Ethylene in Plant Growth

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

STANLEY P. BURG The Fairchild Tropical Garden, and University of Miami, Miami, Florida 33156

ABSTRACT Ethylene inhibits cell division, DNA syn- of ethylene in plant growth destined to be studied intensively thesis, and growth in the meristems of roots, shoots, and axillary buds, without influencing RNA synthesis. Apical again. dominance often is broken when ethylene is removed, ap- Effects of ethylene on cell division parently because the gas inhibits polar auxin transport ir- reversibly, thereby reducing the shoot's auxin content just When etiolated pea seedlings are grown continuously in the as if the apex had been removed. A similar mechanism presence of a trace of ethylene, the stem hardly elongates and may underly ethylene-induced release from dormancy of root growth is inhibited about 60% (refs. 2, 5, 6; Fig. 3). A buds, tubers, root initials, and seeds. Often ethylene inhibits cell expansion within 15 min, but delays differentia- swollen zone develops behind the root tip, root hairs prolifer- tion so that previously expanding cells eventually grow to ate, and the root deflects plageotropically in the gravitational enormous size. These cells grow isodiametrically rather field (5-7); similar changes occur in the stem (2, 5, 6). The than longitudinally because their newly deposited cellu- major cause of the overall growth inhibition is cessation or lose microfibrils are laid down longitudinally rather than retardation of the mitotic process in the meristems of the radially. Tropistic responses are inhibited when ethylene root, reversibly and rapidly prevents lateral auxin transport. In shoot, and axillary buds (5, 6). Within a few hours after most of these cases, as well as certain other instances, ethylene is applied, the number of mitotic figures in the stem ethylene action is mimicked by application of an auxin, apex begins to decline, and within about 10 hr mitosis almost since auxins induce ethylene formation. Regulation by stops. Auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) ethylene extends to abscission, to flower formation and cause fading, and to fruit growth and ripening. Production of the same effect, at least in part by stimulating ethylene ethylene is controlled by auxin and by red light, auxin production in the apex. Ethylene inhibits mitosis in the root acting to induce a labile enzyme needed for ethylene syn- apex by about 60% and 2,4-D has a similar effect, but very thesis and red light to repress ethylene production. Nu- high concentrations of 2,4-D stimulate mitosis in the elon- merous cases in which a response to red light requires an gating zone of the root just as in the elongating zone of the intervening step dependent upon inhibition of ethylene production have been identified. Ethylene action requires stem. These divisions give rise to root initials in both tissues, noncovalent binding of the gas to a metal-containing and ethylene does not interfere with their formation, although receptor having limited access, and produces no lasting it slows their outgrowth. Both auxin (8) and ethylene (5) product. The action is competitively inhibited by C02, and block cell division in meristems at some stage before prophase, requires 02. Ethylene is biosynthesized from carbons 3 and 4 of methionine, apparently by a copper-containing en- and auxins appear to function in this case through an inter- zyme in a reaction dependent upon an oxygen-requiring vening step in which ethylene is produced. Within a few hours step with a Km = 0.2% 02. The oxidative step appears to be after ethylene application, the rate of DNA synthesis from preceded by an energy-requiring step subsequent to me- [3H]thymidine begins to decline not only in the apical meri- thionine formation. stem (Fig. 1; ref. 5), but also even in the elongating zone of Early observations on the effects of ethylene on plant growth the stem where no cell divisions occur (Fig. 2; ref. 5). RNA are contained in a literature, dating to 1858 (1), that describes synthesis from [3H]uridine or [14C]ATP is not affected in the behavior of plants exposed to illuminating gas. In 1901, either tissue (Figs. 1 and 2; ref. 9). DNA synthesis is inhibited the study of a strange growth habit of etiolated pea seedlings because DNA polymerase activity is reduced (10). In roots, raised in laboratory air contaminated with illuminated gas shoots, and lateral buds of the etiolated pea plant there is a revealed that the biologically active component of the gas is quantitative relationship between the inhibitions of DNA ethylene (2). In the presence of ethylene the seedlings undergo synthesis, cell division, and growth caused by ethylene (5). a "triple response," consisting of a thickening of the subapi- Lateral buds are a complex case. After the buds are re- cal portion of the stem, depression in the rate of elongation, leased from apical dominance by removal of the stem apex, and horizontal nutation of the stem. These and numerous their mitotic activity and outgrowth are repressed by applica- other changes in the growth and development of seedlings tion of either ethylene, or enough auxin to induce ethylene might have received immediate attention had not it been production (11, 12). Inclusion of a cytokinin overcomes the learned soon thereafter that ethylene ripens fruits (3). Almost inhibitory action of ethylene or auxin (11-13), but whether all effort was diverted to this economically important aspect this is the manner in which auxin and cytokinin normally of ethylene action, and by the mid-1930s it was established control apical dominance is not resolved. A puzzling thing that ethylene is produced autocatalytically just in advance about ethylene and bud growth is the fact that often apical of fruit ripening (4). The gas became known as the fruit- dominance is broken after an ethylene treatment even though ripening hormone, and not until the early 1960s was the role the gas inhibits the outgrowth of the buds while it is present. No lateral buds grow normally or during a 7-day treatment of Petunia plants with 100 nl/liter of ethylene, but the axillary Abbreviation: 2,4-D, 2,4-dichlorophenoxyacetic acid. buds in the subapical zone of the shoot are released from cor- 591 Downloaded by guest on September 26, 2021 592 Burg Proc. Nat. Acad. Sci. USA 70 (1978)

