Proc. Nat. Acad. Sci. USAVol. 70, No. 2, pp. 591-597, February 1973

Ethylene in Plant Growth

STANLEY P. BURG

The Fairchild Tropical Garden, and University of Miami, Miami, Florida 33156

ABSTRACT Ethylene inhibits cell division, DNA syn-thesis, and growth in the meristems of roots, shoots, andaxillary buds, without influencing RNA synthesis. Apicaldominance often is broken when ethylene is removed, ap-parently because the gas inhibits polar auxin transport ir-reversibly, thereby reducing the shoot's auxin content justas if the apex had been removed. A similar mechanismmay underly ethylene-induced release from dormancy ofbuds, tubers, root initials, and seeds. Often ethylene in-hibits cell expansion within 15 min, but delays differentia-tion so that previously expanding cells eventually grow toenormous size. These cells grow isodiametrically ratherthan longitudinally because their newly deposited cellu-lose microfibrils are laid down longitudinally rather thanradially. Tropistic responses are inhibited when ethylenereversibly and rapidly prevents lateral auxin transport. Inmost of these cases, as well as certain other instances,ethylene action is mimicked by application of an auxin,since auxins induce ethylene formation. Regulation byethylene extends to abscission, to flower formation andfading, and to fruit growth and ripening. Production ofethylene is controlled by auxin and by red light, auxinacting to induce a labile enzyme needed for ethylene syn-thesis and red light to repress ethylene production. Nu-merous cases in which a response to red light requires anintervening step dependent upon inhibition of ethyleneproduction have been identified. Ethylene action requiresnoncovalent binding of the gas to a metal-containingreceptor having limited access, and produces no lastingproduct. The action is competitively inhibited by C02, andrequires 02. Ethylene is biosynthesized from carbons 3 and4 of methionine, apparently by a copper-containing en-zyme in a reaction dependent upon an oxygen-requiringstep with a Km = 0.2% 02. The oxidative step appears to bepreceded by an energy-requiring step subsequent to me-thionine formation.

Early observations on the effects of ethylene on plant growthare contained in a literature, dating to 1858 (1), that describesthe behavior of plants exposed to illuminating gas. In 1901,the study of a strange growth habit of etiolated pea seedlingsraised in laboratory air contaminated with illuminated gasrevealed that the biologically active component of the gas isethylene (2). In the presence of ethylene the seedlings undergoa "triple response," consisting of a thickening of the subapi-cal portion of the stem, depression in the rate of elongation,and horizontal nutation of the stem. These and numerousother changes in the growth and development of seedlingsmight have received immediate attention had not it beenlearned soon thereafter that ethylene ripens fruits (3). Almostall effort was diverted to this economically important aspectof ethylene action, and by the mid-1930s it was establishedthat ethylene is produced autocatalytically just in advanceof fruit ripening (4). The gas became known as the fruit-ripening hormone, and not until the early 1960s was the role

of ethylene in plant growth destined to be studied intensivelyagain.

Effects of ethylene on cell division

When etiolated pea seedlings are grown continuously in thepresence of a trace of ethylene, the stem hardly elongates androot growth is inhibited about 60% (refs. 2, 5, 6; Fig. 3). Aswollen zone develops behind the root tip, root hairs prolifer-ate, and the root deflects plageotropically in the gravitationalfield (5-7); similar changes occur in the stem (2, 5, 6). Themajor cause of the overall growth inhibition is cessation orretardation of the mitotic process in the meristems of the root,shoot, and axillary buds (5, 6). Within a few hours afterethylene is applied, the number of mitotic figures in the stemapex begins to decline, and within about 10 hr mitosis almoststops. Auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D)cause the same effect, at least in part by stimulating ethyleneproduction in the apex. Ethylene inhibits mitosis in the rootapex by about 60% and 2,4-D has a similar effect, but veryhigh concentrations of 2,4-D stimulate mitosis in the elon-gating zone of the root just as in the elongating zone of thestem. These divisions give rise to root initials in both tissues,and ethylene does not interfere with their formation, althoughit slows their outgrowth. Both auxin (8) and ethylene (5)block cell division in meristems at some stage before prophase,and auxins appear to function in this case through an inter-vening step in which ethylene is produced. Within a few hoursafter ethylene application, the rate of DNA synthesis from[3H]thymidine begins to decline not only in the apical meri-stem (Fig. 1; ref. 5), but also even in the elongating zone ofthe stem where no cell divisions occur (Fig. 2; ref. 5). RNAsynthesis from [3H]uridine or [14C]ATP is not affected ineither tissue (Figs. 1 and 2; ref. 9). DNA synthesis is inhibitedbecause DNA polymerase activity is reduced (10). In roots,shoots, and lateral buds of the etiolated pea plant there is aquantitative relationship between the inhibitions of DNAsynthesis, cell division, and growth caused by ethylene (5).

