ethylene-induced gene expression in carnation … · ethylene-induced gene expression in carnation...

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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 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, presenes- cent flowers to 7.5 microliters per liter ethylene for at least 6 hours induced petal in-rolling and premature senescence. Autocatalytic ethylene pro- duction 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 poly- adenylated RNA. At least 6 mRNAs accumulated following ethylene ex- posure. 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. Eth- ylene 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 in- dicate that changes in petal physiology resulting from exposure to ethylene may be the result of rapid changes in gene expression. The senescence of flower petals is often associated with in- creased 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 reg- ulation 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). Further- more, 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 Ex- periment Station. K. A. L. was supported in part by a David Ross Fellowship from Purdue University. events. Little is known about the sequence of events linking ethylene binding to petal senescence. There is increasing evi- dence that responses to ethylene in other tissues are associated with the expression of specific genes (2, 8, 12, 16, 17, 19). Eth- ylene 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 in- duction 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 re- sponses after exposure to exogenous ethylene. Furthermore, we relate these changes to the development of tissue responsiveness to ethylene with increasing age. MATERIALS AND METHODS Plant Material. Carnation (Dianthus caryophyllus L cv White Sim) flowers were harvested from plants grown under green- house conditions as previously described (27). Flowers were har- vested 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 with- out 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 evapo- ration, 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. 498

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Page 1: Ethylene-Induced Gene Expression in Carnation … · Ethylene-Induced Gene Expression in Carnation Petals' RELATIONSHIP TO AUTOCATALYTIC ETHYLENE PRODUCTION AND SENESCENCE ... [35S]methionine

Plant Physiol. (1988) 87, 498-5030032-0889/88/87/0498/06/$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. LAWTONDepartment of Horticulture, Purdue University, West Lafayette, Indiana 47907

ABSTRACT

Exposure of carnation (Dianthus caryophyllus L.) flowers to ethyleneevokes the developmental program of petal senescence. The temporalrelationship of several aspects of this developmental program followingtreatment with ethylene was investigated. Exposure of mature, presenes-cent flowers to 7.5 microliters per liter ethylene for at least 6 hours inducedpetal in-rolling and premature senescence. Autocatalytic ethylene pro-duction was induced in petals following treatment with ethylene for 12 ormore hours. A number of changes in mRNA populations were noted inresponse to ethylene, as determined by in vitro translation of petal poly-adenylated RNA. At least 6 mRNAs accumulated following ethylene ex-posure. The molecular weights of their in vitro translation products were81, 58, 42, 38, 35, and 25 kilodaltons. Significant increases in abundanceof most mRNAs were observed 3 hours following ethylene exposure. Eth-ylene exposure resulted in decreased abundance of another group of mRNAs.Treatment of flowers with competitive inhibitors of ethylene action largelyprevented the induction of these ethylene responses in petals. An increasein flower age was accompanied by an increase in the capacity for ethyleneto induce petal in-rolling, autocatalytic ethylene production, and changesin mRNA populations suggesting that these responses are regulated byboth sensitivity to ethylene and ethylene concentration. These results in-dicate that changes in petal physiology resulting from exposure to ethylenemay be the result of rapid changes in gene expression.

The senescence of flower petals is often associated with in-creased production of the phytohormone ethylene (4, 10, 13, 14,29). In carnations, it is well established that this climacteric risein ethylene plays an important role in the coordination and reg-ulation of petal senescence (4, 10, 15). Treatment of flowers withinhibitors of ethylene biosynthesis or action has been shown todelay the onset of petal senescence (4, 21, 25, 26, 29). Further-more, exposure of preclimacteric flowers to exogenous ethylenehastens the onset of petal senescence and induces autocatalyticethylene 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 ethylenebinding in carnation petals. Of further interest in carnation isthat the responsiveness of petal tissue to ethylene increases withage (10, 13, 15). This increase in sensitivity to ethylene was notlinked to an increase in the concentration of an ethylene receptorsince ethylene binding capacity decreased with petal age (3). Theengagement of ethylene responses could be regulated beyondinitial binding at specific points in the chain of signal-transduction

' Journal paper No. 11,332 of the Purdue University Agricultural Ex-periment Station. K. A. L. was supported in part by a David RossFellowship from Purdue University.

events. Little is known about the sequence of events linkingethylene binding to petal senescence. There is increasing evi-dence that responses to ethylene in other tissues are associatedwith the expression of specific genes (2, 8, 12, 16, 17, 19). Eth-ylene regulation of specific mRNAs has been demonstrated inripening tomato (12) and avocado (24) fruits. Two of these mRNAsencode the proteins cellulase (5) and polygalacturonase (7), whichplay 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 proteinand mRNA populations (27). The ethylene climacteric appearedto be a transition period in relation to these changes. Inhibitorsof protein synthesis have been shown to interfere with the in-duction of petal senescence by ethylene (30). The onset of petalsenescence in Hibiscus rosa-sinensis was found to be associatedwith a transient increase in protein synthesis and a change in thepatterns of proteins synthesized in vivo (28). Taken together,these results indicate petal senescence is regulated at the levelof transcription and/or translation.