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

a 6

0 < F IJ 0 2 0 O

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

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

dominance. In Petunia the effect of applied ethylene on apical C2H4 dominance is quantitatively almost as great as that of excising the apex. A similar mechanism may underlie the breaking of FIG. 1. Effect of ethylene (100 nl/liter) applied to intact dormancy in root initials, buds, seeds, and tubors (14-16) etiolated pea seedlings (7-days old) on the rate of incorporation after brief ethylene treatment. of ['H]thymidine into DNA and [3Hjuridine into RNA. After Several hours after ethylene application, the capacity of the the seedlings were exposed to ethylene for the indicated number of polar auxin transport system begins to decline (17, 18), and hours, either the hook elbow or the apical tip and plumular leaves within 10 hr it is inhibited almost 90% in pea subapical stem were excised and pulse-labeled with a solution containing 1 ,ACi/ml of thymidine or uridine and 50 mM potassium phosphate tissue. As a result, the auxin content of the stem is lowered buffer (pH 7). DNA and RNA were extracted, and isolated; radio- markedly (19-21), possibly in part because auxin synthesis activity was determined. The results were the same regardless of also may be curtailed by ethylene (22). The cause of the block- whether ethylene was present or absent during the pulse-labeling age of auxin transport is not known, but is not enhanced period (Kang and Burg, 1972). i A, hook; O-O, apex-ex- auxin destruction (2, 23) or conjugation (24). In this manner periment 1; *-- , apex-experiment 2. the auxin content of the stem is diminished just as if the natural source of auxin, the apex, had been removed, and it is perhaps for this reason that apical dominance is broken in relative inhibition if the gas is applied for only 2 hr and then some plants when an ethylene treatment is terminated. Other removed (Table 1). When ethylene is removed after an 8- to symptoms of auxin deficiency would be expected and have 12-hr treatment essentially all buds are released from apical been observed when ethylene is applied. This explains in part how the gas causes the abscission of leaves, flowers, and fruits TABLE 1. Ethylene-induced release of apical dominance in (17, 25, 26) for auxin has the opposite effect. Both auxin and 5-week-old Petunia x hybrid Grandiflora calypso seedlings ethylene have been implicated as natural regulators of the (Ramos and Burg, 1972) abscission process (27, 28).

% Buds released from apical dominance Effect of ethylene on cell expansion The overall elongation rate of several intact seedlings, includ- Duration of at indicated time (days) etiolated pea, is inhibited by ethylene within 15 C2H4 treatment 2 4 5 7 ing strongly min (Fig. 3; refs. 19, 29, 30). Inhibition of cell expansion is not 2Hr 0 0 9 18 complete in the growing zone of pea. Instead, in the 4 Hr 0 10 13 23 presence of ethylene, the cells continue to expand, albeit 6 Hr 0 15 30 36 slowly, in an isodiametric manner for a seemingly indefinite 8 Hr 4 27 33 37 time, whereas the same cells in control plants differentiate 12 Hr 7 36 36 43 24 Hr 3 43 46 46 within a few days (6, 31). Ethylene completely prevents 7 Days 0 0 0 0 lignification of fiber elements, and almost stops lignification Control-no C2H4 0 0 0 0 of xylem vessels during a 1-week period (31). Within about 5 days the subapical cells expand to such an extent that their Ethylene (100 ppm) was applied for between 2 and 24 hr during volume exceeds that of the same cells in control tissue (6). the first day, or else continuously for a 7-day period. Bud growth By extending the duration of the growth period, ethylene was appraised continuously throughout the same period. eventually promotes growth of the pea subapical zone, but Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Ethylene in Plant Growth 593