Lateral buds are a complex case. After the buds are re-leased from apical dominance by removal of the stem apex,their mitotic activity and outgrowth are repressed by applica-tion of either ethylene, or enough auxin to induce ethyleneproduction (11, 12). Inclusion of a cytokinin overcomes theinhibitory action of ethylene or auxin (11-13), but whetherthis is the manner in which auxin and cytokinin normallycontrol apical dominance is not resolved. A puzzling thingabout ethylene and bud growth is the fact that often apicaldominance is broken after an ethylene treatment even thoughthe gas inhibits the outgrowth of the buds while it is present.No lateral buds grow normally or during a 7-day treatmentof Petunia plants with 100 nl/liter of ethylene, but the axillarybuds in the subapical zone of the shoot are released from cor-

591

Abbreviation: 2,4-D, 2,4-dichlorophenoxyacetic acid.

Proc. Nat. Acad. Sci. USA 70 (1978)

a 6

<

0

IJ2

C2H4

FIG. 1. Effect of ethylene (100 nl/liter) applied to intactetiolated pea seedlings (7-days old) on the rate of incorporationof ['H]thymidine into DNA and [3Hjuridine into RNA. Afterthe seedlings were exposed to ethylene for the indicated number ofhours, either the hook elbow or the apical tip and plumular leaveswere excised and pulse-labeled with a solution containing 1,ACi/ml of thymidine or uridine and 50 mM potassium phosphatebuffer (pH 7). DNA and RNA were extracted, and isolated; radio-activity was determined. The results were the same regardless ofwhether ethylene was present or absent during the pulse-labelingperiod (Kang and Burg, 1972). i A, hook; O-O, apex-ex-periment 1; *-- , apex-experiment 2.

relative inhibition if the gas is applied for only 2 hr and thenremoved (Table 1). When ethylene is removed after an 8- to12-hr treatment essentially all buds are released from apical

TABLE 1. Ethylene-induced release of apical dominance in5-week-old Petunia x hybrid Grandiflora calypso seedlings

(Ramos and Burg, 1972)

% Buds released from apical dominance

Duration of at indicated time (days)C2H4 treatment 2 4 5 7

2Hr 0 0 9 184 Hr 0 10 13 236 Hr 0 15 30 368 Hr 4 27 33 3712 Hr 7 36 36 4324 Hr 3 43 46 467 Days 0 0 0 0Control-no C2H4 0 0 0 0

Ethylene (100 ppm) was applied for between 2 and 24 hr duringthe first day, or else continuously for a 7-day period. Bud growthwas appraised continuously throughout the same period.

4 8 12 16 20 24HOURS PRETREATMENT WITH 100 tbI/LITER OF C2H4

FIG. 2. Same as Fig. 1, except the subapical 5-mm elongatingzone was excised and pulse-labeled with isotope (Kang and Burg,1972).

dominance. In Petunia the effect of applied ethylene on apicaldominance is quantitatively almost as great as that of excisingthe apex. A similar mechanism may underlie the breaking ofdormancy in root initials, buds, seeds, and tubors (14-16)after brief ethylene treatment.

