periment Station.K. A. L. was supported in part by a David Ross. Fellowship from Purdue University. events. Little is knownabout the sequence of events linking.
Plant Physiol. (1988) 87, 498-503
0032-0889/88/87/0498/06/$01 .00/0
Ethylene-Induced Gene Expression in Carnation Petals' RELATIONSHIP TO AUTOCATALYTIC ETHYLENE PRODUCTION AND SENESCENCE Received for publication September
24. 1987 and in revised form February 29. 1988
WILLIAM R. WOODSON* AND KAY A. LAWTON Department of Horticulture, Purdue University, West Lafayette, Indiana 47907 events. Little is known about the sequence of events linking ethylene binding to petal senescence. There is increasing evidence that responses to ethylene in other tissues are associated with the expression of specific genes (2, 8, 12, 16, 17, 19). Ethylene regulation of specific mRNAs has been demonstrated in ripening tomato (12) and avocado (24) fruits. Two of these mRNAs encode the proteins cellulase (5) and polygalacturonase (7), which play functional roles in the development of ripe fruits. The senescence of carnation petals has been linked to temporal changes in gene expression as evidenced by changes in protein and mRNA populations (27). The ethylene climacteric appeared to be a transition period in relation to these changes. Inhibitors of protein synthesis have been shown to interfere with the induction of petal senescence by ethylene (30). The onset of petal senescence in Hibiscus rosa-sinensis was found to be associated with a transient increase in protein synthesis and a change in the patterns of proteins synthesized in vivo (28). Taken together, these results indicate petal senescence is regulated at the level of transcription and/or translation. In the present work, we examine the relationship between autocatalytic ethylene production, petal senescence, and mRNA populations by following the temporal development of these responses after exposure to exogenous ethylene. Furthermore, we relate these changes to the development of tissue responsiveness to ethylene with increasing age.
ABSTRACT Exposure of carnation (Dianthus caryophyllus L.) flowers to ethylene evokes the developmental program of petal senescence. The temporal relationship of several aspects of this developmental program following treatment with ethylene was investigated. Exposure of mature, presenescent flowers to 7.5 microliters per liter ethylene for at least 6 hours induced petal in-rolling and premature senescence. Autocatalytic ethylene production was induced in petals following treatment with ethylene for 12 or more hours. A number of changes in mRNA populations were noted in response to ethylene, as determined by in vitro translation of petal polyadenylated RNA. At least 6 mRNAs accumulated following ethylene exposure. The molecular weights of their in vitro translation products were 81, 58, 42, 38, 35, and 25 kilodaltons. Significant increases in abundance of most mRNAs were observed 3 hours following ethylene exposure. Ethylene exposure resulted in decreased abundance of another group of mRNAs. Treatment of flowers with competitive inhibitors of ethylene action largely prevented the induction of these ethylene responses in petals. An increase in flower age was accompanied by an increase in the capacity for ethylene to induce petal in-rolling, autocatalytic ethylene production, and changes in mRNA populations suggesting that these responses are regulated by both sensitivity to ethylene and ethylene concentration. These results indicate that changes in petal physiology resulting from exposure to ethylene may be the result of rapid changes in gene expression.
MATERIALS AND METHODS Plant Material. Carnation (Dianthus caryophyllus L cv White
The senescence of flower petals is often associated with increased production of the phytohormone ethylene (4, 10, 13, 14, 29). In carnations, it is well established that this climacteric rise in ethylene plays an important role in the coordination and regulation of petal senescence (4, 10, 15). Treatment of flowers with inhibitors of ethylene biosynthesis or action has been shown to delay the onset of petal senescence (4, 21, 25, 26, 29). Furthermore, exposure of preclimacteric flowers to exogenous ethylene hastens the onset of petal senescence and induces autocatalytic ethylene production (10, 13). The initiation of ethylene responses in plant tissues is thought to first involve ethylene binding to a metalloprotein receptor (23). In this regard, Sisler et al. (21) have demonstrated ethylene binding in carnation petals. Of further interest in carnation is that the responsiveness of petal tissue to ethylene increases with age (10, 13, 15). This increase in sensitivity to ethylene was not linked to an increase in the concentration of an ethylene receptor since ethylene binding capacity decreased with petal age (3). The engagement of ethylene responses could be regulated beyond initial binding at specific points in the chain of signal-transduction ' Journal paper No. 11,332 of the Purdue University Agricultural Experiment Station. K. A. L. was supported in part by a David Ross Fellowship from Purdue University.
