Plant Physiol. (1980) 66, 281-2850032-0889/80/66/028 1/05/$00.00/0

Biosynthesis of Wound Ethylene'Received for publication January 2, 1980 and in revised form March 11, 1980

YEONG-BIAU YU AND SHANG FA YANGDepartment of Vegetable Crops, University of Calfornia, Davis, California 95616

ABSTRACT

Untreated mung bean hypocotyls produced very little C2H4 but, upontreatment with 10 millimolar Cu2+ or 10 millimolar Cu21 + 10 millimolarCa2+, C2H4 production increased 20- and 40-fold, respectively, within 6hours. This increase in C2H4 production was preceded and paraleled by anincrease in 1-aminocyclopropanecarboxylic acid (ACC) content, but thelevel of S-adenosylmethionine (SAM) was unaffected, suggesting that theconversion of SAM to ACC is a key reaction in the production of wound-induced C2H4. This view was further supported by the observation thatapplication of aminethoxyvinylglycine, a known inhibitor of the conversionof SAM to ACC, eliminated the increases in ACC formation and in C2H4production. A significant increase in C2H4 production was observed in thealbedo tissue of orange in response to excision, and it was paralleled by anincrease in ACC content. In columeUa tissue of unripe green tomato fruit,massive increases in the C2H4 production rate (from 0 to 12 nanoliters pergram per hour), in ACC content (from 0.05 to 12 nmoles per gram), and inACC synthase activity (from 0 to 6.4 units per miligram protein) occurredduring the 9-hour incubation period folowing excision. Infiltration with 0.1millimolar cycloheximide, an inhibitor of protein synthesis, completelyblocked wound-induced C2H4 production, ACC formation, and developmentof ACC synthase activity. These data indicate that wounding induces thesynthesis of ACC synthase, which is the rate-controlling enzyme in thepathway of C2H4 biosynthesis and, thereby, causes accumulation of ACCand increase in C2H4 production.

Ethylene is an endogenous plant hormone involved in manyphenomena of growth and senescence (1). Normally C2H4 pro-duction from plant tissue is low. In some tissues, particularlyfruits, a massive C2H4 production at the appropriate time is a partof the normal life cycle (1). In others, a large amount of C2H4 isproduced following trauma caused by chemicals, temperatureextremes, waterlogging, drought, radiation, insect damage, diseaseor mechanical wounding (1, 25). C2H4 produced by plants undersuch conditions is referred to as "wound ethylene" or "stressethylene." It has been suggested that stress C2H4 may enable theplant to cope successfully with the trauma. Some physiologicalconsequences of stress C2H4 are recognized. For example, droughtcauses leaves or fruit to increase C2H4 production, which in turnpromotes abscission and thereby reduces water loss (21). StressC2H4, produced in response to physical stress, may play a role inregulating the growth of the seedling as it emerges through soil(10). Mechanical wounding, such as cutting, abrasion, or bruising,has been shown to cause orange, banana, tomato, apple, and otherfruit tissues to produce large amounts of C2H4. This wound C2H4in turn accelerates fruit ripening and may cause loss of fruitquality during transportation and storage (4, 13, 14, 17-20). The

' This work was supported by National Science Foundation Grant PCM78-09278.

characteristics of cutting-induced C2H4 production in etiolatedpea seedling were recently reported by Saltveit and Dilley (22).The biochemistry of C2H4 formation in wounded tissues has

been studied in a number of plant materials. Abeles and Abeles(2) have shown that the application of toxic compounds, such asCuSO4, endothal, or ozone to bean and tobacco leaves caused arapid increase in C2H4 production and in the conversion ofmethionine to C2H4. Cutting or bruising of rib segments frommorning glory flower has resulted in a remarkable increase inC2H4 production (I 1). Although the tissue utilized methionine asthe substrate, a large dilution of methionine was observed in thewounded tissue. Hyodo (13, 14) observed, in albedo tissue excisedfrom Satsuma mandarin, an increase of C2H4 production whichwas closely accompanied by increased conversion of methionineto C2H4 during a period of 30 h after cutting; neither C2H4production nor conversion of methionine to C2H4 was observed inintact fruit. Although it is generally believed that methionineserves as the precursor of stress C2H4, as it does in ripening fruitor in auxin-treated tissue, understanding of the biosynthesis ofstress C2H4 is very limited.Adams and Yang (3) have studied C2H4 biosynthesis in apple

tissue and established the following biosynthetic pathway: methi-onine -- SAM2 -- ACC -* C2H4. The validity of this pathwayhas since been confirmed in other systems, including auxin-in-duced C2H4 production in vegetative tissues (28). The presentinvestigation was conducted to determine the biochemical step atwhich wounding exerts its effect on the stimulation of C2H4production. The systems examined were Cu2+-induced C2H4 pro-duction in mung bean hypocotyls and cutting-induced C2H4 pro-duction in orange peel and in unripe green tomato.