Several hours after ethylene application, the capacity of thepolar auxin transport system begins to decline (17, 18), andwithin 10 hr it is inhibited almost 90% in pea subapical stemtissue. As a result, the auxin content of the stem is loweredmarkedly (19-21), possibly in part because auxin synthesisalso may be curtailed by ethylene (22). The cause of the block-age of auxin transport is not known, but is not enhancedauxin destruction (2, 23) or conjugation (24). In this mannerthe auxin content of the stem is diminished just as if thenatural source of auxin, the apex, had been removed, and it isperhaps for this reason that apical dominance is broken insome plants when an ethylene treatment is terminated. Othersymptoms of auxin deficiency would be expected and havebeen observed when ethylene is applied. This explains in parthow the gas causes the abscission of leaves, flowers, and fruits(17, 25, 26) for auxin has the opposite effect. Both auxin andethylene have been implicated as natural regulators of theabscission process (27, 28).

Effect of ethylene on cell expansion

The overall elongation rate of several intact seedlings, includ-ing etiolated pea, is strongly inhibited by ethylene within 15min (Fig. 3; refs. 19, 29, 30). Inhibition of cell expansion is notcomplete in the growing zone of pea. Instead, in thepresence of ethylene, the cells continue to expand, albeitslowly, in an isodiametric manner for a seemingly indefinitetime, whereas the same cells in control plants differentiatewithin a few days (6, 31). Ethylene completely preventslignification of fiber elements, and almost stops lignificationof xylem vessels during a 1-week period (31). Within about5 days the subapical cells expand to such an extent that theirvolume exceeds that of the same cells in control tissue (6).By extending the duration of the growth period, ethyleneeventually promotes growth of the pea subapical zone, but

SUBAPEX-INTACT -0-DNA [3H]TWMIDINE--RNA [3HIURIDINE

F 00 O

0-

592 Burg

Proc. Nat. Acad. Sci. USA 70 (1973)

this is not the only way that ethylene may promote growth.In a few cases the rate as well as the duration of growth isenhanced. Thus, ethylene increases the growth rate and dura-tion of growth in rice seedlings (32-34); causes newly dividedcells to begin to expand prematurely in fig fruits (35); andstimulates cells in the upper side of the leaf petiole to expandagain (36, 37) after their growth essentially has stopped,causing an overgrowth or epinasty of the petiole. The mech-anism by which ethylene promotes growth in these cases isunknown, although an auxin assymetry in opposition to thegravity vector has been detected in the case of epinasty (38).That the ethylene-like action of excess auxin on the expan-

sion of cells in the elongating zone is due to auxin-inducedethylene production can be demonstrated by growing plantsunder hypobaric conditions (6). The diffusivity of gases pass-ing through lenticles or stomata is increased as the absolutepressure is decreased, and it follows from Fick's law and canbe directly demonstrated that, at equilibrium, the endog-enous partial pressure of any vapor produced within a plantis directly related to the absolute atmospheric pressure (39).One-fifth of an atmosphere of water-saturated O2, which isequivalent to humid air from which the N2 has been removed,has little effect on the growth of the elongating zone in etio-lated pea plants. However, it reverses almost completely theinhibition of elongation caused by a 2,4-D spray and revealsthe fact that, when all auxin-induced volatile substances areremoved, the main effect of 2,4-D is only to promote growth(6).The cause of the transition from longitudinal to radial

growth in the presence of ethylene is a changed orientationin the direction of deposition of newly formed cellulose micro-fibrils. This change is revealed as an altered optical bire-fringence pattern in the cell wall of tissues treated with ethyl-ene, or excess auxin, benzimidazole, or cytokinin. All areagents that cause cellular swelling (9, 12, 40, 41). Normallythe cellulose microfibrils are deposited in a transverse direc-tion, restricting lateral expansion, but when ethylene (9) orexcess auxin (41, 42) is applied they are deposited instead in alongitudinal direction so that longitudinal expansion may berestricted and radial expansion promoted. As a result of thischanged pattern of cellulose deposition, the epidermal cells,which are not restrained in outward expansion by neighboringcells, bulge out and form hair-like structures both in the root(7) and stem (31).