In the present work, we examine the relationship betweenautocatalytic ethylene production, petal senescence, and mRNApopulations by following the temporal development of these re-sponses after exposure to exogenous ethylene. Furthermore, werelate these changes to the development of tissue responsivenessto ethylene with increasing age.

MATERIALS AND METHODSPlant Material. Carnation (Dianthus caryophyllus L cv White

Sim) flowers were harvested from plants grown under green-house conditions as previously described (27). Flowers were har-vested at anthesis when outer petals were reflexed at 900 anglesto the axis of the calyx except where otherwise noted. Stemswere cut to 10 cm, placed in distilled water, and held in thelaboratory.Chemical Treatment. Flowers were placed in a 20 L container

through which humidified air and ethylene (7.5 ,ul/L) were passedat 500 ml/min. In other experiments where flowers were treatedwith ethylene action inhibitors, flowers were enclosed in 2.5 Ljars, and ethylene was injected to a final concentration of 7.5,u/L. Saturated KOH was placed in the jar with a paper wick toabsorb evolved CO2. Control flowers were enclosed in jars with-out added ethylene. Flowers were treated with 4 mM Ag+ as theanionic complex with sodium thiosulfate (1:4 molar ratio of silvernitrate to sodium thiosulfate) for 1 h then transferred to waterfor 24 h prior to the initiation of ethylene treatment. In anotherexperiment, flowers held in water were placed in jars, and NBD2injected onto a filter paper hung in the jar to facilitate evapo-ration, yielding a final concentration of 2,500 ,ul/L prior to theaddition 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 andequilibrated with air for 15 min following ethylene treatmentafter which 0.5 g of petals were placed in 25 ml serum vials. Thevials were capped, and ethylene was allowed to accumulate for0.5 h, after which a gas sample was removed and analyzed forethylene by GC as previously described (29). The remainingpetals were frozen in liquid N2 immediately following ethyleneexposure and stored at -80° C until subsequent extraction ofRNA.RNA Extraction and Poly(A+), RNA Isolation. RNA was ex-

tracted by a modification of the method of Grierson and Covey(9). Briefly, 10 g of frozen petal tissue were powdered underliquid N2 and were homogenized in equal volumes (50 ml) ofphenol and extraction buffer containing 50 mM Tris-HCl (pH8.4), 5% phenol, 6% sodium p-aminosalicylate, 1% sodium tri-isopropylnaphthalenesulfate, 1% (v/v) 83-mercaptoethanol, and10 mM ribonucleoside vanadyl complexes. Following phase sep-aration and reextraction of the aqueous phase with an equalvolume of phenol:chloroform:isoamyl alcohol (25:24:1, v/v), nu-cleic acids were precipitated by the addition of sodium acetateto 0.2 M and 2 volumes of ethanol at - 20° C overnight. Theprecipitate was resuspended in sterile water and high mol RNAreprecipitated 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 weredissolved in sterile water and poly(A+)RNA isolated by chro-matography over oligo(dT)-cellulose. Samples were scanned witha Beckman DU-8 spectrophotometer from 220 to 320 nm. RNAcontent was calculated assuming an extinction coefficient of 25A260 units/mg RNA.

In Vitro Translation. Poly(A+) RNA was translated in vitrousing the rabbit reticulocyte lysate system of Pelham and Jackson(18). Three ug poly(A+) RNA were translated in the presenceof 10 ,uCi of L-[35S]methionine (>1000 Ci/mmol, from New Eng-land Nuclear) in a final reaction volume of 30 ,ul. Samples wereincubated at 30° C for 1 h. The reaction was stopped on ice, andthe reaction mixtures were stored at - 200C.