MATERIALS AND METHODS

Plant Materials. Etiolated mung bean (Viana radiata [L.] Wil-ezek var. Berkeu) hypocotyls, discs of albedo from Valenciaorange (Citrus sinensis [L.] Osbeck cv. "Valencia") peel, and unripetomato (Lycopersicon esculentum Mill.) fruit were used in thisexperiment.Dry seeds of mung bean obtained from Ekroat Co., Oklahoma,

were grown in Vermiculite for 3.5 days in darkness at 25 C asdescribed previously (28). Lots of 20 2-cm-long hypocotyl seg-ments, cut at I and 3 cm below the hook, were incubated in 5 mlof medium containing 2% sucrose, 50 jig/ml chloramphenicol, and50 mM phosphate buffer (pH 5.3) in a 50-ml Erlenmeyer flask.When indicated, 10 mi Cu2+, 10 mi Ca2+, 25 /LM AVG, and 1.5MCi L-[3-'4Cjmethionine (49 ,iCi/umol) were included. Each flaskwas flushed with air, sealed with a rubber serum cap, and incu-bated at 27 C. At the indicated time, C2H4 in the gas phase wasdetermined and the amount of SAM and ACC in the tissue wasassayed.

2Abbreviations: SAM: S-adenosylmethionine. ACC: I-aminocyclopro-pane-l-carboxylic acid; AVG: aminoethoxyvinylglycine [2-amino-4-(2'-aminoethoxy)-trans-3-butenoic acid]; CHI: cycloheximide; Hepps: 4-(2-hydroxyethyl)- I -piperazinepropanesulfonic acid.

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Plant Physiol. Vol. 66, 1980

Valencia orange albedo discs (1 cm in diameter and about 3mm thick) were prepared by excising the orange peel with a corkborer and cutting off the flavedo tissue with a razor blade. Sixdiscs, weighing 1.6 g, were placed in a 125-ml Erlenmeyer flask;the flask was sealed with a rubber serum cap and incubated at25 C. Every 16 h, one flask was removed to determine C2H4production and ACC content. All flasks were flushed with freshair and sealed again every 16 h.The inner wall of the pericarp (columella) of unripe green

tomato was excised as discs about 0.5 cm in diameter and 0.2 cmin thickness. About 6 g tissue then was placed in a 50-ml flask andincubated at 25 C. C2H4 production, ACC content, and ACCsynthase activity were measured every 3 h. At 30 min beforeindicated time, the flask was flushed with air and sealed with arubber serum cap. The amount of C2H4 accumulated during the30-min period was determined. After C2H4 determination, thetissue was homogenized in buffer for the ACC synthase activityassay or in 3% HC104 for assay of the ACC content. To examinethe effect of protein synthesis inhibitor, tomato slices were sub-merged and vacuum-infiltrated in a solution containing 5% glyc-erol with or without 100 ,UM CHI for 5 min. After blotting on filterpaper, the slices were incubated at 25 C for 9 h. At the end of theincubation period, C2H4 production, ACC content, and ACCsynthase activity were determined.

Chemicals. L-[3-14C]Methionine was purchased from ResearchProducts International; SAM was from Boehringer; pyridoxalphosphate, DTT, and ACC were from Calbiochem; Hepps wasfrom Sigma; and protein standard was from Bio-Rad. AVG wasa gift from J. P. Scannel.

Determination of C2H4. A l-ml gas sample was withdrawn fromthe head space of the flask with a hypodermic syringe, and C2H4was assayed by a gas chromatograph equipped with an aluminacolumn and a flame ionization detector. Radioactive C2H4 wasabsorbed in 250 ,ul 0.25 M Hg(CI04)2 reagent and assayed in aliquid scintillation counter as described previously (28).