this is not the only way that ethylene may promote growth. In a few cases the rate as well as the duration of growth is enhanced. Thus, ethylene increases the growth rate and duration of growth in rice seedlings (32-34); causes newly divided cells to begin to expand prematurely in fig fruits (35); and stimulates cells in the upper side of the leaf petiole to expand again (36, 37) after their growth essentially has stopped, causing an overgrowth or epinasty of the petiole. The mech- -2A E anism by which ethylene promotes growth in these cases is E / PEA SHOOT CABSBAGE SHOOT unknown, although an auxin assymetry in opposition to the gravity vector has been detected in the case of epinasty (38). 3 That the ethylene-like action of excess auxin on the expansion of cells in the elongating zone is due to auxin-induced ethylene production can be demonstrated by growing plants under hypobaric conditions (6). The diffusivity of gases pass- 2 ing through lenticles or stomata is increased as the absolute +C2H;- PEA ROOT pressure is decreased, and it follows from Fick's law and can be directly demonstrated that, at equilibrium, the endogenous partial pressure of any vapor produced within a plant is directly related to the absolute atmospheric pressure (39). I/l . One-fifth of an atmosphere of water-saturated O2, which is equivalent to humid air from which the N2 has been removed, 60 120 180 240 has little effect on the growth of the elongating zone in etio- TIME (Minutes) lated pea plants. However, it reverses almost completely the FIG. 3. Time course of the effect of 100 nl/liter of ethylene on inhibition of elongation caused by a 2,4-D spray and reveals the elongation of etiolated pea and cabbage shoots, and pea roots. the fact that, when all auxin-induced volatile substances are Seedlings were about 3-cm high when they were treated; roots removed, the main effect of 2,4-D is only to promote growth were about 3-cm long. All tissue was preadapted to dim green (6). light for 10-12 hr before the start of the experiment. Measure- The cause of the transition from longitudinal to radial ments were made under dim green light with a sensitive cathe- growth in the presence of ethylene is a changed orientation tometer (Kang and Burg, 1972). in the direction of deposition of newly formed cellulose microfibrils. This change is revealed as an altered optical bire- growth-inhibitory concentration of auxin in the underside of fringence pattern in the cell wall of tissues treated with ethyl- the root as a result of geostimulation. Several studies have ene, or excess auxin, benzimidazole, or cytokinin. All are suggested that it is not auxin per se that causes the growth agents that cause cellular swelling (9, 12, 40, 41). Normally inhibition, but rather some product of auxin action that can the cellulose microfibrils are deposited in a transverse direc- diffuse across the root (44). That ethylene may be this sub- tion, restricting lateral expansion, but when ethylene (9) or stance is indicated by the fact that the geotropic curving of excess auxin (41, 42) is applied they are deposited instead in a roots is slowed by a competitive inhibitor of ethylene action, longitudinal direction so that longitudinal expansion may be CO2 (7, 45), at a concentration that does not change the over- restricted and radial expansion promoted. As a result of this all growth rate (43). Ethylene by itself prevents roots from changed pattern of cellulose deposition, the epidermal cells, curving geotropically (43), and it has this same effect on the which are not restrained in outward expansion by neighboring stem of pea and certain other seedlings (46, 47). The gas also cells, bulge out and form hair-like structures both in the root prevents phototropism in mustard and radish seedlings, as (7) and stem (31). well as the development of a spontaneous curvature in pea- Cell expansion can also be studied by floating stem sections, stem segments (23). The latter curvature develops during the excised from the growing zone, on solutions containing an first few hours of incubation, and is perceptible within 15 min auxin and other factors. It is a relatively easy matter with after the tissue is cut, but is stopped completely before that etiolated pea and certain other light and dark grown tissue time by applied ethylene. Ethylene does not retard elongation to demonstrate under these conditions that the classical bi- of these same stem segments for 2-3 hr (9, 23), so some other phasic growth-response curve to applied auxin is due to a action of the gas underlies its efficacy in preventing tropistic promotive phase caused by induced growth at a low auxin curvature. This function of ethylene has been examined with concentration, and an inhibitory phase due to induced ethyl- [I4C]indole-3-acetic acid, and has been found to be based on ene production at a high auxin concentration (11, 12, 23). the ability of the gas to completely, rapidly and reversibly In many tissues a very high concentration of auxin, especially inhibit the auxin lateral transport system that is sensitive to a synthetic nondegradable type, causes an additional inhibi- gravity (23). As soon as ethylene is removed, normal curvature tion, the herbicidal effect, which occurs independent of and again develops. In this way ethylene may exert feedback in addition to ethylene action (5, 7, 12, 43). Auxin-induced control over the lateral transport of auxin under certain condi- inhibition of growth in pea roots also is explained in terms of tions (48). induced ethylene production, and under certain conditions an additional, direct, herbicidal auxin effect (7, 43). This has Other processes influenced by ethylene relevance to the geotropic curving of roots that, according to Regulation by ethylene extends to the stages of flower forma- the Cholodny-Went theory, is caused by accumulation of a tion, sex expression, flower fading, and fruit growth and Downloaded by guest on September 26, 2021 594 Burg Proc. Nat. Acad. Sci. USA 70 (1973)

