for time O (TO) before any treatment, excluding flowers with BKS below 50 g, in flower explants after incubation for 48 h in air (48 h AIR), and in flower explants incubated in 1'3 pL/L ethylene for 48 h (48 h C,H,).
from time 0. The NA population (4%) decreased substantially, and there was a corresponding increase in the INTM category (63%). Moreover, there was a de novo formation of the Abzos that were weak and AB (20%). ST Abzos composed 13% of the explants. This suggests that there is a sequential passage of the Abzo through BKS categories. This also indicates that in air most of the NA population is becoming weaker at the abscission site but is not yet ready to separate. When explants were incubated in ethylene for 48 h, 75% abscised with no measurable force and none broke outside the Abzo. The remaining 25% of explants broke at the Abzo with intermediate BKS (50-200 g) and less than 1%were ST Abzos (200400 g). This complete loss of the NA population indicates that the transition or passage between stages is accelerated in the presence of ethylene in the entire population of explants, independent of the starting BKS. Also, these results suggest that the BKS categories established empirically are related to the development of abscission in tomato flowers. Furthermore, they indicate that prior to shedding FAZs become weaker at the abscission site but are not yet ready to separate. Effect of Ethylene plus IAA on BKS
There is strong evidence that implicates ethylene and auxin (IAA) in the regulation of abscission in dicotyledonous plants (Sexton and Roberts, 1982). Roberts et al. (1984) showed that remova1 of the flower accelerated abscission and postulated that the inhibitor factor coming from the flower was IAA. To analyze the effect of IAA on tomato flower abscission, the flower was removed and replaced by an agar plug containing 50 p~ IAA. The explants were then incubated in ethylene for 48 h. Figure 3 shows that when explants were incubated in ethylene for 48 h, in the presente of IAA, the main effect was to almost completely inhibit the ethylene-induced abscission of tomato pedicels (only 3% abscised readily). The proportions of Abzos with
Cellulase activity has been found to correlate with the initiation and progress of abscission in severa1 species. In particular, the increase in the pI 9.5 cellulase has been found to closely correlate with the decline in BKS in abscission of bean leaf. Thus, cellulase activity was monitored in Abzos of naturally abscising tomato flowers, sorted according to BKS category. Figure 4 shows that Abzos from NA and ST categories contain some cellulase activity, and the activity increases 4-fold in the naturally AB category. When activity was analyzed in flower explants treated in air for 40 h, the cellulase activity in NA and ST categories was the same or slightly larger. However, the activity found in the AB category was almost 3 times higher than the corresponding AB category in naturally abscising flowers. Furthermore, if the flowers were treated with ethylene for 40 h, cellulase activity in the abscising flower explants was even higher. This suggests an overexpression of the abscission cellulase in the explant system and / or that additional cellulase(s) are being induced in the FAZ in addition to the abscission cellulase(s). ldentification of Abscission-Specific Cellulases
The results presented above suggest that the BKS categories represent abscission stages. We next identified cell-
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Breakstrength (9) Figure 3. Distribution of BKS in tomato flower pedicels at time O (TO) before any treatment, excluding flowers with BKS below 50 g, in flower explants after incubation for 48 h in 13 pL/L ethylene (48 h C,H,), and in flower explants incubated with 13 pL/L ethylene in the presence of 50 p~ IAA (48 h IAA + C,H,).
Abscission of Tomato Flowers
INTM
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corresponding region of Ce13 and only about 43% identity with Cell, Ce12, and Ce14. Comparison of the deduced amino acid sequence of the new tomato cellulases with bean and avocado fruit cellulases over their corresponding regions showed that Ce15 shares a higher homology (80% identity) with avocado fruit cellulase than with bean pI 9.5 cellulase (63% identity), whereas Ce16 is distantly related to both the avocado and bean genes, sharing a 43 and 39% identity, respectively.
Analysis of Cellulase mRNA Levels in Planta in Relation to BKS Distribution
Treatment Figure 4. Cellulase activity in Abzos of tomato flowers classified according to BKS categories. Comparison between flowers that abscised in planta (P) and flower explants that were induced to abscise by 40 h of incubation in air (A) or ethylene (E).