Cell expansion can also be studied by floating stem sections,excised from the growing zone, on solutions containing anauxin and other factors. It is a relatively easy matter withetiolated pea and certain other light and dark grown tissueto demonstrate under these conditions that the classical bi-phasic growth-response curve to applied auxin is due to apromotive phase caused by induced growth at a low auxinconcentration, and an inhibitory phase due to induced ethyl-ene production at a high auxin concentration (11, 12, 23).In many tissues a very high concentration of auxin, especiallya synthetic nondegradable type, causes an additional inhibi-tion, the herbicidal effect, which occurs independent of andin addition to ethylene action (5, 7, 12, 43). Auxin-inducedinhibition of growth in pea roots also is explained in terms ofinduced ethylene production, and under certain conditionsan additional, direct, herbicidal auxin effect (7, 43). This hasrelevance to the geotropic curving of roots that, according tothe Cholodny-Went theory, is caused by accumulation of a

-2AEE / PEA SHOOT CABSBAGE SHOOT

3

2

+C2H;- PEA ROOT

I/l .60 120 180 240

TIME (Minutes)

FIG. 3. Time course of the effect of 100 nl/liter of ethylene onthe elongation of etiolated pea and cabbage shoots, and pea roots.Seedlings were about 3-cm high when they were treated; rootswere about 3-cm long. All tissue was preadapted to dim greenlight for 10-12 hr before the start of the experiment. Measure-ments were made under dim green light with a sensitive cathe-tometer (Kang and Burg, 1972).

growth-inhibitory concentration of auxin in the underside ofthe root as a result of geostimulation. Several studies havesuggested that it is not auxin per se that causes the growthinhibition, but rather some product of auxin action that candiffuse across the root (44). That ethylene may be this sub-stance is indicated by the fact that the geotropic curving ofroots is slowed by a competitive inhibitor of ethylene action,CO2 (7, 45), at a concentration that does not change the over-all growth rate (43). Ethylene by itself prevents roots fromcurving geotropically (43), and it has this same effect on thestem of pea and certain other seedlings (46, 47). The gas alsoprevents phototropism in mustard and radish seedlings, aswell as the development of a spontaneous curvature in pea-stem segments (23). The latter curvature develops during thefirst few hours of incubation, and is perceptible within 15 minafter the tissue is cut, but is stopped completely before thattime by applied ethylene. Ethylene does not retard elongationof these same stem segments for 2-3 hr (9, 23), so some otheraction of the gas underlies its efficacy in preventing tropisticcurvature. This function of ethylene has been examined with[I4C]indole-3-acetic acid, and has been found to be based onthe ability of the gas to completely, rapidly and reversiblyinhibit the auxin lateral transport system that is sensitive togravity (23). As soon as ethylene is removed, normal curvatureagain develops. In this way ethylene may exert feedbackcontrol over the lateral transport of auxin under certain condi-tions (48).

Other processes influenced by ethylene

Regulation by ethylene extends to the stages of flower forma-tion, sex expression, flower fading, and fruit growth and

Ethylene in Plant Growth 593

Proc. Nat. Acad. Sci. USA 70 (1973)

ripening. Flowering of all Bromeliads, including the commer-cially important pineapple, is stimulated by very brief exposureto ethylene or enough auxin to stimulate ethylene production(49). In many flowers, ethylene acts as a flower-fading hor-mone (50, 51). A corrolary to this behavior is the fading thatensues after pollination of certain flowers. The pollen is a richsource of auxin, and releases sufficient growth hormone tostimulate ethylene production in the stigma. [14C]Indole-3-acetic acid remains localized there, but because ethyleneproduction is autocatalytic in flowers just as it is in fruits,each cell in the stigma gases its neighbors, causing it to pro-duce ethylene, and in this way the stimulus for flower-fadingspreads to the outer appendages (50).

