Journal of Experimental Botany, Vol. 49, No. 329, pp. 1987–1997, December 1998
Possible involvement of abscisic acid in senescence of daylily petals Tadas Panavas, Elsbeth L. Walker and Bernard Rubinstein1 Biology Department and Plant Biology Graduate Program, University of Massachusetts, Amherst, MA 01003–5810, USA Received 26 May 1998; Accepted 4 August 1998
Abstract
Introduction
Daylily flowers (Hemerocallis hybrid, cv. Stella d’Oro) senesce and die autonomously over a 24 h period after opening. Investigations were performed to determine some of the mechanisms that lead to death of the petals. The flowers are insensitive to ethylene, but exogenous ABA prematurely upregulates events that occur during natural senescence, such as loss of differential membrane permeability, increases in lipid peroxidation and the induction of proteinase and RNase activities. Furthermore, the same patterns of proteinase and RNase activities appearing on activity gels during natural senescence are induced prematurely by ABA. The mRNA profile from ABA-treated, prematurely senescing petals visualized by differential display shows a high degree of similarity to the mRNA profile of naturally senescing petals 18 h later. In addition, endogenous ABA increases before flower opening and continues to increase during petal senescence. An osmotic stress by sorbitol increases endogenous levels of ABA and upregulates the same parameters of senescence as those occurring during natural senescence and after application of ABA. The mRNA profile from sorbitol-treated, prematurely senescing petals shows a high degree of similarity to mRNA from naturally senescing petals, but somewhat less similarity to mRNA from ABA-treated petals. The possibility is discussed that ABA is a constituent of the signal transduction chain leading to programmed cell death of daylily petals.
The death of entire, functioning organs is largely unique to plants and is an important component of their life cycle. The period of senescence preceding cell death involves active processes that are initiated at a particular developmental phase or by an environmental cue (Nooden and Guiamet, 1996). During senescence, endogenous signals upregulate certain genes whose products show high homology to enzymes known to degrade proteins, RNA, lipids, and chlorophyll (BuchananWollaston, 1997). This results in a breakdown and relocation of essential cellular constituents, destruction of differential membrane permeability and, ultimately, death of the cells comprising the organ. Flowers of the daylily (Hemerocallis sp.) provide an excellent model system to study the signals leading to programmed cell death of a plant organ (Lay-Yee et al., 1992; Bieleski and Reid, 1992). The flowers senesce in a rapid, highly predictable fashion without an external stress, and cell deterioration is obvious just 24 h after opening. Furthermore, the changes that occur in petals of the intact daylily flower are inhibited by the protein synthesis inhibitor cycloheximide and are seen in isolated petals or petal segments (Bieleski and Reid, 1992; Courtney et al., 1994; Stephenson and Rubinstein, 1998). Many morphological and biochemical changes occur in daylily petals both before and after 0 h, the time at which the flower is opening. From −36 h to +12 h, there are striking alterations in cell size, shape and cell-to-cell attachment (Stead and Van Doorn, 1994; Panavas et al., 1998). Ion leakage increases just after +12 h (Lay-Yee et al., 1992; Panavas and Rubinstein, 1998). This loss of membrane permeability is accompanied by alterations of phospholipid metabolism (Bieleski and Reid, 1992), and
Key words: Abscisic acid, daylily, differential display, Hemerocallis, organ senescence, programmed cell death.
1 To whom correspondence should be addressed. Fax: +1 413 545 3243. E-mail:
[email protected] Abbreviations: ABA, abscisic acid; RT, reverse transcription; TBARS, thiobarbituric acid reactive substances. © Oxford University Press 1998
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is preceded by increases in lipoxygenase activity (from −24 h), peroxidized lipid (from −24 h) and H O (from 2 2 −12 h) (Panavas and Rubinstein, 1998). There are also extensive decreases in protein levels per petal after 0 h. This loss of protein is likely due to the up-regulation at 0 h of several classes of proteinases (Stephenson and Rubinstein, 1998). Thus, within a relatively short time and before senescence of daylily petals becomes evident (i.e. from −12 h to +12 h), there is a striking upregulation of a range of degradative enzyme activities. The signals that initiate the degradative changes during senescence are largely unknown for daylily. In some groups of plants (e.g. Brassicaceae, Convolvulaceae, Orchidaceae, Caryophyllaceae), floral senescence is regulated at least in part by increases in ethylene production. The daylily, however, belongs to another group of plants (e.g. Amaryllidaceae, Liliaceae, Iridaceae, Asteraceae) whose flowers are insensitive to ethylene ( Van Doorn and Stead, 1994). Thus, floral death is not hastened by physiological levels of this hormone, nor is death delayed by preventing ethylene synthesis or inhibiting its action (Lukaszewski and Reid, 1989). Abscisic acid (ABA) is a possible candidate for a hormonal trigger for death of flowers such as the daylily that do not respond to ethylene. There are reports that exogenously applied ABA accelerates leaf senescence (Smart, 1994; Quiles et al., 1995; Yeh et al., 1995; Hung and Kao, 1997) and that ABA level increases during senescence (Even-Chen and Itai, 1975; Philosoph-Hadas et al., 1993), although some of these results may be related to the stresses imposed to induce senescence in leaves (Becker and Apel, 1993). In rose and carnation flowers, ABA stimulates senescence and ABA levels increase during senescence (Borochov and Woodson, 1989; Bianko et al., 1991; LePage-Degivry et al., 1991; Garello et al., 1995). However, in these flowers, ABA applications led to an early increase in ethylene evolution. If ABA were involved in daylily cell death, it would not be through an ethylene pathway since ethylene evolution does not increase until late in senescence (Lay-Yee et al., 1992). Evidence will be presented that exogenous ABA prematurely causes senescence-associated changes, such as lipid peroxidation and ion leakage as well as the appearance of proteinase and RNase activities in petals of the ethyleneinsensitive daylily. Furthermore, the pattern of mRNA regulation in ABA-treated, prematurely senescing petals, as visualized by differential display, is very similar to that of naturally senescing petals. Moreover, the amount of endogenous ABA increases during senescence. Finally, imposition of a water stress that increases the endogenous ABA level causes premature senescence of daylily petals.