Determination of SAM. Mung bean hypocotyls which had beenincubated for 6 h in a medium containing L-[3-'4Cjmethioninewere homogenized and extracted with 6% HC104. After centrifu-gation, the supernatant was neutralized to pH 4.5 at 0 C by slowlyadding solid KHCO3. The precipitated salt was discarded and thesupernatant was passed through an ion-exchange resin (Bio-Rex70, H' form) column (23). The column was washed with wateruntil neutral, and the effluent was used to determine the total andradioactive ACC content. [14CJSAM was eluted from the Bio-Rexcolumn with 0.1 N HCI. This eluate was lyophilized and dissolvedin 1O0o acetic acid. The identification of radioactive SAM wascarried out by paper co-electrophoresis with authentic SAM at pH2.2 (10%1o acetic acid). SAM was visualized under UV light. Radio-activity on the paper was quantitated with a radioscanner. Toquantitate total SAM based on UV absorption, it is essential thatthe fraction is free from S-adenosylhomocysteine and other inter-fering UV-absorbing material. The method of Glazer and Peales(9) was followed. The HC104 extract of separate samples contain-ing no radioactivity was mixed with a known amount of [14CJ-SAM as internal standard to monitor recovery during the purifi-cation procedures. After addition of solid KHCO3 to pH 4.5, thesupernatant was passed through a sulfopropyl (SP)-Sephadexcolumn previously equilibrated with 10 mm HCI. After washingthe column with 150 mm HCI until no absorption was detectableat 260 nm, SAM was eluted with 500 mM HCI and the eluate wascollected in fractions of 3 ml. The fractions which containedradioactivity were pooled. The concentration of SAM was deter-mined spectrophotometrically, assuming a molar adsorptivity of15,000 cm-1 M-1 at 260 nm. Recovery based on the radioactivityof SAM ranged from 55 to 65%.

Determination of ACC. The HC104 extract, or the effluent fromthe Bio-Rex column, as described above, was adjusted to neutral

with I N NaOH and the ACC content was determined by themethod of Lizada and Yang (16), based on the conversion ofACCto C2H4 with NaOCl reagent. The amount of C2H4 liberated wasdetermined by GC. The efficiency of the conversion of ACC toC2H4 was estimated by adding a known amount of authentic ACCas internal standard to a separate sample, which then was degradedto C2H4 by the same method. The yield was usually between 70and 85%. The amount of ACC was calculated as the quotient ofC2H4 liberated and the conversion efficiency. For the determina-tion of [14C]ACC, 250 nmol authentic ACC was added to theeffluent obtained from the Bio-Rex 70 column, and the solutionwas passed through a cation-exchange column (Dowex 50, H+).Amino acids, including ACC, then were eluted with 2 N NH40H.After concentration, the residue was chromatographed on What-man 3MM paper and developed with 1-butanol-acetic acid-H20(4:1:1, v/v). The radioactive region corresponding to ACC waseluted with water and then degraded to C2H4 by the method ofLizada and Yang (16). The C2H4 liberated was transferred to anevacuated 25-ml scintillation vial as described previously (28). A0.5-ml gas sample was withdrawn from the vial to determine theamount of C2H4. The remainder of C2H4 was absorbed byHg(C104)2 and radioactivity was determined by liquid scintilla-tion. Radioactive ACC (nCi) was calculated as the specific radio-activity of C2H4 (nCi/nmol) x 250 nmol.

Assay of ACC Synthase. Preparation and assay of ACC syn-thase were similar to those described previously (7, 26). Tomatoslices were homogenized in 50 mm Hepps buffer (pH 8.5) contain-ing 0.5 uM pyridoxal phosphate and 4 mm DTT. The extract wasdialyzed overnight with two changes of solution containing 2 mMHepps (pH 8.5), 0.5 ylM pyridoxal phosphate, and 0.1 mm DTT.ACC synthase activity was determined in a reaction mixturecontaining 0.4 ml enzyme solution, 30,umol Hepps buffer, and 70nmol SAM in a total volume of 0.6 ml. After incubation for 3 hat 30 C, the ACC formed was assayed by the method of Lizadaand Yang (16). One unit of enzyme activity is defined as theamount of enzyme which catalyzes the formation of I nmol ACCin 3 h under the conditions specified above. The content of proteinwas determined by the Bradford method (5) using Bio-Rad proteinstandard II as standard.