ripening. Flowering of all Bromeliads, including the commer- moval of ethylene under hypobaric conditions, in which case cially important pineapple, is stimulated by very brief exposure the cell-division frequency triples (5). to ethylene or enough auxin to stimulate ethylene production (49). In many flowers, ethylene acts as a flower-fading hor- Mechanism of ethylene action mone (50, 51). A corrolary to this behavior is the fading that To act like ethylene a molecule must have a terminal carbon ensues after pollination of certain flowers. The pollen is a rich adjacent to an unsaturated bond (45). Substituents that with- source of auxin, and releases sufficient growth hormone to draw electrons from the unsaturated bond or sterically in- stimulate ethylene production in the stigma. [14C]Indole-3- hibit an approach to it, reduce activity. A quantitative rela- acetic acid remains localized there, but because ethylene tionship exists between ability to bind metal and biological production is autocatalytic in flowers just as it is in fruits, activity, and a known metal binder, carbon monoxide, will each cell in the stigma gases its neighbors, causing it to pro- replace ethylene in all its actions at a concentration of several duce ethylene, and in this way the stimulus for flower-fading hundred nl/liter. It has been concluded that ethylene binds to spreads to the outer appendages (50). a metal-containing receptor having limited access of approach, with a Km = 6 X 10-10 M (45). The binding must be through Factors controlling ethylene synthesis a noncovalent bond for no exchange of deuterium occurs when deuterated ethylene is applied to responsive plants (58, 59). Two natural factors controlling the rate of ethylene synthesis The transient nature of the binding is also revealed by the have been identified, auxin and red light. Induction of ethyl- fact that many responses to ethylene are rapidly reversible; ene synthesis by an auxin occurs after a lag of 30-60 min, and for example growth inhibition in the etiolated pea stem (Fig. according to inhibitor studies must involve de novo synthesis 3) or root (43) and the action of the gas on lateral transport of a requisite enzyme (52, 53). The enzyme is labile, so that if (23). These same studies also indicate that no lasting product cycloheximide is added after ethylene production has been of ethylene action is produced as a result of binding to its stimulated by an auxin, the production stops within a few receptor. hours. Auxin must be continuously present in vegetative Amongst the active compounds substituting for ethylene is tissue for ethylene to be produced, and the rate of production allene, a close analogue of C02. Because C02 inhibits fruit is proportional to the endogenous content of indole-3-acetic ripening, the possibility was investigated that it might act acid (24, 43, 52, 53). If auxin is removed from the bathing as a competitive inhibitor of ethylene action through its solution, or if the tissue is induced to develop an auxin- similarity to allene. Competition between ethylene and C02 conjugating system, whereby the endogenous auxin content was demonstrated by use of the Lineweaver-Burke plots, and is lowered, ethylene production is diminished proportionately. subsequently has been established for essentially all actions In etiolated seedlings, ethylene is produced primarily in the of ethylene (7, 45). By the same approach it was shown that apex (11, 12, 54), the site of auxin production and therefore ethylene action requires 02. As the 02 concentration is di- the tissue richest in auxin content. After a seedling is exposed minished, the amount of ethylene required for a half-maximal to red light, ethylene production in the apex declines pro- response is increased (45). 02 also is needed for ethylene gressively, at least in part because the ability of auxin to production (Figs. 4 and 5; refs. 60, 61). These interactions ex- stimulate ethylene production is repressed (12, 48, 54-57). plain why controlled atmospheres low in 02 and high in C02 If a response to red light requires an intervening step depen- extend the storage life of many fruits. A simpler and better dent upon inhibition of ethylene production, it can be simu- method of commodity preservation is the operation of a hypo- lated in darkness by application of the competitive inhibitor baric system to remove ethylene, supplying it with water-satu- of ethylene activity, C02, or by removal of ethylene in a rated flowing air to maintain a preselected low level of 02 (62). hypobaric atmosphere. In this manner it has been demon- Extensive laboratory studies have revealed that this method strated that endogenous ethylene production is responsible greatly prolongs the storage life of many fruits, cut flowers, for the formation of the seedling hook, which protects the vegetables, potted plants, and stem cuttings (51, 62). Proto- young leaves or cotyledons from mechanical damage during type shipping containers embodying the method have been their emergence from the soil (12, 57). When the seedling constructed and tested commercially, and the first storage reaches the light, ethylene production is suppressed, the hook warehouses will be in operation within the forthcoming year. opens, and the leaves expand. In darkness the hook opens if the seedlings are placed in a hypobaric chamber or exposed to Biosynthesis of ethylene C02 (5, 54, 55, 57). In fact, the hook never forms in darkness The in vivo precursor of ethylene in fruits and vegetative tis- if the seedling is continuously grown under either of these sue is methionine (61, 63, 64). Ethylene arises from carbons conditions (57). Even after the hook has opened it can be re- 3 and 4, carbon 1 is converted to C02, carbon 2 yields formate closed by application of ethylene or allowing the plant to pro- but no C02, and the S-methyl is retained in the tissue in a non- duce its own ethylene in response to certain treatments (12, volatile form (61, 63, 65). During ethylene synthesis the S- 57). Other responses to red light mediated by repressed methyl is transferred intact, or incorporated as dimethyl ethylene production are stimulation of carotinoid and antho- mercaptan, into homoserine to form homocysteine, which is cyanin synthesis (55, 57), and enhancement of geotropic recycled through several steps to methionine (65). Model sensitivity (48). It takes only 0.1 nl/liter of ethylene to half- systems producing ethylene from methionine and other com- inhibit hook opening in the light, and the same amount of pounds have been described including one that utilizes Cu+ gas to half-inhibit cell division in the dark, so if there is nor- and ascorbate or peroxide (66). A peroxidase system, requiring mally enough ethylene present in the etiolated plant to cause Mn++ (or peroxide), S03--, and monophenol, degrades the hook formation, there must also be enough present to regu- 2-keto analogue of methionine, 2-keto4-methylthiobutyrate, late cell division. This can be directly demonstrated by re- to ethylene, forming C02 from carbons 1 and 2, and dimethyl Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Ethylene in Plant Growth 595