Materials and methods Treatment of flowers Daylily (Hemerocallis hybrid, cv. Stella D’Oro) plants were grown in a growth chamber under a 16 h photoperiod as
described earlier (Panavas et al., 1998). The tepals from the inner whorl (referred to hereafter as ‘petals’) were placed in vials containing aqueous solutions of the desired additive and incubated in the chamber with the intact plants. The data presented are from one typical experiment that had been repeated one or more times. Ion leakage Measurement of ion leakage from petal discs was performed as described previously (Panavas and Rubinstein, 1998). Disks 8 mm in diameter were cut from each side of the central vein of the petal about half-way from the tip (2 discs per petal ) and placed on 1.5 ml distilled water in 35 mm Petri dishes (8 discs per dish). After a 3–6 h wash to remove ions from cut surfaces, the water was aspirated and the appropriate solutions added. Conductivity of the solutions was measured with a YSI Model 31 conductivity bridge and the solutions were changed after each measurement. Peroxidized lipids Peroxidized lipids were determined as thiobarbituric acidreactive substances (TBARS) as described previously (Panavas and Rubinstein, 1998). Petals were homogenized in 1.25 ml grinding buffer [50 mM TRIS-MES, (pH 6.5), 0.1% (w/v) butylated hydroxytoluene] for every 1.0 g tissue. After the addition of 0.2 ml of a freshly prepared solution of 8.1% (w/v) SDS, 1.5 ml 20% sodium acetate pH 3.5, and 1.5 ml 0.8% (w/v) thiobarbituric acid, the solution was heated at 95 °C for 30 min, cooled and centrifuged at 10 000 g for 10 min. The absorbance at 532 nm was compared with a standard curve for malondialdehyde. Protein extraction Petals were homogenized in liquid N and 1 ml of 100 mM 2 sodium phosphate buffer (pH 6.1) was added for each petal. The slurry was centrifuged for 20 min at 10 000 g. Protein concentration was measured with the Bradford reagent (BioRad) using BSA as a standard. Proteinase activity Azocasein assays were performed using the method of Torrigiani et al. (1996) with some modifications. Sample tubes containing 250 ml of 100 mM sodium phosphate buffer pH 6.1, 150 ml of 1% (w/v) azocasein, and 100 mg of total daylily protein in a 100 ml volume were incubated for 4.5 h at 37 °C. Reference tubes were prepared by incubating azocasein and petal homogenate in separate tubes. Reactions were stopped by adding 800 ml of 10% (w/v) trichloroacetic acid and the precipitate was pelleted by spinning in a microcentrifuge at top speed for 2.5 min at 4 °C. Absorbance of the supernatant was measured at 340 nm as described previously (Stephenson and Rubinstein, 1998). Activity gels SDS-PAGE was performed by the system of Laemmli (1970), except that the resolving gel also contained 1% (w/v) gelatin for proteinases or 3 mg ml−1 yeast RNA for RNases. An 8% polyacrylamide gel was used for proteinases and a 16% polyacrylamide gel for RNases. Samples were prepared by mixing 4 parts of the protein extract with 1 part of 5× sample buffer [5% (w/v) SDS; 25% (v/v) glycerol and 0.06% (w/v) bromophenol blue in 0.25 M TRIS-HCl buffer (pH 6.8)]. 5 mg of protein was loaded into each lane. Following SDS-PAGE, the SDS was removed by washing the gels twice for 30 min in 2.5% (v/v) Triton X-100
ABA and daylily flower senescence 1989 buffered with 0.1 M TRIS-HCl (pH 7.0). The gel slabs were then transferred to 0.1 M glycine in 0.1 M TRIS-HCl pH 7.0 for proteinases or 0.1 M imidazole pH 6.0 for RNases and kept at 37 °C with gentle agitation. After a 1 h incubation, proteinase gels were stained with 0.1% (w/v) Coomassie in methanol5acetic acid5water (40510550, by vol.) and destained in the same solution without the dye. Gels containing RNA were stained with 0.1% toluidine blue and destained in water. Endogenous ABA Petals were homogenized in 80% methanol, adding 2 ml per petal, and shaken 16 h in the dark at 0 °C. The homogenate then was centrifuged for 20 min at 10 000 g. The supernatant was filtered through a Nylon-66 filter (Alltech) and passed through a Sep-Pak C18 cartridge ( Waters) (Grossmann et al., 1994). ABA was quantified by an ELISA assay using Phytodetek ABA Immunoassay Kit (Idetec, Sunnyvale, CA). GCMS was performed according to Dunlap and Binzel (1996). RNA isolation Total RNA was isolated using guanidinium thiocyanate followed by centrifugation in CsCl as described by Chirgwin et al. (1979) with some modifications. Briefly, petals were homogenized in liquid nitrogen with a pestle and mortar and then 2 ml of guanidinium thiocyanate buffer [5 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% (w/v) sarcosyl and 5% (v/v) b-mercaptoethanol ] were added for