RESULTS AND DISCUSSION

Cu2' has been shown to induce wound C2H4 in a number oftissues (1, 7). Lau and Yang (15) reported that in mung beanhypocotyls Cu2+ stimulated C2H4 production with a lag period ofabout 2 h. When Cu2+ was applied with Ca2+, a remarkablesynergistic stimulation of C2H4 production was observed. We haveexamined the changes in ACC content in relation to C2H4 pro-duction in mung bean hypocotyls. The time course of ACCaccumulation in relation to C2H4 production is shown in Figure1. The control tissue contained very little ACC and produced verylittle C2H4 during the course of incubation. Treatment with 10 mmCu2+ greatly increased both C2H4 production and ACC content,and the increase of C2H4 production was synergistically enhancedby the addition of 10 mm Ca2+. The mechanism of the synergismwas not investigated. Trace studies by Lau and Yang ( 15) indicatedthat Cu2' enhanced the uptake of Ca2+, and this increase in Ca2+uptake paralleled the enhancement of C2H4 production. Thepresent observation that the increase in C2H4 production waspreceded and paralleled by the increase of ACC content suggeststhat the induction ofACC formation may play an important rolein the production of wound C2H4 (Fig. 1; Table I).

Since ACC is biosynthesized from methionine via SAM (3), wehave studied the effect of Cu2" or Cu2+ + Ca2+ on the conversionof methionine to SAM, ACC, and C2H4, on the level of endoge-nous SAM and ACC, and on C2H4 production in mung beanhypocotyls. The results are summarized in Table I. Whereas Cu2+or Cu2+ + Ca2+ treatment greatly stimulated both C2H4 and ACC

282 YU AND YANG

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BIOSYNTHESIS OF WOUND ETHYLENE

Table I. Influence of Cu2+ or Cu2+ + Ca2+ on Conversion of Methionine to SAM, A CC, and C2H4, on SAM andACC Content, and on C2H4 Production in Mung Bean Hypocotyl

Twenty hypocotyl segments (about 1.4 g) were incubated in 5 ml medium containing 50 mm phosphate buffer(pH 5.3), 2% sucrose, 50 lig/ml chloramphenicol, 10 mM Cu2+, 10 mm Ca2+ as indicated, and 1.5 uCi (30 nmol)of L-l3-'4Clmethionine. At the end of6-h incubation, total and radioactive C2H4 were determined. The hypocotylsthen were homogenized for assay of [14C]SAM, I'4C]ACC, and total ACC. Total SAM was determined in separatesamples. The uptake of [14CImethionine during the incubation period for control, Cu2", and Cu2+ + Ca2+treatments was 18, 13, and 13%, respectively.

Treatment C2H4 ACC SAM

nmol nCi nCi/nmol nmol nCi nCi/nmol nmol nCi nCi/nmolControl 0.2 0.4 2 0.4 0.24 0.6 30 17 0.6Cu2+ 8.4 0.4 0.05 12.9 0.47 0.04 33 1.1 0.03Cu2+ + Ca2+ 15.6 0.8 0.05 21.3 0.64 0.03 33 1.4 0.04

400

Xt200 0LP Ci~~~~/2+

100

' Control0

14-'2 A

212 A*4 +C2+

0E

8AL) -0-

/ Cu2~6/

4 /

A'

2 Control

O t4 i I---0 2 4 6 8

INCUBATION TIME (h)

FIG. 1. Time course of C2H4 production (A) and of changes in ACCcontent (B) of mung bean hypocotyls treated with 10 mM Cu21 or 10 mMCu2+ + 10 mM Ca2+.

production, the incorporation of label from labeled methionineinto ACC and into C2H4 was not greatly affected. Cu2+ or Cu2++ Ca2+ treatment greatly inhibited the conversion of labeledmethionine to labeled SAM but did not alter the quantity of totalSAM (Table I). When the conversions of methionine to C2H4,ACC, and SAM were compared with respect to specific radioac-tivity, it was apparent that all were significantly inhibited by eitherCu2+ or Cu2+ + Ca2+. These data indicate that the increases in thequantity of total SAM, ACC, and C2H4 in response to Cu2+treatment did not parallel the change in the incorporation of labelfrom exogenous methionine into these metabolites. These resultsare in contrast with the C2H4 production systems induced by IAA(28) or Ca2+ + cytokinin (24), in which the exogenous methioninewas extensively incorporated into ACC and C2H4 in parallel withthe increase in the production of ACC and C2H4. These resultsindicate that the failure to stimulate the incorporation of exoge-