TABLE 2. Dependence of ethylene production on oxygen concentration in the absence of a liquid phase (Imaseki and Burg, 1972)

Relative ethylene Oxygen concentration (%) production at 250 o o 0.5 73 1.0 95 2.0 98 20.0 100 100.0 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 filter paper, and placed in a 25-ml Erlenmeyer flask. The flasks were flushed with N2 containing less than 0.002% 02 until ethylene production ceased for 1 hr. Measured quantities of 02 were then injected, and rates of ethylene production were determined in 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 provides information about the nature of the oxidative step it is important to determine why such different results have been obtained. The solution to this question is afforded by a study on the 0, dependency of respiration in the Aroid spadix (72), Time (Hours) which shows that the presence of a liquid-phase shifts the FIG. 4. Effect of air, N2, and various O2 concentrations on apparent Km to 16% 02, whereas the value is 0.2%02 in the ethylene production by apple discs. Tissue was prepared and incu- case of dry discs maintained in a moist atmosphere. When bated as described in the footnote to Table 3 (Imaseki and Burg, dry apple discs are flushed with N2, ethylene production 1972). immediately stops, but if the discs are floated on a liquid phase through which the N2 is bubbled, ethylene production does not stop for at least one hour (Fig. 4) due to the slow mercaptan from the S-methyl (67). This system also works escape of 02 trapped within the tissue by the liquid phase. with methional, N-acetyl methionine and C-terminal methi- Once ethylene production has ceased under anaerobic condi- onine peptides, but not with methionine itself (68). Isolation tions in the presence of a liquid phase, the Km for the process of a transaminase converting methionine to 2-keto4-methyl- can be determined by addition of 02 to the gas phase. Under thiobutyrate has been reported (69), but the latter cannot be an intermediate in ethylene production by apples because in vivo carbon 2 of methionine forms formate, whereas with 2-keto-4-methylthiobutyrate it yields CO. Moreover in apples, [14C]2-keto-4-methylthiobutyrate is converted less efficiently than [14C]methionine to ethylene and then only after conversion to methionine (64). Objection also has been raised to the proposed role of 2-keto-4-methylthiobutyrate in ethylene production by other tissues (70). Peroxidase systems oxidatively decarboxylate methionine and other amino acids in the presence of Mn++, pyridoxal- phosphate, and monophenol. Nonenzymatic oxidation also occurs, especially with excess pyridoxal and Mn++ at alkaline pH, and transamination to form 2-keto-4-methylthiobutyrate can be effected in model systems with pyridoxal and various metals. When these systems are coupled to the peroxidase system producing ethylene in the presence of S03--, a signifi- * 0.25 0.50 0.75 cant conversion of methionine to ethylene is observed, but K'm = 20%02 1/' (%0°2)1 there is no proof that this test-tube system functions in vivo. To the contrary, in vegetative tissue peroxidase is not the FIG. 5. Lineweaver-Burke plot of data from Fig. 4, in which tissue pretreated with N2 for several hours, until ethylene pro- enzyme induced by auxin when it stimulates ethylene produc- duction had stopped, was exposed to various concentrations of 02 tion (53). to start ethylene production again. The rates are initial values Ethylene production is an aerobic process. Estimates of its taken during the first 40 min after O2 was readded, but were linear dependency on pO2 range from a very high affinity similar to for several hours (Imaseki and Burg, 1972). Downloaded by guest on September 26, 2021 596 Burg Proc. Nat. Acad. Sci. USA 70 (1973)