Factors controlling ethylene synthesis

Two natural factors controlling the rate of ethylene synthesishave been identified, auxin and red light. Induction of ethyl-ene synthesis by an auxin occurs after a lag of 30-60 min, andaccording to inhibitor studies must involve de novo synthesisof a requisite enzyme (52, 53). The enzyme is labile, so that ifcycloheximide is added after ethylene production has beenstimulated by an auxin, the production stops within a fewhours. Auxin must be continuously present in vegetativetissue for ethylene to be produced, and the rate of productionis proportional to the endogenous content of indole-3-aceticacid (24, 43, 52, 53). If auxin is removed from the bathingsolution, or if the tissue is induced to develop an auxin-conjugating system, whereby the endogenous auxin contentis lowered, ethylene production is diminished proportionately.In etiolated seedlings, ethylene is produced primarily in theapex (11, 12, 54), the site of auxin production and thereforethe tissue richest in auxin content. After a seedling is exposedto red light, ethylene production in the apex declines pro-gressively, at least in part because the ability of auxin tostimulate ethylene production is repressed (12, 48, 54-57).If a response to red light requires an intervening step depen-dent upon inhibition of ethylene production, it can be simu-lated in darkness by application of the competitive inhibitorof ethylene activity, C02, or by removal of ethylene in ahypobaric atmosphere. In this manner it has been demon-strated that endogenous ethylene production is responsiblefor the formation of the seedling hook, which protects theyoung leaves or cotyledons from mechanical damage duringtheir emergence from the soil (12, 57). When the seedlingreaches the light, ethylene production is suppressed, the hookopens, and the leaves expand. In darkness the hook opens ifthe seedlings are placed in a hypobaric chamber or exposed toC02 (5, 54, 55, 57). In fact, the hook never forms in darknessif the seedling is continuously grown under either of theseconditions (57). Even after the hook has opened it can be re-closed by application of ethylene or allowing the plant to pro-duce its own ethylene in response to certain treatments (12,57). Other responses to red light mediated by repressedethylene production are stimulation of carotinoid and antho-cyanin synthesis (55, 57), and enhancement of geotropicsensitivity (48). It takes only 0.1 nl/liter of ethylene to half-inhibit hook opening in the light, and the same amount ofgas to half-inhibit cell division in the dark, so if there is nor-mally enough ethylene present in the etiolated plant to causehook formation, there must also be enough present to regu-

moval of ethylene under hypobaric conditions, in which case

the cell-division frequency triples (5).

Mechanism of ethylene action

To act like ethylene a molecule must have a terminal carbonadjacent to an unsaturated bond (45). Substituents that with-draw electrons from the unsaturated bond or sterically in-hibit an approach to it, reduce activity. A quantitative rela-tionship exists between ability to bind metal and biologicalactivity, and a known metal binder, carbon monoxide, willreplace ethylene in all its actions at a concentration of severalhundred nl/liter. It has been concluded that ethylene binds toa metal-containing receptor having limited access of approach,with a Km = 6 X 10-10 M (45). The binding must be througha noncovalent bond for no exchange of deuterium occurs whendeuterated ethylene is applied to responsive plants (58, 59).The transient nature of the binding is also revealed by thefact that many responses to ethylene are rapidly reversible;for example growth inhibition in the etiolated pea stem (Fig.3) or root (43) and the action of the gas on lateral transport(23). These same studies also indicate that no lasting productof ethylene action is produced as a result of binding to itsreceptor.Amongst the active compounds substituting for ethylene is