nously supplied methionine into ACC and C2H4 was due not toCa2" but to Cu2+.One of the distinctive characteristics of C2H4 production from

methionine is that it is inhibited by AVG, which blocks theconversion of SAM to ACC (3, 6). As shown in Table II, AVGstrongly inhibited Cu2+-induced ACC production and C2H4 pro-duction, although the inhibition was not complete. There is noevidence suggesting that ACC and C2H4 could be formed in thesesystems from sources other than methionine. Since Cu2+ inhibitedthe incorporation of exogenous methionine into SAM, it mustexert its effect at, or prior to, the conversion of methionine toSAM. Two explanations are possible which account for the lowconversion efficiency (specific radioactivity of ["4CJC2H4 pro-duced/specific radioactivity of ['4C]methionine administered) inthe wounded tissue: (a) Cu2+ caused a disruption in the normalcompartmentation and, thus, increased the metabolic pool size ofendogenous methionine and (b) Cu2+ interfered with the utiliza-tion of exogenous methionine. Biosynthesis of wound C2H4 in ribsegments of morning glory flower buds following excision wasstudied by Hanson and Kende (I 1). Although wound C2H4 pro-duction was inhibited by AVG, the specific radioactivity of C2H4evolved from wounded tissue was lower than that from un-wounded tissue. They therefore suggested that wounding causeda temporary breakdown in compartmentation between two me-thionine pools. If a large pool of methionine was present in thevacuole and if Cu2+ caused a breakdown of the tonoplast, then alarge dilution of radioactive methionine by endogenous methio-nine would have occurred in the cytoplasm. It should be notedthat wounding resulted in ACC accumulation (Table I). Thus, inwounded tissues the incorporation of labeled methionine intoC2H4 would be greatly diluted by the endogenous ACC accumu-lated, even if there is no breakdown in compartmentation ofmethionine pools. The other explanation (b) is that Cu2+ mayhave chelated the administered ["4C]methionine, decreasing itsavailability or mobilization and, thus, greatly reducing its futhermetabolism into SAM, ACC, and C2H4. The formation of a

Table II. Effect ofA VG on Cu2+-induced C2H4 Production and onFormation ofACC in Mung Bean Hypocotyl

The incubation media and conditions were as those described in TableI except that L-[3-14CJmethionine was not included. The AVG concentra-tion was 25 tM. C2H4 production and ACC content were determined atthe end of a 6-h incubation period.

Treatment C2H4 ACC

nl nmolControl 7.8 0.4Cu2+ 180 11.8Cu2+ + AVG 53 2.6Cu2+ + Ca2+ 331 15.9Cu2+ + Ca2+ + AVG 103 3.7

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284 YU AND YANG

Cu2+-amino acid chelate complex has been recognized (8). Thisexplanation might also account for the incomplete inhibition byAVG of Cu2+- or Cu2+ + Ca2+-induced C2H4 and ACC produc-tion. In these systems, AVG at 25 1&m inhibited C2H4 productionand ACC formation only about 75% (Table II), whereas at thissame or lower concentration AVG almost completely blocked theauxin-induced (27, 28) and Ca2+ + cytokinin-induced C2H4 pro-duction (Y. Yu, unpublished results). This was probably due to acomplex formation between AVG, an amino acid, and Cu2+,resulting in decreased reactivity of AVG. When the influence ofCu2+ on the level of intermediates in the biosynthetic sequencewas examined, it was revealed that Cu2+ did not affect the level ofSAM but did cause a remarkable increase in the ACC and C2H4production rates. These data clearly indicate that the particularstep which resulted in the increased C2H4 production was theenhanced conversion of SAM to ACC.The stress-C2H4 production caused by Cu2+ or other toxic

chemicals has been shown to be effectively blocked by the appli-cation of CHI (2). This was taken to suggest that the inductionrequires synthesis of a new enzyme (2). If so, it is reasonable toassume that Cu21 stimulates C2H4 production by inducing thesynthesis of ACC synthase. The conversion of SAM to ACC hasbeen identified as the rate-limiting step in the biosynthesis ofC2H4in vegetative tissue as well as in fruit tissue (24, 26, 28), and auxin-induced synthesis of ACC synthase has been shown to be the keyreaction for auxin-induced C2H4 production (28). The inductionof C2H4 production by Cu2+ treatment is thus similar, if notidentical, to the induction by auxin.