Sim) flowers were harvested from plants grown under greenhouse conditions as previously described (27). Flowers were harvested at anthesis when outer petals were reflexed at 900 angles to the axis of the calyx except where otherwise noted. Stems were cut to 10 cm, placed in distilled water, and held in the laboratory. Chemical Treatment. Flowers were placed in a 20 L container through which humidified air and ethylene (7.5 ,ul/L) were passed at 500 ml/min. In other experiments where flowers were treated with ethylene action inhibitors, flowers were enclosed in 2.5 L jars, and ethylene was injected to a final concentration of 7.5 ,u/L. Saturated KOH was placed in the jar with a paper wick to absorb evolved CO2. Control flowers were enclosed in jars without added ethylene. Flowers were treated with 4 mM Ag+ as the anionic complex with sodium thiosulfate (1:4 molar ratio of silver nitrate to sodium thiosulfate) for 1 h then transferred to water for 24 h prior to the initiation of ethylene treatment. In another experiment, flowers held in water were placed in jars, and NBD2 injected onto a filter paper hung in the jar to facilitate evaporation, yielding a final concentration of 2,500 ,ul/L prior to the addition of ethylene. 2
Abbreviations: NBD, 2,5-norbornadiene; STS, silver thiosulfate;
poly(A +), polyadenylated.
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ETHYLENE-REGULATED GENE EXPRESSION AND PETAL SENESCENCE Ethylene Measurement. Petals were detached from flowers and equilibrated with air for 15 min following ethylene treatment after which 0.5 g of petals were placed in 25 ml serum vials. The vials were capped, and ethylene was allowed to accumulate for 0.5 h, after which a gas sample was removed and analyzed for ethylene by GC as previously described (29). The remaining petals were frozen in liquid N2 immediately following ethylene exposure and stored at -80°C until subsequent extraction of RNA. RNA Extraction and Poly(A+), RNA Isolation. RNA was extracted by a modification of the method of Grierson and Covey (9). Briefly, 10 g of frozen petal tissue were powdered under liquid N2 and were homogenized in equal volumes (50 ml) of phenol and extraction buffer containing 50 mM Tris-HCl (pH 8.4), 5% phenol, 6% sodium p-aminosalicylate, 1% sodium triisopropylnaphthalenesulfate, 1% (v/v) 83-mercaptoethanol, and 10 mM ribonucleoside vanadyl complexes. Following phase separation and reextraction of the aqueous phase with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, v/v), nucleic acids were precipitated by the addition of sodium acetate to 0.2 M and 2 volumes of ethanol at - 20°C overnight. The precipitate was resuspended in sterile water and high mol RNA reprecipitated by the addition of 3 volumes of 4 M sodium acetate (pH 6.0) for 1.5 h at 0°C. The precipitated nucleic acids were dissolved in sterile water and poly(A+)RNA isolated by chromatography over oligo(dT)-cellulose. Samples were scanned with a Beckman DU-8 spectrophotometer from 220 to 320 nm. RNA content was calculated assuming an extinction coefficient of 25 A260 units/mg RNA. In Vitro Translation. Poly(A+) RNA was translated in vitro using the rabbit reticulocyte lysate system of Pelham and Jackson (18). Three ug poly(A+) RNA were translated in the presence of 10 ,uCi of L-[35S]methionine (>1000 Ci/mmol, from New England Nuclear) in a final reaction volume of 30 ,ul. Samples were incubated at 30°C for 1 h. The reaction was stopped on ice, and the reaction mixtures were stored at - 200C. Electrophoresis of in Vitro Translation Products. Samples containing equal amounts of TCA-precipitable [35S]methionine were brought to the concentration of Laemmli's (11) sample buffer and were boiled for 3 min prior to electrophoresis in 12.5% polyacrylamide gels containing 1% SDS (11). Following electrophoresis gels were fixed in 50% methanol (v/v) and 12.5% acetic acid (v/v) containing 0.025% (w/v) Coomassie blue to visualize protein standards. Gels were destained in 40% methanol and 10% acetic acid then in 5% methanol and 7% acetic acid. Gels were equilibrated with 7% acetic acid, impregnated with FluoroHance (Research Products International Corp., Mount Prospect, IL), vacuum-dried onto Whatman 3MM filter paper, and exposed to Kodak XAR-5 film at - 80°C. The resulting fluorographs were scanned densitometrically with a Beckman DU-8 spectrophotometer.