allene, a close analogue of C02. Because C02 inhibits fruitripening, the possibility was investigated that it might actas a competitive inhibitor of ethylene action through itssimilarity to allene. Competition between ethylene and C02was demonstrated by use of the Lineweaver-Burke plots, andsubsequently has been established for essentially all actionsof ethylene (7, 45). By the same approach it was shown thatethylene action requires 02. As the 02 concentration is di-minished, the amount of ethylene required for a half-maximalresponse is increased (45). 02 also is needed for ethyleneproduction (Figs. 4 and 5; refs. 60, 61). These interactions ex-

plain why controlled atmospheres low in 02 and high in C02extend the storage life of many fruits. A simpler and bettermethod of commodity preservation is the operation of a hypo-baric system to remove ethylene, supplying it with water-satu-rated flowing air to maintain a preselected low level of 02 (62).Extensive laboratory studies have revealed that this methodgreatly prolongs the storage life of many fruits, cut flowers,vegetables, potted plants, and stem cuttings (51, 62). Proto-type shipping containers embodying the method have beenconstructed and tested commercially, and the first storagewarehouses will be in operation within the forthcoming year.

Biosynthesis of ethylene

The in vivo precursor of ethylene in fruits and vegetative tis-sue is methionine (61, 63, 64). Ethylene arises from carbons3 and 4, carbon 1 is converted to C02, carbon 2 yields formatebut no C02, and the S-methyl is retained in the tissue in a non-

volatile form (61, 63, 65). During ethylene synthesis the S-methyl is transferred intact, or incorporated as dimethylmercaptan, into homoserine to form homocysteine, which isrecycled through several steps to methionine (65). Modelsystems producing ethylene from methionine and other com-

pounds have been described including one that utilizes Cu+and ascorbate or peroxide (66). A peroxidase system, requiringMn++ (or peroxide), S03--, and monophenol, degrades the2-keto analogue of methionine, 2-keto4-methylthiobutyrate,

late cell division. This can be directly demonstrated by re-

594 Burg

to ethylene, forming C02 from carbons 1 and 2, and dimethyl

Proc. Nat. Acad. Sci. USA 70 (1973)

Time (Hours)

FIG. 4. Effect of air, N2, and various O2 concentrations onethylene production by apple discs. Tissue was prepared and incu-bated as described in the footnote to Table 3 (Imaseki and Burg,1972).

mercaptan from the S-methyl (67). This system also workswith methional, N-acetyl methionine and C-terminal methi-onine peptides, but not with methionine itself (68). Isolationof a transaminase converting methionine to 2-keto4-methyl-thiobutyrate has been reported (69), but the latter cannotbe an intermediate in ethylene production by apples becausein vivo carbon 2 of methionine forms formate, whereas with2-keto-4-methylthiobutyrate it yields CO. Moreover inapples, [14C]2-keto-4-methylthiobutyrate is converted lessefficiently than [14C]methionine to ethylene and then onlyafter conversion to methionine (64). Objection also has beenraised to the proposed role of 2-keto-4-methylthiobutyratein ethylene production by other tissues (70).

Peroxidase systems oxidatively decarboxylate methionineand other amino acids in the presence of Mn++, pyridoxal-phosphate, and monophenol. Nonenzymatic oxidation alsooccurs, especially with excess pyridoxal and Mn++ at alkalinepH, and transamination to form 2-keto-4-methylthiobutyratecan be effected in model systems with pyridoxal and variousmetals. When these systems are coupled to the peroxidasesystem producing ethylene in the presence of S03--, a signifi-cant conversion of methionine to ethylene is observed, butthere is no proof that this test-tube system functions in vivo.To the contrary, in vegetative tissue peroxidase is not theenzyme induced by auxin when it stimulates ethylene produc-tion (53).Ethylene production is an aerobic process. Estimates of its

dependency on pO2 range from a very high affinity similar to

TABLE 2. Dependence of ethylene production on oxygenconcentration in the absence of a liquid phase

(Imaseki and Burg, 1972)