Intact citrus fruits produce very little C2H4 unless subjected tosome form of stress, such as chemical wounding (7) or cutting (13,14). Chemically induced stress-C2H4 production is now beingcommercially exploited to enhance fruit abscission for the me-chanical harvest of oranges. The time courses of C2H4 productionand of changes in ACC content in excised Valencia orange albedoare shown in Figure 2. Both the ACC content and the C2H4production rate increased progressively during the incubationperiod, and a very close relationship between the two quantitiesis apparent. This result is similar to those obtained with the Cu2+-treated mung bean hypocotyls described above, and with unripetomato slices which will be discussed subsequently. The close

I

0

cwv

9.0

7.5

6.0 7T0'EC

4.5 -

004

3.0

i.5

*0

INCUBATION TIME (h)FIG. 2. Comparison of changes in C2H4 production rate and in ACC

content of excised Valencia orange albedo. Each C2H4 production valuerepresents the accumulation during the individual 16-h incubation period.

15

12L

99C

.-6ItN

03

0

7-8

.

E

4I

.-__ 2

cn

04 O

I

Plant Physiol. Vol. 66, 1980

AC2H4

0_ - ° ACC

B

A

A'-,~~A

0 . . 9

0 3 6 9

8

6-f

4 EC

0

2 4

0

INCUBATION TIME (h)

FIG. 3. Time courses of C2H4 production,.ACC formation (A), anddevelopment of ACC synthase (B), by excised columella tissue of unripetomato fruit.

Table III. Influence of Cycloheximide on Wound-induced C2H4Production, ACC Formation, and Development ofACC Synthase in Unripe

Tomato FruitColumella tissue of immature green tomato was sliced and vacuum-

infiltrated with a solution containing 5% glycerol with or without 100I,MCHI. Assays were done after a 9-h incubation period at 25 C.

Inhibitor C2H4 ACC ACC synthase

nl/g h nmol/g unit/mg proteinNone 8.5 8.2 8.4CHI 0 0.05 0

relationship between ACC content and the rate of C2H4 produc-tion suggest that induction of the enzyme for ACC formation mayplay one of the most important roles in wound C2H4 production.

If the synthesis of ACC is the key rate-limiting step in theinduction of the synthesis of wound C2H4, it is expected that ACCsynthase activity should parallel the rate of ACC accumulation.The columella tissue of immature green tomato fruit was chosenfor this study because it produces little C2H4 when freshly excisedbut much C2H4 during subsequent incubation and because ACCsynthase in active form is readily extracted from it. Time coursesfor changes in rate of C2H4 production, ACC content, and ACCsynthase activity following excision are shown in Figure 3. In thefreshly prepared tissue, no C2H4 production, ACC, or ACC syn-thase activity could be detected. Over the 9-h incubation period,C2H4 production increased from 0 to 12 nl g-' h-1, the ACCcontent increased from 0.05 to 12 nmol/g, and ACC synthaseactivity increased from 0 to 6.4 unit/mg protein. The close corre-lation between ACC synthase activity, ACC content, and rate ofC2H4 production clearly supports the view that the induction ofACC synthase is a key step for wound C2H4 production. It hasbeen suggested that protein synthesis is required for the induction

A C2 4

r

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BIOSYNTHESIS OF WOUND ETHYLENE

of wound C2H4 production (2). If ACC synthase is newly synthe-sized as a result of wounding and is essential for the accumulationof ACC and the consequent C2H4 production, then an inhibitor ofprotein synthesis would be expected to block each of the threerelated processes. We therefore administered CHI to green tomatocolumella tissue and measured its effect upon the production ofwound C2H4, the accumulation of ACC, and the development ofACC synthase activity during a 9-h incubation period (Table III).CHI treatment did indeed block each of these processes. Immaturecantaloupe fruit, like green tomato, produced very little C2H4 (lessthan 0.5 nl g-' h-'). The rate of C2H4 production increased rapidlyfollowing excision; within 6 h, the rate increased by a factor ofmore than 10 and reached 180 nl g- h-' in 24 h (N. E. Hoffmanand M. L. Bliss, unpublished results). The freshly cut cantaloupetissue did not convert measurable amounts of labeled methionineto C2H4, but the incubated tissue, producing C2H4 at a high rate,successfully converted methionine into C2H4. As in the greentomato tissue, development of ACC synthase activity and accu-mulation of ACC in the excised cantaloupe tissue paralleled theincrease in C2H4 production.