RESULTS Effects of Ethylene Exposure Duration. Flowers harvested at anthesis and exposed to 7.5 ,uIIL ethylene for 6 or more h showed
evidence of initiation of senescence (Fig. 1). A brief ethylene exposure of 3 h did not induce premature petal senescence. Petal in-rolling, an early indication of the onset of senescence (10), was exhibited by flower petals following an ethylene treatment of at least 6 h (data not shown). When flowers were removed from the ethylene atmosphere after 6 h this in-rolling was reversed. However, petals exposed to ethylene for more than 12 h did not recover from in-rolling following transfer to air and were fully wilted after 24 h (Fig. 1). A response to exogenous ethylene exposure in many flowers and climateric fruits is the induction of autocatalytic ethylene production (10, 12, 13, 24). Ethylene production by petals was measured following removal
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FIG. 1. Effect of ethylene exposure on the longevity of carnation petals. Flowers were exposed to 7.5 AtIIL ethylene for various durations. Petal wilting and necrosis were used to indicate senescence. Following ethylene treatment, petals were isolated and equilibrated in air for 15 min, and their ethylene production was measured. Means of five petal
samples + SE. from the ethylene atmosphere. Treatment of flowers with 7.5 ,ul/ L ethylene for 12 h or more resulted in the induction of auto-
catalytic ethylene production (Fig. 1). In an attempt to relate changes in petal gene expression to other ethylene responses, poly(A+) RNA was isolated from petals after exposure to ethylene for various lengths of time and translated in vitro in a rabbit reticulocyte lysate system. In vitro translation products were separated by SDS-PAGE, visualized by fluorography, and quantified densitometrically. By this assay, at least 6 mRNAs were more abundant in ethylene-treated petals as compared to those held in air (Fig. 2). The mol wt of their translation products were 81, 58, 42, 38, 35, and 25 kD. In addition, another group of mRNAs decreased in abundance following exposure to ethylene. The duration of ethylene exposure influenced changes in mRNA populations. Most ethylene-in-
duced mRNAs exhibited a marked increase in abundance following 3 h of ethylene treatment (Fig. 3). The accumulation of mRNAs with 81 and 38 kD translation products was most pronounced. The 81, 42, 38, and 35 kD translation products reached apparent steady state levels following 6 h of ethylene. One mRNA species with an apparent 58 kD translation product showed transient expression, increasing with 6 h of ethylene treatment, then declining with further exposure. Thus, the duration of ethylene exposure was reflected in the abundance of ethylene-induced mRNAs. Effect of Ethylene Action Inhibitors. To further investigate ethylene responses in carnation petals, flowers were treated with the ethylene action inhibitors STS and NBD. These unrelated chemicals have been shown to compete with ethylene for binding sites (1, 20, 22). Relative longevity of petals following treatment with action inhibitors and ethylene is shown in Table I. Both STS and NBD prevented the induction of premature petal senescence by 7.5 ,ul/L ethylene. Flowers treated with STS exhibited increased longevity over NBD-treated and control flowers. This is likely due to the continued presence of STS following ethylene treatment, thus preventing the induction of senescence by endogenous ethylene. In contrast, NBD is applied as a gas only during the period of ethylene exposure and therefore its effects are lost following the return of flowers to air. The effect of action inhibitors on the capacity of exogenous ethylene to induce autocatalytic ethylene production was determined (Table I). Treatment with STS and NBD reduced autocatalytic ethylene production following ethylene exposure. Of the two inhibitors tested, NBD was more effective in preventing the induction of
ethylene biosynthesis by ethylene. The effects of ethylene action inhibitors were also reflected in
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FIG. 2. Fluorograph of carnation of petal poly(A+) RNA in vitro translation products separated by SDS-PAGE (12.5%). Poly(A+) RNA was extracted from petals isolated from flowers following exposure to 7.5 ,ul/L ethylene for various durations. The left lane ( mRNA) contains 35S-met labeled rabbit reticulocyte lysate translation products in the absence of added carnation mRNA. Ethylene-induced polypeptides are indicated by arrows in kD. -
the population of mRNAs (Fig. 4). Treatment with both STS and NBD reduced or prevented the accumulation of ethyleneinduced mRNAs in response to ethylene exposure (Fig. 5). As was the case with autocatalytic ethylene production, NBD was more effective at preventing these changes. These results indicate a relationship exists between ethylene responses such as autocatalytic ethylene induction and gene expression. Ethylene Responsiveness during Petal Development. Carnation petals change in their responsiveness to ethylene during aging such that petals from young flowers require greater doses of exogenous ethylene to induce premature senescence as compared to petals from more mature flowers (13, 15). We followed this change in responsiveness by determining the capacity of exogenous ethylene to induce autocatalytic ethylene production and changes in gene expression at the following stages of development: stage 1, petals emerged 10 mm from calyx; stage 2, petals separated and forming a 300 angle with respect to the axis of the calyx; and stage 3, fully open flowers with petals forming a 90° angle with respect to the axis of the calyx. Flowers were exposed to 7.5 ,Il/L ethylene for 12 h. An increase in flower age was associated with an increase in the capacity for ethylene to induce autocatalytic ethylene production (Table II). Very young flowers
ETHYLENE EXPOSURE (h) FIG. 3. Abundance of major in vitro translation products from carnation petal poly(A+) RNA in response to ethylene exposure. (0), 81 kD; (A), 58 kD; (LI), 42 kD; (0), 38 kD; and (A) 35 kD translation products, respectively. Results were obtained by scanning the fluorograph in Figure 2 densitometrically. Peaks were integrated and peak areas are presented.