Electrophoresis of in Vitro Translation Products. Samples con-taining equal amounts of TCA-precipitable [35S]methionine werebrought to the concentration of Laemmli's (11) sample bufferand were boiled for 3 min prior to electrophoresis in 12.5%polyacrylamide gels containing 1% SDS (11). Following electro-phoresis gels were fixed in 50% methanol (v/v) and 12.5% aceticacid (v/v) containing 0.025% (w/v) Coomassie blue to visualizeprotein standards. Gels were destained in 40% methanol and10% acetic acid then in 5% methanol and 7% acetic acid. Gelswere equilibrated with 7% acetic acid, impregnated withFluoroHance (Research Products International Corp., MountProspect, IL), vacuum-dried onto Whatman 3MM filter paper,and exposed to Kodak XAR-5 film at - 80° C. The resultingfluorographs were scanned densitometrically with a BeckmanDU-8 spectrophotometer.

RESULTSEffects of Ethylene Exposure Duration. Flowers harvested at

anthesis and exposed to 7.5 ,uIIL ethylene for 6 or more h showedevidence of initiation of senescence (Fig. 1). A brief ethyleneexposure of 3 h did not induce premature petal senescence. Petalin-rolling, an early indication of the onset of senescence (10),was exhibited by flower petals following an ethylene treatmentof at least 6 h (data not shown). When flowers were removedfrom the ethylene atmosphere after 6 h this in-rolling was re-versed. However, petals exposed to ethylene for more than 12h did not recover from in-rolling following transfer to air andwere fully wilted after 24 h (Fig. 1). A response to exogenousethylene exposure in many flowers and climateric fruits is theinduction of autocatalytic ethylene production (10, 12, 13, 24).Ethylene production by petals was measured following removal

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Ethylene exposure (h)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. Followingethylene treatment, petals were isolated and equilibrated in air for 15min, and their ethylene production was measured. Means of five petalsamples + 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 toother ethylene responses, poly(A+ ) RNA was isolated from pet-als after exposure to ethylene for various lengths of time andtranslated in vitro in a rabbit reticulocyte lysate system. In vitrotranslation products were separated by SDS-PAGE, visualizedby fluorography, and quantified densitometrically. By this assay,at least 6 mRNAs were more abundant in ethylene-treated petalsas compared to those held in air (Fig. 2). The mol wt of theirtranslation products were 81, 58, 42, 38, 35, and 25 kD. Inaddition, another group of mRNAs decreased in abundance fol-lowing exposure to ethylene. The duration of ethylene exposureinfluenced changes in mRNA populations. Most ethylene-in-duced mRNAs exhibited a marked increase in abundance fol-lowing 3 h of ethylene treatment (Fig. 3). The accumulation ofmRNAs with 81 and 38 kD translation products was most pro-nounced. The 81, 42, 38, and 35 kD translation products reachedapparent steady state levels following 6 h of ethylene. One mRNAspecies with an apparent 58 kD translation product showed tran-sient expression, increasing with 6 h of ethylene treatment, thendeclining with further exposure. Thus, the duration of ethyleneexposure was reflected in the abundance of ethylene-inducedmRNAs.

Effect of Ethylene Action Inhibitors. To further investigateethylene responses in carnation petals, flowers were treated withthe ethylene action inhibitors STS and NBD. These unrelatedchemicals have been shown to compete with ethylene for bindingsites (1, 20, 22). Relative longevity of petals following treatmentwith action inhibitors and ethylene is shown in Table I. BothSTS and NBD prevented the induction of premature petal se-nescence by 7.5 ,ul/L ethylene. Flowers treated with STS exhib-ited increased longevity over NBD-treated and control flowers.This is likely due to the continued presence of STS followingethylene treatment, thus preventing the induction of senescenceby endogenous ethylene. In contrast, NBD is applied as a gasonly during the period of ethylene exposure and therefore itseffects are lost following the return of flowers to air. The effectof action inhibitors on the capacity of exogenous ethylene toinduce autocatalytic ethylene production was determined (TableI). Treatment with STS and NBD reduced autocatalytic ethyleneproduction following ethylene exposure. Of the two inhibitorstested, NBD was more effective in preventing the induction ofethylene biosynthesis by ethylene.The effects of ethylene action inhibitors were also reflected in

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WOODSON AND LAWTON

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FIG. 2. Fluorograph of carnation of petal poly(A+) RNA in vitrotranslation products separated by SDS-PAGE (12.5%). Poly(A+) RNAwas extracted from petals isolated from flowers following exposure to7.5 ,ul/L ethylene for various durations. The left lane ( - mRNA) contains35S-met labeled rabbit reticulocyte lysate translation products in the ab-sence of added carnation mRNA. Ethylene-induced polypeptides are

indicated by arrows in kD.

the population of mRNAs (Fig. 4). Treatment with both STSand NBD reduced or prevented the accumulation of ethylene-induced mRNAs in response to ethylene exposure (Fig. 5). Aswas the case with autocatalytic ethylene production, NBD wasmore effective at preventing these changes. These results indicatea relationship exists between ethylene responses such as auto-catalytic ethylene induction and gene expression.