each 1 g petal tissue. The homogenate was centrifuged for 10 min at 10 000 g and the supernatant was overlaid on 3 ml of 5.7 M CsCl, 0.1 M EDTA (pH 7.5) and 5 mg ml−1 ethidium bromide. After a 24 h spin at 30 000 rpm in the SW41 rotor, the supernatant was aspirated and the RNA pellet dissolved in 7 M urea-sarcosyl (2%, w/v), then purified with phenolchloroform (1:1), ethanol precipitated overnight at −20 °C and resuspended in an appropriate amount of water. Differential display Differential display was performed by the methods described by Liang et al. (1994) using the kit obtained from GenHunter (Nashville, TN ). Total RNA was prepared as above from petals extracted at three time points: 12 h before flower opening (−12 h), at opening (0 h) and 12 h after opening (+12 h). In addition, RNA was extracted at −6 h from petals treated with 100 mM ABA or 0.3 M sorbitol for 24 h starting at 30 h before flower opening. Two separate extractions were made for each sample. Next, cDNAs were prepared from the mRNA of the RNA preparations using reverse transcriptase, and these were amplified by PCR. The PCR reactions were run using combinations of three different anchor primers: H-T C, 11 H-T A, H-T G, and six arbitrary 13-nucleotide primers 11 11 manufacturer and defined as H-AP1, H-AP2, designed by the H-AP3, H-AP4, H-AP5, and H-AP8. An anchor primer anneals to the polyA tail plus a single nucleotide upstream, and an arbitrarily chosen primer binds at a random area upstream. The amplified cDNAs that resulted were separated on 6% ‘Long Ranger’ (FMC Bioproducts, Rockland, ME) gels. Quantitative data that describe trends in band appearance or disappearance and that compare experimental treatments to controls were made by a naive observer.
Results Exogenous ABA stimulates parameters associated with senescence Discs cut from petals 24 h before the flowers open and floated on distilled water show a constant, slow rate of
ion leakage until +12 h. After that time, the leakage rate increases steeply until cells lose their pigment and appear dead about 12 h later (Bieleski and Reid, 1992; Panavas and Rubinstein, 1998). When ABA is added at −21 h to the solution upon which petal discs are floating, ion leakage is stimulated in a concentration-dependent manner after a lag period of about 8–12 h (Fig. 1A). This lag period is approximately the same even if ABA is added at −12 h (data not shown). Leakage is stimulated by concentrations of ABA as small as 5 mM, but the accelerated rate of ion leakage with 20 mM and 50 mM ABA is similar to the rate of controls 24 h later (Fig. 1A). To see if ABA accelerates the appearance of other senescence-associated parameters, the accumulation of peroxidized lipid was measured as thiobarbituric acidreactive substances ( TBARS) (Ohkawa et al., 1979). Intact petals were detached from flowers 30 h before opening and incubated in distilled water or 100 mM ABA. This concentration of ABA was selected because previous results showed it was necessary to use a concentration of an inhibitor or stimulator for intact petals that was approximately 5 times higher than that used for petal discs to get the same response (unpublished results). Presumably the higher concentration is required because uptake is more restricted in intact petals versus petal discs. ABA at 100 mM has also been used frequently to induce senescence in detached leaves ( Williams et al., 1994; Oh et al., 1996). The ratio of TBARS per petal in untreated controls increases slowly up to −12 h, and then increases more rapidly until +18 h (Fig. 1B), which is similar to the kinetics for TBARS in undetached petals (Panavas and Rubinstein, 1998). ABA causes a premature increase of TBARS ( Fig. 1B), so that by −12 h the amount of TBARS is significantly higher in the hormone-treated petals than in petals incubated in water. The difference between the two treatments becomes larger during the course of the experiment, so that by 0 h, the ABA-treated petals already have a level of TBARS similar to that of untreated, normally senescing petals at +18 h. An increase in proteinase activity is another characteristic of senescing systems (Quiles et al., 1995), including daylily (Stephenson and Rubinstein, 1998). When petals are removed 30 h before flower opening and incubated in water, the specific activity of proteinases using azocasein as a substrate remains low until about 0 h, the time at which an intact flower would be opening, and then the activity increases steadily (Stephenson and Rubinstein, 1998; Fig. 1C ). When 100 mM ABA is added at −30 h, however, the proteinase activity increases after a lag period of about 6–10 h; proteinase activity at 0 h in ABA is similar to that seen about 20 h later in untreated petals ( Fig. 1C ). Proteinase activity was separated on activity gels and divided into three zones ( Fig. 2A). The activity in zones