Relative ethyleneOxygen concentration (%) production at 250

o o0.5 731.0 952.0 98

20.0 100100.0 53

Four McIntosh apple discs, 1-mm thick X 1-cm diameter(fresh weight = 1 g) were rinsed in 0.1 M Tris *HCl buffer (pH 7)containing 0.55 M glycerol and 50 mM CaCl2, blotted on filterpaper, and placed in a 25-ml Erlenmeyer flask. The flasks wereflushed with N2 containing less than 0.002% 02 until ethyleneproduction ceased for 1 hr. Measured quantities of 02 werethen injected, and rates of ethylene production were determinedin the interval 2-3 hr later.

that for respiration (60) to so low an affinity that even 100%02 is stimulatory (66). Since the 02 dependency providesinformation about the nature of the oxidative step it is im-portant to determine why such different results have beenobtained. The solution to this question is afforded by a studyon the 0, dependency of respiration in the Aroid spadix (72),which shows that the presence of a liquid-phase shifts theapparent Km to 16% 02, whereas the value is 0.2%02 in thecase of dry discs maintained in a moist atmosphere. Whendry apple discs are flushed with N2, ethylene productionimmediately stops, but if the discs are floated on a liquidphase through which the N2 is bubbled, ethylene productiondoes not stop for at least one hour (Fig. 4) due to the slowescape of 02 trapped within the tissue by the liquid phase.Once ethylene production has ceased under anaerobic condi-tions in the presence of a liquid phase, the Km for the processcan be determined by addition of 02 to the gas phase. Under

* 0.25 0.50 0.75K'm = 20%02 1/' (%0°2)1

FIG. 5. Lineweaver-Burke plot of data from Fig. 4, in whichtissue pretreated with N2 for several hours, until ethylene pro-duction had stopped, was exposed to various concentrations of 02to start ethylene production again. The rates are initial valuestaken during the first 40 min after O2 was readded, but were linearfor several hours (Imaseki and Burg, 1972).

Ethylene in Plant Growth 595

Proc. Nat. Acad. Sci. USA 70 (1973)

TABLE 3. Effect of arsenate and phosphate on ethyleneproduction (Imaseki and Burg, 1972)

Relative ethylene production

0-2 hr 2-4 hr

Control 100 100AsO4 (30 mM) 102 75AsO4 + P04 (100mM) 98 91AsO4 (50 mM) 68 39As04 + P04 (100mM) 98 82As04 + Met (10 mM) 68 43

Conditions are similar to those described in the footnote inTable 2, except that the apple discs were floated on 2 ml of therinsing solution contained in each Erlenmeyer flask. Whenarsenate + 100 mM phosphate (pH 7) were added, the 100 mMTris.HCl buffer (pH 7) was omitted, and the result was com-pared to that with a control having 100 mM phosphate bufferbut no arsenate.

these conditions the apparent Km is 20%02 and stimulationby 100% 02 occurs for several hours (Figs. 4 and 5). It issignificant that in this case 100% 02 only stimulates ethylenesynthesis if the tissue was preincubated in N2 (Fig. 4) for itis known that some substance accumulates under anaerobicconditions that causes ethylene production to be acceleratedfor several hours after air is readmitted (60). This substance,probably methionine, can limit the rate of ethylene synthesisso that in the presence of a liquid phase, 100% 02 is onlystimulatory when the other factor is present in sufficientconcentration. In the absence of a liquid phase, the Km forthe 02 dependency of ethylene production is about 0.2%(Table 2). These data indicate that respiration and ethyleneproduction have the same high affinity for 02, and since thereare no known oxidases other then cytochrome oxidase withthis characteristic (72), the 02 dependency in both casesmust reflect involvement of the respiratory electron-transportsystem rather then an oxidase specific to ethylene synthesis.Oxidation must occur close to the terminal step in ethylenebiosynthesis, for immediately after 02 is supplied to N2-treated tissue ethylene is produced at a linear rate (Fig. 4,refs. 60, 72). In apples evolution of 14CO2 from [1-14C]methi-onine does not occur under anaerobic conditions, nor does itoccur from [U-'4C]methionine (71), so the decarboxylation isan oxidative process. When air is readded, for several hourseach 14CO2 derived from [1-14C]methionine is accompanied byone ['4C]ethylene derived from [U-14C]methionine, but afterseveral hours when the initially high rate of ethylene synthesischaracteristic of N2-treated tissue subsides to the normalaerobic rate, stoichiometry is no longer maintained and someof the decarboxylated methionine does not yield ethylene.A close connection between oxidative decarboxylation ofmethionine and evolution of ethylene also can be shown bymeans of inhibitors. Ethylene production is inhibited bydiethyldithiocarbamate (66), suggesting that a copper-containing enzyme may be involved, although other metalscannot be excluded. With 500 ,uM diethyldithiocarbamate theinhibition is fairly specific to ethylene formation, reducingthe rate by 90% without interfering with respiratory CO2evolution or 02 consumption. Under these conditions forma-tion of 14CO2 from [1-14C]methionine is also reduced 90%.Similarly, 50 AM dinitrophenol inhibits both ethylene produc-