wall hydrolase genes whose expression was correlated with the progression of Abzos through the different BKS categories. Cellulase gene fragments in FAZ treated with ethylene were isolated by RT-PCR using degenerate primers to conserved amino acid sequences from known plant cellulases. These primers have been used previously to identify four related endo-/3-(1,4)-glucanase cDNA clones from a ripe tomato fruit cDNA library (Lashbrook and Bennett, 1992).These previously identified tomato cellulase cDNAs are Cell, Ce12 (Lashbrook et al., 1994), Ce13 (Brummel1 et al., 1994), and Ce14, which was found to be identical with tomato TPP18, an endoglucanase expressed predominantly in pistils of young flowers (Milligan and Gasser, 1995). Cell and Ce12 are expressed in FAZ explants exposed to ethylene. Hybridization analysis of the Abzo RTPCR revealed that 25 to 30% of the PCR products crosshybridized with Cell, 8% of the clones cross-hybridized with Ce12,6% of the clones cross-hybridized with Ce13, and 2% cross-hybridized with Ce14. PCR products of the correct size that failed to cross-hybridize with any known fruitderived cellulases were considered to represent putative new abscission cellulases. Two new tomato cellulase cDNA fragments, Ce15 and Ce16, were identified and subjected to further analysis.
The accumulation of Ce15, Ce16, and Cell genes in flower abscission was analyzed using the RPA. This method is an extremely sensitive procedure for the detection and quantitation of low-abundance mRNAs (Friedberg et al., 1990). Figure 6A shows the levels of mRNA accumulation of Ce15 and Ce16 in the Abzo of flowers from plants growing in the field (time O) in relation to the distribution of BKS. Results showed that accumulation of Ce15 mRNA at time O was extremely low for NA, ST, and INTM but increased 22-fold in the AB population. In contrast, expression of Ce16 at time O was high for NA and ST, and as high or even higher for INTM, but was more than 90% lower in the AB population. When Cell mRNA levels were analyzed, there were detectable levels of expression in the NA, ST, and INTM BKS categories, but mRNA levels were not elevated in the AB population.
cell f
d in
7n
***** * * * * * * * * *** ***** ***
Analysis of Putative New Abscission Cellulase Cenes
Figure 5 shows the deduced amino acid sequence of a11 cellulase cDNA fragments found in the Abzo, including the new cellulases referred to as Ce15 and Ce16. Alignment of their amino acid sequence showed that within a11 cellulases there are 36 invariant amino acids in addition to the conserved regions that were used to generate the primers. The alignment also shows that the deduced amino acid sequence of the Ce15 fragment shares 63 to 73% identity with the corresponding regions of Cell, Ce12, and Ce14 and only 45% identity with Ce13. In contrast, the deduced amino acid sequence of the Ce16 fragment shares approximately 80% identity with the
. . . . . . .......... ;e14
ADKYRGSYSW G L X W C P Y Y CSVSGYFDEL LIC G . . . . . . .
Cei2
ANWRGAYSS SLHSAVCPFY CDFNGYODEL L W A . . . . . . S*****
...___.... _ _ .. . .
Celj M ) a R G S Y S D S L S S W C P F Y CSYSGYKIIEL L ? ? A . . . . . . . . . . .
Cell
....___._.
cell r
Amino acid sequences of ali cellulase gene fragments found in tomato FAZs. Amino acids were deduced from t h e nucleotide sequences. A m i n o acid sequences have been aligned for maximal homology, with asterisks (*) inserted to denote gaps introduced into either sequence. Figure 5.
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Figure 6. mRNA accumulation of Cel5, Cel6, and Cell homolog in the Abzo (Absc Zones) of tomato flowers (cv Castlemart) growing in the field: A, in relation to the distribution of BKS; B, in flower explants incubated in ethylene, in air, and in ethylene in the presence of IAA and in stem explants before and after incubation with ethylene; and C, in relation to two developmental stages, young, unopen flowers (Y) and older, senescent flowers (O). Each mRNA was measured by the RPA.