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Panavas et al. 2 and 3 is present even in unopened flower petals, i.e. at −12 h (Fig. 2A) and earlier (data not shown), and is thus a likely contributor to the basal proteinase activity level observed in the biochemical assay ( Fig. 1C ). It is not possible to tell how many proteinases contribute to zones 2 and 3, but the activity in these zones increases in senescing petals. The proteinases with activity bands in zone 1 may be senescence inducible, since their activities appear as senescence progresses ( Fig. 2A). When petals are treated with 0.1 mM ABA from −30 h to −6 h, the proteinase activities in all of the zones are strongly upregulated, so that the extracts from naturally senescing petals at +12 h and from ABA-treated, prematurely senescing petals at −6 h give almost identical banding patterns ( Fig. 2A). RNase activities were also examined using activity gels and divided into four zones (Fig. 2C ). The activities in zones 2 and 4 are already present at −12 h, and then these activities increase markedly during senescence. The activities in zones 1 and 3 appear to be induced as senescence progresses. As with proteinase activities ( Fig. 2A), ABA treatment prematurely increases/induces the RNase activities observed during natural senescence (e.g. the patterns of −6 h ABA and +12 h control are similar). Thus, the sequence of expression and the banding pattern is identical in both naturally senescing and ABAtreated, prematurely senescing petals (Fig. 2C ). For both proteinase and RNase activity gels, equal amounts of protein were loaded, but similar trends were observed when the lanes were loaded on an equal petal basis. ABA prematurely alters levels of mRNAs
Fig. 1. Effect of exogenous ABA on ion leakage (A), lipid peroxidation (B) and proteinase activity (C ) in daylily petals. Ion leakage (A) from 8 petal discs, 8 mm in diameter, floating on water (O), or on 5 mM ABA (%), 20 mM ABA (6), or 50 mM ABA (B) was determined by solution conductivity. Peroxidized lipids (B) were measured as thiobarbituric acid reactive substances ( TBARS ) per petal from petals incubated in water (O) or in 100 mM ABA (6). Data were quantified using malondialdehyde as a standard. Specific proteinase activity ( mg azocasein mg−1 total protein h−1) using azocasein as a substrate (C ). Proteinase activity in petals incubated in water (O); proteinase activity in petals incubated in 100 mM ABA (6). Values represent the means and SE from three independent experiments.
Given that ABA can accelerate the appearance of various enzyme activities, the effect of ABA on the appearance (or disappearance) of specific mRNAs was tested using the procedure of differential display. This method involves amplification of specific sets of mRNA by reverse transcription (RT ) followed by amplification of the resulting cDNAs by PCR (Liang et al., 1994; see Materials and methods). Figure 3A, B show the mRNA profiles resulting from two sets of primer pairs. The number of bands that increased, decreased or stayed the same for the water controls from −12 h to +12 h was determined, using only those bands that showed the same pattern in duplicate lanes. There were just three or four bands apparent when the RT step was omitted and these bands were not considered for analysis. For clarity, only a few bands in each category are marked in Fig. 3, but many others may be easily scored. The analysis ( Table 1) shows that out of the 1093 bands that were distinguishable on the gels (a mean of 62±13 for each of the 18 primer pairs), 41% showed no change from −12 h to +12h, i.e. during natural senescence, 31% increased in intensity and 28% decreased. Thus, 59% of the bands changed during senescence.
ABA and daylily flower senescence 1991
Fig. 2. Activity gels of daylily proteinases (A, B) and RNAses (C, D). Petals were excised from flowers at −30 h and incubated until −12 h, −6 h, 0 h, +12 h, or +24 h in water (control ), or in 0.1 mM ABA (A, C ) or in 0.3 M sorbitol (B, D) for the same time periods. A +24 h point was not used for ABA and sorbitol treatments because of severe deterioration of the petals. Homogenates were prepared at the times indicated and 5 mg of protein were separated on SDS-PAGE with either gelatin (A, B) or yeast RNA (C, D) co-polymerized into the gel. After a 1 h incubation, the gels were stained with Coomassie (A, B) or toluidine blue (C, D). Clear areas in the gels represent proteinase (A, B) and RNase (C, D) activity.