tion from [U-'4C]methionine and 14CO2 production from [1-IC ]methionine by 50% without altering the evolution of

respiratory CO2. Ethylene production in apples is insensitiveto cycloheximide even when slices are treated for 6 hr, so therate-limiting enzymes in fruit tissue are not labile as they arein vegetative tissue induced to produce ethylene by an auxin.N-acetyl methionine is a substrate for ethylene production inthe peroxidase model system, but at a concentration of 10-100 mM it profoundly inhibits ethylene synthesis in vivo,suggesting that the peroxidase system is not the normal path-way. Arsenate inhibits ethylene formation (Table 3) and theinhibition is reversed by phosphate. These data and the factthat dinitrophenol and respiratory poisens inhibit ethylenebiosynthesis (60, 66) suggest the existance of a high-energystep in the conversion of methionine to ethylene. S-Adenosylmethionine is a possible intermediate since it is formed ingood yield from [14C]methionine applied to apple discs (63),has a tendency to split-off its S-methyl, and we find it is con-verted to ethylene by the Cu+-ascorbate model system (butnot the peroxidase system), perhaps because the adenosinemoeity coveys a positive charge to the sulfur just as Cu+ isproposed to do.

The- substrate for ethylene production, methionine, isformed from organic acids produced in the mitochondria.To be converted to ethylene, energy supplied by the mito-chondria appears to be required, and electrons released frommethionine have to be carried by a cofactor to the respiratoryelectron-transport system. Presumably because of thesenumerous interactions between the mitochondria and theethylene producing system, it has not yet been possible toassemble a cell-free system capable of evolving the gas.

This work was supported by National Science FoundationGrant GB-27424.1. Fahnstock, G. W. (1858) Proc. Acad. Nat. Sci. Phila., 1.2. Neljubow, D. (1901) Bot. Centr. 10, 128.3. Sievers. A. F. & True, R. H. (1912) U.S. Dept. Agr. Bur.

Plant Ind. Bull. 232, 1.4. Gane, R. (1934) Nature 134, 1008.5. Apelbaum, A. & Burg, S. P. (1972) Plant Physiol. 50, 117.6. Apelbaum, A. & Burg, S. P. (1972) Plant Physiol. 50, 125.7. Chadwick, A. V. & Burg, S. P. (1967) Plant Physiol. 42,

415.8. Webster, P. L. & Davidson, D. (1967) Amer. J. Bot. 54, 633.9. Eisinger, W. & Burg, S. P. (1972) Plant Physiol., 50, 510.

10. Sfakiotakis, E. (1972) Doctoral thesis, Michigan StateUniversity.

11. Burg, S. P. & Burg, E. A. (1968) Plant Physiol. 43, 1069.12. Burg, S. P. & Burg, E. A. (1967) in Biochemistry & Physiol-

ogy of Plant Growth Substances (Runge Press, Ottawa, Can-ada), p. 1275.

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