Boller et al. (6) have recently examined ACC levels in tomatodiscs prepared from fruits at different ripening stages and foundthat tissues prepared from the mature-green stage contained asmuch ACC (1.7 nmol g-') as those prepared from breaker stage.Their results are in contrast with those of Hoffman and Yang (12),who found that mature green tomatoes contained very little ACC(0.05 nmol g-1) but increased to more than 1 nmol g-1 at breakerstage. The high ACC level observed by Boller et al. (6) in maturegreen tissue was undoubtedly due to the wounding effect becausethey assayed ACC after the prepared pericarp disks were incu-bated overnight.Our present results indicate that ACC synthase is the rate-

limiting enzyme in the pathway of C2H4 biosynthesis. Woundinginduces the synthesis ofACC synthase which is in turn responsiblefor the accumulation ofACC and the increase in C2H4 production.The evidence indicates that the pathway of the biosynthesis ofwound C2H4, and its induction, are similar, if not identical, tothose of auxin-induced C2H4. The biochemical mechanism bywhich wounding causes induction of ACC synthase remains un-known.

LITERATURE CITED

1. ABELES FB 1973 Ethylene in Plant Biology. Academic Press, New York, pp. 87-102

2. ABELES AL, FB ABELES 1972 Biochemical pathway of stress-induced ethylene.Plant Physiol 40: 496-498

3. ADAMS DO, SF YANG 1979 Ethylene biosynthesis: identification of I-aminocy-clopropane-l-carboxylic acid as an intermediate in the conversion of methio-nine to ethylene. Proc Nat Acad Sci USA 76: 170-174

4. ADATO I, S GAZIT 1974 Water-deficit stress, ethylene production, and ripeningin avocado fruits. Plant Physiol. 53: 45-46

5. BRADFORD MM 1976 A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem 72: 248-254

6. BOLLER T, RC HERNER, H KENDE 1979 Enzymatic formation of an ethyleneprecursor, I-aminocyclopropane-l-carboxylic acid. Planta 145: 293-303

7. COOPER WC, GK RAsMUSSEN, BJ ROGERS, PC REECE, WH HENRY 1965 Controlof abscission in agricultural crops and its physiological basis. Plant Physiol 43:1560-1576

8. FRUTON JS, S SIMMONDS 1958 General Biochemistry. John Wiley & Sons, NewYork, p 108

9. GLAZER RI, AL PEALE 1978 Measurement of S-adenosyl-L-methionine level bySP-Sephadex chromatography. Anal Biochem 91: 516-520

10. GOESCHL JD, L RAPPAPORT, HK PRATr 1966 Ethylene as a factor regulating thegrowth of pea epicotyls subjected to physical stress. Plant Physiol 41: 877-884

11. HANSON AD, H KENDE 1976 Biosynthesis of wound ethylene in morning-gloryflower tissue. Plant Physiol 57: 538-541

12. HOFFMAN NE, SF YANG 1980 Changes of l-aminocyclopropane-l-carboxylicacid content in ripening fruits in relation to their ethylene production rates.Proc Am Soc Hort Sci. In press

13. HYoDO H 1977 Ethylene production by albedo tissue of Satsuma mandarin(Citrus unshiu Marc.) fruit. Plant Physiol 59: 111-113

14. HYoDO H 1978 Ethylene production by wounded tissue of citrus fruit. Plant CellPhysiol 19: 545-551

15. LAU OL, SF YANG 1976 Stimulation of ethylene production in the mung beanhypocotyls by cupric ion, calcium ion, and kinetin. Plant Physiol 57: 88-92

16. LIZADA MCC, SF YANG 1979 A simple and sensitive assay for l-aminocyclopro-pane-l-carboxylic acid. Anal Biochem 100: 140-145

17. LOUGHEED EC, EW FRANKLIN 1974 Ethylene production increased by bruisingof apples. Hortscience 9: 192-193

18. MAcLEOD RF, AA KADER, LL MORRIS 1976 Stimulation of ethylene and CO2production of mature-green tomatoes by impact bruising. Hort Sci I1: 604-606

19. McGLAssON WB 1969 Ethylene production by slices of green banana fruit andpotato tuber tissue during the development of induced respiration. Austr J BiolSci 22: 489-491

20. McGLAssON WB, HK PRATT 1964 Effects of wounding on respiration andethylene production by cantaloupe fruit tissue. Plant Physiol 39: 128-132

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