Ethylene Responsiveness during Petal Development. Carnationpetals change in their responsiveness to ethylene during agingsuch that petals from young flowers require greater doses ofexogenous ethylene to induce premature senescence as comparedto petals from more mature flowers (13, 15). We followed thischange in responsiveness by determining the capacity of exog-enous ethylene to induce autocatalytic ethylene production andchanges in gene expression at the following stages of develop-ment: stage 1, petals emerged 10 mm from calyx; stage 2, petalsseparated and forming a 300 angle with respect to the axis of thecalyx; and stage 3, fully open flowers with petals forming a 90°angle with respect to the axis of the calyx. Flowers were exposedto 7.5 ,Il/L ethylene for 12 h. An increase in flower age wasassociated with an increase in the capacity for ethylene to induceautocatalytic ethylene production (Table II). Very young flowers

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nation petal poly(A+) RNA in response to ethylene exposure. (0), 81kD; (A), 58 kD; (LI), 42 kD; (0), 38 kD; and (A) 35 kD translationproducts, respectively. Results were obtained by scanning the fluoro-graph in Figure 2 densitometrically. Peaks were integrated and peakareas are presented.

Table I. Effect of Ethylene Action Inhibitors on Ethylene-Induced PetalSenescence and Ethylene Production

Flowers were treated with 4 mm Ag+ as STS or 2500 uIl/L NBD thenexposed to 7.5 uIl/L ethylene for 12 h. Petal longevity of five separateflowers was determined following ethylene exposure. Petals were con-sidered senescent when wilting and necrosis occurred. Following ethyleneexposure, petals were equilibrated in air for 15 min then enclosed inserum vials for ethylene production measurements. Means of five petalsamples ± SE.

Treatment Petal Longevity (d) Ethylene Productionnl g fresh wt- I h-'

Air 6.4 - 0.9 0.5 ± 0.1C2H4 1.3 + 0.2 62.4 + 6.1STS + C2H4 12.0a 5.1 ± 1.4NBD + C2H4 6.1 ± 0.7 0.6 ± 0.2

a Experiment terminated after 12 d at which time STS-treated flowersexhibited no evidence of the onset of senescence.

(stage 1) showed no evidence of induction of ethylene productionfollowing a 12 h exposure to 7.5 ,AIL ethylene. Further evidencefor this increase in responsiveness to ethylene was seen at thelevel of gene expression. The accumulation of most ethylene-induced mRNAs increased with petal age (Figs. 6 and 7). Indeed,petals from stage 1 flowers did not accumulate messages for the58, 38, or 25 kD proteins and accumulated other ethylene-in-duced messages at very reduced levels as compared to moremature petals in response to ethylene exposure. One ethylene-induced message with a 42 kD translation product showed littlechange in abundance during petal maturation in response toethylene treatment.

DISCUSSIONEthylene is a pleiotropic plant growth modulator which is known

to initiate many developmentally coordinated programs such asabscission (19), fruit ripening (6, 12, 24), and petal senescence(10). There is growing evidence that in many cases ethyleneexposure results in changes in gene expression (2, 8, 12, 16). Forexample, ethylene has been shown to regulate the expression ofseveral genes in ripening fruit (12, 24). The results presentedhere indicate that ethylene also modulates gene expression in

500 Plant Physiol. Vol. 87, 1988

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ETHYLENE-REGULATED GENE EXPRESSION AND PETAL SENESCENCE

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FIG. 4. Fluorograph of carnation petal poly (A+) RNA translated invitro and separated by SDS-PAGE (12.5%). Flowers were treated with4 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 labeledrabbit 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-inducedmRNAs are similar to those which were previously shown toaccumulate during natural senescence concomitant with the eth-ylene climacteric as estimated by the molecular weights of theirin vitro translation products (27). The ethylene-induced changesin gene expression in mature, presenescent flower petals wererapid with many mRNAs showing evidence of accumulation fol-lowing 3 h of 7.5 ,ul/L ethylene exposure (Fig. 3). In contrast,the expression of several ethylene-regulated fruit ripening genesrequires prolonged exposure to ethylene (24). However, induc-tion of gene expression following an ethylene exposure of 0.5 hhas 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 howits effects are partitioned. To this end, we have attempted torelate the temporal development of several ethylene responsesin carnation petals. One of the earliest responses to ethylene incarnation petals is the induction of petal in-rolling (10, 13). In-deed, petal in-rolling was evident following 6 h of ethylene ex-posure and occurred prior to the induction of autocatalytic eth-ylene production. This in-rolling was reversible following removalof flowers to an ethylene-free atmosphere. Reversibility of petalin-rolling has been previously reported (13). In spite of the re-versible nature of in-rolling and the lack of autocatalytic ethyleneproduction, these petals senesced prior to the air-treated controls