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NATURALLY ABSCISSING Absc Zones Time Zero NA
ST
INTM
AB
EXPLANTS Absc Zones C2H4
Air
Stem
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B Analysis of Cellulase mRNA Levels in Flower Explants: Effect of Ethylene and IAA
Figure 6B shows the mRNA accumulation levels in Abzos and stem explants treated with ethylene and in Abzos treated with ethylene in the presence of IAA. Results showed that after treatment of Abzo explants with ethylene for 48 h (75% of the Abzos abscised) the level of expression of Cel5 mRNA in Abzos increased 120-fold over the expression of nonabscising flowers at time 0. In contrast, when stem explants were treated with ethylene for 24 h, no expression of Cel5 was observed either before or after the treatment. Treatment of the Abzo explants for 48 h in air (20% of Abzos abscised) resulted in a 15-fold increase in Cel5 expression, a smaller increase than in explants treated with ethylene (Fig. 6A). The presence of IAA in the ethylene treatment reduced the levels of expression of Cel5 by 99%, consistent with the low levels of abscission (3%). Note that the level of Cel5 mRNA in Abzo explants treated with ethylene was 5 times higher than the level found in flowers that shed naturally. This suggests that wounding and external ethylene causes the accumulation of this RNA above the amount necessary to cause shedding. Cel6 mRNA levels were low in both air- and ethylenetreated Abzos. The lowest level was in explants incubated in air for 48 h, but there was some increase in this message when IAA was added in addition to ethylene. It is interesting that expression of Cel6 was high in stems and remained high in stem explants treated with ethylene for 24 h. Thus, the down-regulation of Cel6 was specific to the Abzo. Cell mRNA in Abzos of flower explants treated with ethylene for 48 h increased 25-fold over the expression at time 0 of the nonabscising flowers (Fig. 6B). The presence of IAA also reduced Cell expression by 99%, as would be expected for an abscission cellulase. However, expression
of Cell was not correlated with BKS in naturally abscising flowers (Fig. 6A). Expression of Cell in Abzo explants incubated in air or ethylene was higher than expression in Abzos of naturally abscising flowers. Thus, Cell may be more important for abscission in explants than in intact flowers. Cell mRNA levels were very low in stem tissue and increased slightly after treatment of stems with ethylene for 24 h. Expression in the Abzo of these cellulase genes was also analyzed with respect to the developmental stage of the flower (Fig. 6C). In general, mRNA levels of Cel5 and Cell in Abzos of young buds and older flower tissue was very low. In contrast, expression of Cel6 was significantly higher in old flowers compared to expression in young buds. Old flowers bearing small fruit are characterized by a thicker pedicel, particularly at the distal side of the Abzo, which is actively expanding laterally during the first stages of fruit set. Spatial Distribution of Cellulase Gene Expression in Tomato Flower
Table I shows the level of expression of Cel5 and Cel6 in FAZs and the regions above (distal side) and below (proximal side) the Abzo. The expression of Cel5 outside the Table I. Tissue distribution of cellulase gene expression in abscising flower explants after 24 h of treatment with ethylene Each mRNA was measured by RPA, and quantitation of the primary protected fragment was accomplished with a phosphorimager analyzer. Location
Cel5
Cel6
Distal Abzo Proximal
0.27 20.90 6.81
9.1 12.6 17.8
Abscission of Tomato Flowers Abzo was very low in AB pedicels (BKS = O), whether they were treated for 24 h in air or ethylene. The expression was confined to the Abzo. The expression of Ce16 was low in AB pedicels and had no specific distribution with respect to the Abzo, although it was somewhat lower on the distal side. In contrast, Cell mRNA was equally abundant or slightly higher on the distal side of the pedicel than in the Abzo (Lashbrook et al., 1994). DlSCUSSlON
In this study a quantitative method was used to monitor abscission in naturally abscising flowers. The method was developed for the analysis of tomato flowers, which are induced to abscise in planta if pollination has failed. Thus, any collection of flowers at time O would be expected to have some flowers already committed to abscise and others that are strongly attached. The progress of abscission was determined by comparing the force needed to break the pedicel at the abscission site (BKS) with the overall strength of the pedicel as a whole. Using this method, we established that abscission can be divided into stages and that prior to shedding Abzos become progressively weaker at the abscission site but are not yet ready to separate. In addition, we demonstrated that the transition of flowers toward weaker BKS categories is accelerated by ethylene. Therefore, we postulate that abscission and the decline in BKS that correlates with abscission occurs in stages characterized by a progressive decline in cell-wall integrity. The progress of abscission