To study the effects of ABA treatment on mRNA profiles, excised petals were treated with 100 mM ABA at −30 h and RNA was extracted at −6 h to be analysed by differential display. The resulting cDNA bands at −6 h from petals treated with ABA for 24 h were compared to cDNA bands from +12 h controls, that had either increased or decreased in intensity ( Table 2). In other words, if the intensity of a particular band increased from −12 h to +12 h and there was a band of corresponding intensity in the −6 h ABA lanes, it was scored as a similarity. A similarity also included a band that disappeared from −12 h to +12 h on control lanes and was not present on the −6 h ABA lanes. The +12 h control time point was chosen to compare with −6 h ABA treatment because ABA appeared to accelerate a variety of senescence parameters by approximately 18 h ( Figs 1, 2). Thus, it was possible that petals in ABA from −30 h to −6 h were at the same developmental state as +12 h controls. The data in Table 2 show that 529 bands were scored from gels prepared from 15 different primer pairs and 81% of the bands from extracts made at −6 h from petals incubated for 24 h in 100 mM ABA had the same intensity as the corresponding bands from petals incubated in
water and extracted 12 h after flower opening ( Table 2). The other 19% of the bands did not correspond to those of untreated petals, i.e. a band that increased in intensity from −12 h to +12 h on the control lanes was not present on the ABA lanes; or, a band that disappeared from −12 h to +12 h on the control lanes was present on the ABA lanes. For control analyses, the mRNA profile of two sets of primer pairs from untreated petals at +12 h was compared to petals at −6 h after a heat shock (from −9 h to −6 h), a stress which is optimal for prolonging the life of daylily petals (Panavas et al., 1998). In this case, only 38% of the bands had a similar intensity. What is more, when the mRNAs of heat-shocked petals at −6 h were compared to mRNAs of ABA-treated petals at −6 h, only 19% of the bands were similar. Endogenous levels of ABA increase during daylily petal senescence Amounts of ABA in developing petals were determined by ELISA ( Fig. 4). ABA levels increase 2-fold between −36 h and −12 h when expressed on a per petal basis. From −12 h, ABA accumulates at a much faster rate,
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Fig. 3. Differential display of mRNA from daylily petals using two different primer pairs. Petals were excised from flowers at −30 h and incubated either in water until −12 h, 0 h or +12 h, or in 0.1 mM ABA (A, B) or in 0.3 M sorbitol (B) until −6 h when RNA was extracted. Total RNA was used for RT-PCR and the cDNAs were separated on a 6% ‘Long Ranger’ gel. A segment of each gel is shown. Some of the most distinct bands that either increase, decrease or do not change are marked, so that arrows indicate the bands (cDNAs) representing mRNAs that increase, arrowheads indicate the bands representing mRNAs that decrease, and dark circles indicate bands representing mRNAs that do not change during daylily flower senescence. Each lane represents a separate RNA extraction and the time point for every treatment was replicated. Only bands that were identical in replicate lanes were scored. These data are presented numerically in Table 1. The presence or absence of bands at −12 h that had either increased or decreased from −12 h to +12 h were then compared to the presence or absence of bands at −6 h from petals treated with ABA or sorbitol from −30 h to −6 h. These comparisons are presented numerically in Table 2.
ABA and daylily flower senescence 1993 Table 1. Patterns of cDNAs on differential display during ageing of daylily petals Counts were made of cDNA bands from control petals on gels like those illustrated in Fig. 3, dividing into categories those bands whose intensity stayed the same, increased, or decreased over a 24 h period from 12 h before flower opening to 12 h after opening. The data for band number were obtained by pooling the counts from 18 different primer pairs. The mean values represent the averages of the different categories from the 18 primer pairs with the SE of the mean in parentheses. The percentage values are calculated by dividing each of the different categories of bands by the total number of bands scored. Category
Band number
Mean (SE )
Percentage
Bands unchanged Bands increasing Bands decreasing
445 342 306
25 (6) 19 (8) 17 (8)
41 31 28
Table 2. Patterns of cDNAs on differential display from untreated controls at +12 h compared to ABA- and sorbitol-treated petals at −6 h The cDNA bands on the +12 h lane of control petals that increased or decreased from −12 h to +12 h (as categorized in Table 1) were compared to the −6 h lanes of cDNA extracted from petals exposed to 100 mM ABA or 0.3 M sorbitol for 24 h. A band was scored ‘similar’ if it increased to a detectable level by +12 h and was of equal intensity in the ABA or sorbitol lanes. A band was also scored ‘similar’ if it decreased over the 24 h period in controls and was of equal intensity or undetectable in the ABA or sorbitol lanes. Bands scored ‘dissimilar’ were either present in the +12 h lanes of controls but faint or absent in the ABA or sorbitol lanes, or the inverse was true. Total band numbers obtained for comparisons of ABA treatments with controls are also shown as the mean value for 15 different primer pairs with the SE of the mean in parentheses. Total band numbers obtained for comparisons of sorbitol treatments with controls and with ABA treatments are also shown as the mean value for three different primer pairs with the SE of the mean in parentheses. Percentage values were obtained by dividing the total bands scored for each comparison into the number that were similar or dissimilar.