Table I. Effect of Ethylene Action Inhibitors on Ethylene-Induced Petal Senescence and Ethylene Production with 4 mm Ag+ as STS or 2500 uIl/L NBD then treated Flowers were exposed to 7.5 uIl/L ethylene for 12 h. Petal longevity of five separate flowers was determined following ethylene exposure. Petals were considered senescent when wilting and necrosis occurred. Following ethylene exposure, petals were equilibrated in air for 15 min then enclosed in serum vials for ethylene production measurements. Means of five petal samples ± SE. Treatment Petal Longevity (d) Ethylene Production nl g fresh wt- I h-' 0.5 ± 0.1 Air 6.4 - 0.9 62.4 + 6.1 1.3 + 0.2 C2H4 12.0a 5.1 ± 1.4 STS + C2H4 0.6 ± 0.2 NBD + C2H4 6.1 ± 0.7 a Experiment terminated after 12 d at which time STS-treated flowers exhibited no evidence of the onset of senescence.
(stage 1) showed no evidence of induction of ethylene production following a 12 h exposure to 7.5 ,AIL ethylene. Further evidence for this increase in responsiveness to ethylene was seen at the level of gene expression. The accumulation of most ethyleneinduced mRNAs increased with petal age (Figs. 6 and 7). Indeed, petals from stage 1 flowers did not accumulate messages for the 58, 38, or 25 kD proteins and accumulated other ethylene-induced messages at very reduced levels as compared to more mature petals in response to ethylene exposure. One ethyleneinduced message with a 42 kD translation product showed little change in abundance during petal maturation in response to ethylene treatment.
DISCUSSION Ethylene is a pleiotropic plant growth modulator which is known to initiate many developmentally coordinated programs such as abscission (19), fruit ripening (6, 12, 24), and petal senescence (10). There is growing evidence that in many cases ethylene exposure results in changes in gene expression (2, 8, 12, 16). For example, ethylene has been shown to regulate the expression of several genes in ripening fruit (12, 24). The results presented here indicate that ethylene also modulates gene expression in
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FIG. 4. Fluorograph of carnation petal poly (A+) RNA translated in vitro and separated by SDS-PAGE (12.5%). Flowers were treated with 4 mM Ag+ as STS prior to or 2500 ,ul/L NBD during exposure to 7.5 ,ul/ L ethylene for 12 h. The left lane ( - mRNA) contains 35S-met labeled rabbit reticulocyte lysate in the absence of added carnation mRNA. Ethylene-induced polypeptides are indicated by arrows in kD.
carnation petals. Furthermore, most of the ethylene-induced mRNAs are similar to those which were previously shown to accumulate during natural senescence concomitant with the ethylene climacteric as estimated by the molecular weights of their in vitro translation products (27). The ethylene-induced changes in gene expression in mature, presenescent flower petals were rapid with many mRNAs showing evidence of accumulation following 3 h of 7.5 ,ul/L ethylene exposure (Fig. 3). In contrast, the expression of several ethylene-regulated fruit ripening genes requires prolonged exposure to ethylene (24). However, induction of gene expression following an ethylene exposure of 0.5 h has been reported in tomato fruits (12). Of critical importance in understanding the mode of ethylene's action in the regulation of petal senescence is to determine how its effects are partitioned. To this end, we have attempted to relate the temporal development of several ethylene responses in carnation petals. One of the earliest responses to ethylene in carnation petals is the induction of petal in-rolling (10, 13). Indeed, petal in-rolling was evident following 6 h of ethylene exposure and occurred prior to the induction of autocatalytic ethylene production. This in-rolling was reversible following removal of flowers to an ethylene-free atmosphere. Reversibility of petal in-rolling has been previously reported (13). In spite of the reversible nature of in-rolling and the lack of autocatalytic ethylene production, these petals senesced prior to the air-treated controls
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