TABLE 3. Effect of arsenate and phosphate on ethylene tion from [U-'4C]methionine and 14CO2 production from [1- production (Imaseki and Burg, 1972) IC ]methionine by 50% without altering the evolution of respiratory CO2. Ethylene production in apples is insensitive Relative ethylene production to cycloheximide even when slices are treated for 6 hr, so the 0-2 hr 2-4 hr rate-limiting enzymes in fruit tissue are not labile as they are in vegetative tissue induced to produce ethylene by an auxin. Control 100 100 N-acetyl methionine is a substrate for ethylene production in AsO4 (30 mM) 102 75 the peroxidase model system, but at a concentration of 10- AsO4 + P04 (100 mM) 98 91 100 mM it profoundly inhibits ethylene synthesis in vivo, AsO4 (50 mM) 68 39 As04 + P04 (100mM) 98 82 suggesting that the peroxidase system is not the normal path- As04 + Met (10 mM) 68 43 way. Arsenate inhibits ethylene formation (Table 3) and the inhibition is reversed by phosphate. These data and the fact Conditions are similar to those described in the footnote in that dinitrophenol and respiratory poisens inhibit ethylene Table 2, except that the apple discs were floated on 2 ml of the biosynthesis (60, 66) suggest the existance of a high-energy rinsing solution contained in each Erlenmeyer flask. When step in the conversion of methionine to ethylene. S-Adenosyl arsenate + 100 mM phosphate (pH 7) were added, the 100 mM methionine is a possible intermediate since it is formed in Tris.HCl buffer (pH 7) was omitted, and the result was com- good yield from [14C]methionine applied to apple discs (63), pared to that with a control having 100 mM phosphate buffer has a tendency to split-off its S-methyl, and we find it is con- but no arsenate. verted to ethylene by the Cu+-ascorbate model system (but not the peroxidase system), perhaps because the adenosine these conditions the apparent Km is 20%02 and stimulation moeity coveys a positive charge to the sulfur just as Cu+ is by 100% 02 occurs for several hours (Figs. 4 and 5). It is proposed to do. significant that in this case 100% 02 only stimulates ethylene The- substrate for ethylene production, methionine, is synthesis if the tissue was preincubated in N2 (Fig. 4) for it formed from organic acids produced in the mitochondria. is known that some substance accumulates under anaerobic To be converted to ethylene, energy supplied by the mito- conditions that causes ethylene production to be accelerated chondria appears to be required, and electrons released from for several hours after air is readmitted (60). This substance, methionine have to be carried by a cofactor to the respiratory probably methionine, can limit the rate of ethylene synthesis electron-transport system. Presumably because of these so that in the presence of a liquid phase, 100% 02 is only numerous interactions between the mitochondria and the stimulatory when the other factor is present in sufficient ethylene producing system, it has not yet been possible to concentration. In the absence of a liquid phase, the Km for assemble a cell-free system capable of evolving the gas. the 02 dependency of ethylene production is about 0.2% (Table 2). These data indicate that respiration and ethylene This work was supported by National Science Foundation production have the same high affinity for 02, and since there Grant GB-27424. are no known oxidases other then cytochrome oxidase with 1. Fahnstock, G. W. (1858) Proc. Acad. Nat. Sci. Phila., 1. this characteristic (72), the 02 dependency in both cases 2. Neljubow, D. (1901) Bot. Centr. 