Control (+12 h) Total bands Mean (SE) Percentage Sorbitol (−6 h) Total bands Mean (SE) Percentage
ABA (−6 h)
Sorbitol (−6 h )
Similar
Dissimilar
Similar
Dissimilar
431 29 (9) 81
98 7 (3) 19
102 34 (12) 80
25 8 (1) 20
79 26 (2) 65
44 15 (5) 35
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and that rate continues until 24 h after flower opening when ABA levels are more than six times higher than those seen at −36 h. When the amount of ABA is expressed on a fresh weight basis, an increase is still observed, but the difference between the lowest and the highest points is 2-fold (data not shown). The level of ABA in daylily petals is similar to the range of ABA levels in other systems (Feldman and Sun, 1986; Holappa and Walker-Simmons, 1995). To confirm the results from the ELISA assay indicating that ABA levels were indeed rising, daylily petal extracts from three time points (−32 h, −24 h and −12 h) were
Fig. 4. Changes of endogenous ABA levels in daylily petals from −36 h to +24 h as determined by ELISA. Inset: ABA levels at −12 h and 0 h in control petals (C, open bars) incubated in water, compared with ABA levels at −12 h of petals in 0.3 M sorbitol from −24 h to −12 h (S, shaded bar). Values represent the means and SE from three independent experiments.
analysed by GCMS (performed by the J Dunlap laboratory at Texas Agricultural Experimental Station, College Station, TX ). The absolute values for ABA measured by GCMS are one-third less than those obtained by ELISA, but the ratio of the data obtained by the two methods is the same for all three time points (data not shown). Osmotic stress induces accumulation of endogenous ABA and accelerates cell death To test for an effect of endogenous ABA on petal senescence, the concentration of the hormone was increased by imposing an osmotic stress. Cut ends of petals were placed in 0.3 M sorbitol, a concentration that decreases but does not eliminate water uptake (Panavas et al., 1998). After incubation from −24 h to −12 h, ABA accumulation is over 2-fold higher in sorbitol-treated petals than in water controls at −12 h, and is at the same level as the 0 h controls ( Fig. 4, inset). When applied from −12 h to 0 h, the level of ABA in sorbitol-treated petals is also over 2-fold higher than petals on water (data not shown). The sorbitol-induced osmotic stress prematurely stimulates parameters associated with natural senescence in a manner similar to ABA additions. When petal discs are floated on 0.3 M sorbitol, the ion leakage rate is significantly higher than from control discs floated on water, although the difference is not apparent until +12 h ( Fig. 5A). Sorbitol also causes a concentration-dependent accumulation of peroxidized lipids (TBARS ) in the petals after a lag period of less than 12 h. The largest effect is with 0.3 M sorbitol, but 0.2 M and 0.1 M sorbitol also lead to significant increases in TBARS compared to the control ( Fig. 5B).
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Panavas et al. Further evidence that the sorbitol-induced senescence is similar to natural senescence and to that induced by ABA is shown in Fig. 5C. Proteinase activity, using azocasein as the substrate, increases markedly 12 h after incubation in 0.3 M sorbitol (at −12 h). The very steep increase in proteinase specific activity in sorbitol-treated petals occurs at least 24 h earlier than similar increases in proteinase activity from untreated petals and the level of proteinase activity after sorbitol treatment at 0 h is similar to control values at about +18 h. What is more, the patterns of both proteinase ( Fig. 2B) and RNase ( Fig. 2D) activities (i.e. the appearance of, or increases in, the various zones) are similar and appear prematurely on activity gels. Thus, the bands and their intensities in the 0 h lane of the sorbitol treatment resemble those of the +12 h control. There is also a strong similarity on the gels between the banding patterns of sorbitol-treated and ABA-treated petals for both proteinase ( Fig. 2A versus Fig. 2B) and RNAse (Fig. 2C versus Fig. 2D) activities. The mRNA pattern of petals treated with 0.3 M sorbitol from −30 h to −6 h was compared to that of petals incubated in water and in ABA ( Table 2). The same criteria for comparison were used as mentioned above for ABA, i.e. the presence or absence of cDNA bands from control petals at +12 h that either increased or decreased from −12 h were compared to cDNAs from petals extracted at −6 h. Of the 127 bands scored from three sets of primer pairs (one set of which is shown in Fig. 3B), the intensity of bands that either increased or decreased during normal petal ageing that is detectable at +12 h is similar to 80% of the bands from sorbitoltreated petals that were extracted 18 h earlier (at −6 h). When the same bands from the sorbitol treatments are compared to the bands from ABA-treated petals extracted at −6 h, 66% of the bands are similar.