could be explained by a simple model by which transitions between different stages occur through the cumulative activity of one abscission hydrolase. Indeed, in the primary leaves of bean, this appears to be the case because the activity of pI 9.5 cellulase is closely correlated with the decrease in BKS at the abscission site (Sexton et al., 1980; Durbin et al., 1981). The mRNA levels of three cellulase genes were analyzed in relation to transition of abscission stages in naturally abscising flowers and in explants treated with ethylene and IAA. The results showed that one cellulase gene, Ce15, is up-regulated in the latest stages of abscission (shedding); another cellulase, Ce16, is present in nonabscising flowers, remains high or increases slightly during weakening, and is down-regulated during shedding; and cellulase Cell mRNA is detectable at low levels in a11 abscission stages and does not increase consistently in the population that sheds spontaneously (Fig. 6A). Thus, in tomato flowers in which multiple cellulase genes are expressed, an alternative model is necessary to explain the progress of abscission. The new model could explain changes in BKS by the phased expression of several cellulase genes by which some are activated and some are repressed during the process. In addition, a polygalacturonase gene was recently isolated from tomato leaf Abzos, which may also coordinate with cellulases in flower abscission (Kalaitzis et al., 1995). This model of abscission is consistent with prevailing views regarding the complexity
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and dynamics of the primary cell wall (Carpita and Gibeaut, 1993). The disassembly of the complex cell-wall network is hypothesized to require different glucanohydrolases with distinct temporal and spatial regulation. The need for multiple hydrolases is particularly relevant in tomato, since the fracture plane comprises several types of cells, such as epidermis, parenchyma, and pith cells, which are likely to have some differences in their primary cellwall structure. If abscission occurs through successive and interdependent changes in the cell wall of the abscission cells, then in parallel with the cell-wall breaking process, which involves up-regulated hydrolases, there may be competing cell-wall biosynthetic processes, which may have to be down-regulated for abscission to take place. In this context it is interesting to note that Ce16 is specifically down-regulated in the Abzo at the shedding stage. It is interesting that in bean leaves the activity of a membrane-associated cellulase isoenzyme, referred to as pI 4.5 cellulase, which is present before the induction of abscission, was also found to decline during abscission prior to the accumulation of the abscission of pI 9.5 cellulase (de1 Campillo et al., 1988). Expression of Cell and Ce12 (Lashbrook et al., 1994) and Cell protein (C. Gonzalez-Bosch, E. de1 Campillo, and A.B. Bennett, unpublished data) has been correlated with abscission in tomato flowers explants. We found here that Cell mRNA levels did not correlate with abscission in naturally abscising flowers (Fig. 6A). Thus, Cell expression may be more important for abscission in explants than for the intact flowers. In addition, Morgan et al. (1990) noted that explant systems tend to react differently to shock stresses than intact plants. Explant systems are subjected to several stresses such as excision and handling, which may promote an increase in the sensitivity to ethylene and, in turn, increase the magnitude of the effects observed compared with changes that occur in plants under normal growing conditions. In fact, we found that the cellulase activity in abscising flower explants treated with air or ethylene for 40 h was at least 3 times higher than the corresponding abscising flowers in planta. Similarly, Oberholster et al. (1991) showed that cytological and ultrastructural changes in soybean flower abscission are different in naturally abscising flowers versus in flowers that were induced to abscise by treatment with ethephon. A11 of these data suggest that, if multiple cellulases are needed to cause abscission in tomato flowers, the relative importance of each cellulase in the process may depend on the physiological conditions under which abscission takes place. More importantly, these data suggest that gene expression in natural abscission is different from ethylene-induced abscission. This report correlates BKS stages with gene regulation during abscission of tomato flowers. The definition of abscission stages adds a new dimension to the analysis of this process and offers the opportunity to distinguish genes involved in cell-wall weakening versus genes involved in catastrophic cell-wall disruption. In the future, we will use BKS to evaluate the role of multiple cellulases in natural
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systems using abscission-defective mutants generated by transformation with antisense cellulase genes.