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Table II. Influence of Stage of Petal Development on Ethylene-InducedAutocatalytic Ethylene Production

Flowers were harvested at the following stages: stage 1, petals emerged10 mm from calyx; stage 2, petals reflexed at 300 angles with respect tothe axis of the calyx; stage 3, petals reflexed at 90° angles with respectto the axis of the calyx. Flowers were exposed to air or 7.5 ,ul/L ethylenefor 12 h. Following ethylene exposure, petals were equilibrated in airfor 15 min then enclosed in serum vials for ethylene production meas-urements. Means of five petal samples ± SE.

Ethylene ProductionStage of Development ArEhln

Air Ethylenenlgfresh wt-I h-'

1 0.9 ± 0.1 0.8 ± 0.22 0.5 ± 0.2 19.2 ± 3.93 0.5 ± 0.1 68.7 ± 8.9

(Fig. 1). Ethylene exposure has been shown to increase the de-velopment of sensitivity to ethylene (10). Therefore, the pre-mature senescence resulting from a relatively brief exposure toethylene may be interpreted as an increase in tissue responsive-ness to ethylene such that the petals senesce in response to lowbasal levels of endogenous ethylene prior to the untreated con-trols. Clearly, one of the earliest events in petals in response toethylene exposure was the accumulation of new mRNAs (Fig.2). In most cases, the abundance of these ethylene-induced mRNAs

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WOODSON AND LAWTON

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sponse to ethylene. Stages are as described in Table II. Results were

obtained by densitometrically scanning the fluorograph in Figure 6. Peakswere integrated and peak areas are presented.

increased with the duration of ethylene exposure and reachedsteady state levels between 6 and 12 h of ethylene treatment (Fig.3). Whether the accumulation of ethylene-induced mRNAs isthe result of increased transcription, changes in mRNA pro-cessing, and/or mRNA stability is not known at the present time.However, these results indicate that changes in petal physiologyin response to ethylene may be the result of rapid alterations ingene expression.The evocation of ethylene responses are thought to be me-

diated initially through the binding of ethylene to specific re-ceptors (23). Not surprisingly, therefore, treatment of flowerswith the competitive inhibitors of ethylene binding, STS, andNBD largely prevented ethylene-induced responses in carnationpetals (Table I; Figs. 4 and 5). The simplest method of regulatingdevelopmental responses to ethylene would be at the level ofperception (i.e. binding). However, given the diversity of eth-ylene responses, it is more likely that ethylene-induced eventsare controlled in some cases beyond ethylene binding.

In many cases, the responsiveness to ethylene is develop-mentally regulated (10, 12, 19). In carnations, petals from youngflowers are less sensitive to ethylene as compared to more maturepetals (10). This increased responsiveness in older petals wasreflected in the capacity for ethylene to induce ethylene pro-duction, petal senescence, and changes in mRNA populations.While very young flowers exhibited no petal in-rolling, ethyleneproduction, or premature senescence following ethylene expo-sure, they did accumulate, although at a reduced level, the 81,42, and 35 kD in vitro translation product (Fig. 7). Interestingly,an 81 kD translation product was previously shown to accumulateimmediately prior to the increase in ethylene production asso-

ciated with petal senescence (27). It is possible that the inductionof this mRNA by ethylene during fower development occurs inresponse to ethylene, not as a result of increased ethylene pro-duction but because of increased ethylene sensitivity. The inhi-bition of the appearance of the 81 kD message by ethylene actioninhibitors is consistent with this hypothesis. Another mRNA witha 42 kD translation product accumulated to similar levels inresponse to ethylene exposure regardless of tissue age. This in-dicates the change in tissue sensitvity to ethylene is regulated ata point beyond a change in overall capacity to respond to ethylene(i.e. ethylene binding). Perhaps these changes are associated withbranch pathways off the main signal transduction pathway. Theinduction of the 58 kD message by ethylene was only apparentin older tissue which exhibited evidence of the onset of petalsenescence and ethylene production in response to ethylene.While the function of this polypeptide is unknown, the patternof its expression is suggestive of a functional role in senescenceor ethylene biosynthesis.

Acknowledgments-We thank Drs. R. A. Bressan, J. H. Cherry. P. B. Golds-brough, and A. K. Handa for critical reading of the manuscript and Dr. D. Kuhnfor the rabbit reticulocyte lysate used in these experiments.

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