Discussion
Fig. 5. Effect of 0.3 M sorbitol on ion leakage (A), lipid peroxidation (B) and proteinase activity (C ) in daylily petals. Ion leakage (A) was determined from 8 petal discs, 8 mm in diameter floating on water (O), or on 0.3 M sorbitol (6) as a change in solution conductivity. Amount of peroxidized lipids (B) was measured as thiobarbituric acid reactive substances ( TBARS ) from petals incubated either in water (O), or 0.1 M (6), 0.2 M (%) or 0.3 M (B) sorbitol. Data were quantified using malondialdehyde as a standard. Specific activity of proteinases ( mg mg−1 total protein h−1) measured using azocasein as a substrate (C ). Petals were incubated in water (O) or in 0.3 M sorbitol (6). Sorbitol was added at −24 h in (A) and at −30 h in ( B) and (C ). Values represent the means and SE from three independent experiments.
Programmed cell death in daylily petals is preceded by increases in ion leakage (Bieleski and Reid, 1992), reactive oxygen species, and lipid peroxidation (Panavas and Rubinstein, 1998), and the upregulation of hydrolytic enzymes such as proteinases ( Valpuesta et al., 1995, Stephenson and Rubinstein, 1998) and RNases (Fig. 2C, D). The experiments reported here were designed to determine the factor or factors that initiate these processes leading to death of the petal cells. Ethylene has been implicated as an hormonal trigger of programmed cell death in leaves (Smart, 1994), and while ethylene also activates senescence-related processes in several flowers ( Woltering and van Doorn, 1988), the participation of this hormone in daylily senescence appears unlikely (Lay-Yee et al., 1992). ABA is another hormone that may be implicated in senescence. Exogenous
ABA and daylily flower senescence 1995 ABA accelerates senescence in detached rice leaves as determined by loss of total protein and chlorophyll ( Yeh et al., 1995). ABA also induces the premature appearance of proteinase activity in barley leaves (Quiles et al., 1995) with a pattern on activity gels that is very similar to the pattern observed in naturally senescing leaves. However, the antisense suppression of a phospholipase essential for ABA-stimulated senescence of Arabidopsis leaves, has no effect on the normal course of senescence ( Fan et al., 1997), thereby implying that senescence of these leaves need not involve ABA. In the daylily flower, ABA additions can accelerate the appearance of many of the biochemical and molecular changes that take place during senescence. For example, ion leakage ( Fig. 1A) and lipid peroxidation as measured by TBARS ( Fig. 1B), as well as proteinase activity ( Fig. 1C ) occur prematurely in petals treated with ABA. Furthermore, the patterns of proteinase and RNase activities that are detected on activity gels during ABA treatments are similar to those occurring later during normal senescence ( Fig. 2A, B). Another important consideration when comparing the effects of ABA applications with natural senescence in daylily petals is the levels of mRNAs. Using differential display, there is a striking resemblance (81%) at +12 h between the pattern of messages that had appeared or disappeared during natural senescence and the pattern occurring 18 h earlier (at −6 h) after a 24 h ABA treatment (Fig. 3; Table 2). On the other hand, there is much less correspondence (38%) when the mRNAs from heatshocked petals at −6 h are compared to control petals at +12 h, so the effect of ABA on patterns of mRNA visualized by differential display is probably not entirely due to a non-specific stress. There are several reasons why some dissimilarities (19%) exist between cDNAs of the 24 h ABA treatment at −6 h and controls at +12 h. The cells may be responding to an external ABA concentration that is not identical to normal levels, or the time periods chosen for comparison (i.e. −6 h ABA versus +12 h control ) may not correspond precisely to the same degree of senescence. There are also processes in control petals that are not observed, or occur only slowly, in ABA treatments, for example, increases in cell size (data not shown). It is possible, therefore, that some of the mRNAs that appear or disappear in controls may be related to growth. The differential display technique also provides an interesting view of the dynamics of natural petal senescence at the mRNA level. It appears that the steady-state levels of 40% of the cDNA bands detected on the gels do not change during the early stages of senescence (from −12 h to +12 h), and of the remaining bands, about 50% increase and 50% decrease ( Table 1). Any analysis of banding patterns on the gels must take into consideration the likelihood of false positives, for