ACKNOWLEDCMENTS E.d.C. would like to thank Larry Smart for providing help with the use of the phosphorimager and Coralie Lashbrook for providing the primers and useful suggestions in these studies. E.d.C. thanks Dr. Paul Bottino for facilitating the continuation of this work in Maryland. Received November 6, 1995; accepted April 1, 1996. Copyright Clearance Center: 0032-0889/96/ 111/0813/08. LITERATURE ClTED
Abeles FB (1968) The role of RNA and protein synthesis in abscission. Plant Physiol 43: 1577-1586 Brummell DA, Lashbrook CC, Bennett AB (1994) Plant endo-P1,4-glucanases: structure, properties and physiological function. Am Chem SOCSymp Ser 566: 100-129 Carpita CN, Gibeaut DM (1993) Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J 3: 1-32 Craker LE, Abeles FB (1969) Abscission. Quantitative measurement with a recording abscissor. Plant Physiol 8: 1139-1143 de1 Campillo E, Durbin M, Lewis LN (1988) Changes in two forms of membrane-associated cellulase during ethyleneinduced abscission. Plant Physiol 88: 904-909 de1 Campillo E, Lewis LN (1992) Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones. Plant Physiol 98: 955-961 de1 Campillo E, Reid PD, Sexton R, Lewis LN (1990) Occurrence and localization of 9.5 cellulase in abscising and nonabscising tissues. Plant Cell 2: 245-254 Durbin ML, Lewis LN (1988) Cellulases in Phaseolus vulgaris. Methods Enzymol 160: 342-351 Durbin ML, Sexton R, Lewis LN (1981)The use of immunological methods to study the activity of cellulase isozymes ( P 1:4 glucan 4-glucan hydrolase) in bean leaf abscission. Plant Cell Environ 4 67-73 Eyal Y, Meller Y, Lev-Yadum S, Fluhr R (1993) A basic-type promoter directs ethylene responsiveness, vascular and abscission zone-specific expression. Plant J 4: 225-234 Friedberg T, Grassow MA, Oerch F (1990) Selective detection of mRNA forms encoding the major phenobarbital inducible cytochrome P450 and other members of the P450 IIB family by the RNAses A protection assay. Arch Biochem Biophys 279: 167 Jensen TE, Valdovinos JG (1967) Fine structure of abscission zones. I. Abscission zones of the pedicels of tobacco and tomato flowers at anthesis. Planta 77: 298-318 Kalaitzis P, Koehler SM, Tucker ML (1995) Cloning of a tomato polygalacturonase expressed in abscission. Plant Mo1 Biol 28: 647-656
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Kendall JN (1918) Abscission of flowers and fruits in the Solanacea,with special reference to Nicotiana, Vol 5. University of California Publications in Botany, Berkeley, CA, pp 347428 Lashbrook CC, Bennett AB (1992) Functional analysis of Cxcellulase (endo-P-1,4-glucanase) gene expression in transgenic tomato fruit. In JC Pech, A Latche, C Balague, eds, Cellular and Molecular Aspects of the Plant Hormone Ethylene. Kluwer Academic Publishers, Boston, pp 123-128 Lashbrook CC, González-Bosch C, Bennett AB (1994). Two divergent endo-P-1,4-glucanase genes exhibit overlapping expression in ripening fruit and abscising flowers. Plant Cell 6: 1485-1493 Lewis LN, Varner JE (1970) Synthesis of cellulase during abscission of Phaseolus vulgaris leaf explants. Plant Physiol46: 194-199 Morgan PW, Chuan-Jin H, De Greef JA, D e Proft MP (1990)Does water stress promote ethylene synthesis by intact plants? Plant Physiol 9 4 1616-1624 Milligan SB, Gasser CS (1995) Nature and regulation of pistilexpressed genes in tomato. Plant Mo1 Biol 28: 691-711 Oberholster SD, Peterson CM, Dute RR (1991) Pedicel abscission of soybean: cytological and ultrastructural changes induced by auxin and ethephon. Can J Bot 69: 2177-2186 Rick CM, Dempsey WH (1969)Position of the stigma in relation to fruit setting of the tomato. Bot Gaz 130: 180-186 Roberts JA, Shindler BC, Tucker GA (1984) Ethylene-promoted tomato flower abscission and the possible involvement of an inhibitor. Planta 160: 159-163 Schaffner W, Weissmann C (1973) A rapid, sensitive and specific method for the determination of protein in dilute solutions. Ana1 Biochem 56: 502-514 Sexton R, Durbin ML, Lewis LN, Thomson WW (1980) Use of cellulase antibodies to study leaf abscission. Nature 283: 873-874 Sexton R, Lewis LN, Trewavas AJ, Kelly P (1985) Ethylene and abscission. In JA Roberts, GA Tucker, eds, Ethylene and Plant Development. Butterworths, London, pp 173-196 Sexton R, Roberts JA (1982) Cell biology of abscission. Annu Rev Plant Physiol 33: 133-162 Sexton R, Tucker ML, de1 Campillo E, Lewis LN (1989) The cell biology of bean leaf abscission. In DJ Osborne, MB Jackson, eds, Proceedings of the NATO Advanced Research Workshop on Cell Separation in Plants. Springer-Verlag, Berlin, pp 69-78 Taylor JE, Tucker GA, Lasslett Y, Smith CJS, Arnold CM, Watson CF, Achuch W, Grierson D, Roberts JA (1990) Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants. Planta 183: 133-138 Thompson DS, Osborne DJ (1994) A role for the stele in intertissue signaling in the initiation of abscission in bean leaves (Pkaseolus vulgaris L). Plant Physiol 105: 341-347 Tucker GA, Schindler CB, Roberts JA (1984) Flower abscission in mutant tomato plants. Planta 160: 164-167 Tucker ML, Sexton R, de1 Campillo E, Lewis LN (1988) Bean abscission cellulase. Characterization of a cDNA and regulation of gene expression by ethylene and auxin. Plant Physiol 88: 1257-1262