example, when bands arise from the same mRNA. But there should be no preferential amplification of particular mRNAs, so the pattern analysis, presented here as a percentage, should not be affected. Banding patterns may also be altered by DNA that contaminates the RNA preparations. The purity of the RNA presented here was verified by the fact that only three or four bands were seen in controls without RT; these bands were not used for the analyses. The reliability of differential display is further supported by the fact that six different cDNA bands were cloned that increased from −12 h to +12 h in control petals and were also upregulated prematurely after ABA treatments. After analysis by the RNase protection assay, all six clones increased over time and all were upregulated by ABA (Panavas et al., unpublished results). These results tend to validate the interpretations made from a pattern analysis of differential display. The authors are unaware of similar analyses of mRNA patterns using differential display. It would be interesting to compare the data generated here for senescing daylily petals with other developmental systems. The increases in endogenous levels of ABA appear to provide support for the suggestion that ABA is an important component of normal senescence of daylily petals. Amounts of ABA increase in detached, senescing tobacco ( Even-Chen and Itai, 1975) and rice (Philosoph-Hadas et al., 1993) leaves. ABA also increases in rose petals, but only during advanced stages of senescence (LePageDegivry et al., 1991). When expressed per petal, ABA levels of daylily petals measured both by ELISA and by GCMS increase steadily from the first point measured at −36 h to −12 h and then rise more sharply after −12 h ( Fig. 4), which is before hydrolytic enzyme activity increases (Figs 1, 5). While these data are suggestive of a causative role for ABA, it is not possible to determine if the elevated levels of this hormone are high enough to initiate responses related to senescence in daylily cells. However, hormonal effects need not be dependent on endogenous levels, since cell receptivity is also an important determinant (Hetherington and Quatrano, 1991; Trewavas and Jones, 1991). To test further for an involvement of ABA in daylily petals, an attempt was made to increase the level of endogenous ABA prematurely by imposing an osmotic stress, a response commonly reported in leaves or roots (Ingram and Bartels, 1996). The osmotic stress induced by 0.3 M sorbitol causes an easily measurable elevation of ABA in the petals (Fig. 4, inset), and these petals die prematurely, demonstrating many of the same changes that appear during natural senescence ( Figs 2B, D, 5). What is more, after a 24 h sorbitol treatment from −30 h to −6 h, 80% of the mRNAs at −6 h on the differential display are similar to the mRNAs up- or down-regulated
1996
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in naturally senescing petals from −12 h to +12 h ( Table 2). The lack of a 100% correspondence between mRNAs in sorbitol-treated and control petals may be due to the same factors mentioned above for ABA—the times chosen for comparison may not be perfectly analogous and sorbitol is likely to initiate other responses that do not occur in naturally senescing petals. Furthermore, when the bands of cDNAs from 24 h sorbitol treatments at −6 h are compared to the 24 h ABA treatments at −6 h, only 66% are similar ( Table 2). These data indicate that the osmotic stress causes ABA-independent changes in mRNA levels. Indeed, signal transduction after osmotic stress has both ABA-dependent and ABA-independent pathways (Hetherington and Quatrano, 1991; Trewavas and Jones, 1991; Gosti et al., 1995). Thus, ABA and sorbitol appear to affect certain mRNA levels similarly to natural senescence, but the osmotic stress and exogenous applications of the hormone also cause changes in mRNA levels that are unrelated to senescence and to each other. The involvement of ABA in a physiological process depends in part on accurately mimicking the responses after applications of ABA ( Trewavas and Jones, 1991). As shown above for daylily, both exogenous ABA and an osmotic stress that elevates endogenous ABA levels prematurely bring about many of the same changes (e.g. loss of differential membrane permeability, accumulation of peroxidized lipids and up-regulation of proteinase and RNase activities) that occur during normal senescence, including appearance and loss of particular mRNAs. Still, as pointed out by Trewavas and Jones (1991), ABA may only be inducing changes because of an artificially elevated concentration. Furthermore, there may be other, as yet undetermined factors involved with the natural response that are not regulated by ABA. Another means of accumulating evidence for a role of ABA is to reduce the levels of this hormone with a herbicide such as norflurazon or floradone that blocks synthesis of carotenoids, a precursor for ABA (Zeevart and Creelman, 1988). However, it was not possible to detect any change in ABA levels in daylily petals after treatment with 1 mM norflurazon. There was also no change in the kinetics or amount of ion leakage from petal discs (Panavas et al., unpublished results). It is possible that the high amount of carotenoids already present in the petals during the herbicide treatment provides ample substrate for ABA synthesis ( Kende and Zeevaart, 1997). No ABA mutants are available in daylily, but it is possible to clone genes that are up-regulated naturally during senescence and also prematurely by ABA. The sequences and functions of these genes as well as an examination of their regulatory regions may shed more
light on the possible involvement of ABA in daylily programmed cell death.
Acknowledgements This work was partially supported by grants to TP from the American Hemerocallis Society and Sigma Xi and by an Internal Grant Award to BR. We would like to thank John Mistler for help with several experiments and Paul Stephenson for useful advice. We are grateful to Ron Beckwith for growing the daylily plants.
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