10, 128. 3. Sievers. A. F. & True, R. H. (1912) U.S. Dept. Agr. Bur. must reflect involvement of the respiratory electron-transport Plant Ind. Bull. 232, 1. system rather then an oxidase specific to ethylene synthesis. 4. Gane, R. (1934) Nature 134, 1008. Oxidation must occur close to the terminal step in ethylene 5. Apelbaum, A. & Burg, S. P. (1972) Plant Physiol. 50, 117. biosynthesis, for immediately after 02 is supplied to N2- 6. Apelbaum, A. & Burg, S. P. (1972) Plant Physiol. 50, 125. at a linear rate 4, 7. Chadwick, A. V. & Burg, S. P. (1967) Plant Physiol. 42, treated tissue ethylene is produced (Fig. 415. refs. 60, 72). In apples evolution of 14CO2 from [1-14C]methi- 8. Webster, P. L. & Davidson, D. (1967) Amer. J. Bot. 54, 633. onine does not occur under anaerobic conditions, nor does it 9. Eisinger, W. & Burg, S. P. (1972) Plant Physiol., 50, 510. occur from [U-'4C]methionine (71), so the decarboxylation is 10. Sfakiotakis, E. (1972) Doctoral thesis, Michigan State an oxidative process. When air is readded, for several hours University. 11. Burg, S. P. & Burg, E. A. (1968) Plant Physiol. 43, 1069. each 14CO2 derived from [1-14C]methionine is accompanied by 12. Burg, S. P. & Burg, E. A. (1967) in Biochemistry & Physiol- one ['4C]ethylene derived from [U-14C]methionine, but after ogy of Plant Growth Substances (Runge Press, Ottawa, Can- several hours when the initially high rate of ethylene synthesis ada), p. 1275. characteristic of N2-treated tissue subsides to the normal 13. Wickson, M. & Thimann, K. V. (1958) Physiol. Plant. 11, is no maintained and some 62. aerobic rate, stoichiometry longer 14. Vacha, G. A. & Harvey, R. B. (1927) Plant Physiol. 2, 187. of the decarboxylated methionine does not yield ethylene. 135. Michener, H. D. (1935) Science 82, 551. A close connection between oxidative decarboxylation of 16. Michener, H. D. (1942) Amer. J. Bot. 29, 558. methionine and evolution of ethylene also can be shown by 17. Morgan, P. W. & Gausman, H. W. (1968) Plant Physiol. 41, means of inhibitors. Ethylene production is inhibited by 44. 18. Burg, S. P. & Burg, E. A. (1967) Plant Physiol. 42, 1224. diethyldithiocarbamate (66), suggesting that a copper- 19. Michener, H. D. (1938) Amer. J. Bot. 25, 711. containing enzyme may be involved, although other metals 20. Burg, S. P., Apelbaum,A., Eisinger, W. & Kang, B. G. (1971) cannot be excluded. With 500 ,uM diethyldithiocarbamate the HortScience 6, 7. inhibition is fairly specific to ethylene formation, reducing 21. Valdovinos, J. G., Ernest, L. C. & Henry, E. W. (1967) with CO2 Plant Physiol. 42, 1803. the rate by 90% without interfering respiratory 22. Ernest, L. C. & Valdovinos, J. G. (1971) Plant Physiol. 48, evolution or 02 consumption. Under these conditions forma- 402. tion of 14CO2 from [1-14C]methionine is also reduced 90%. 23. Burg, S. P. & Burg, E. A. (1966) Proc. Nat. Acad. Sci. USA Similarly, 50 AM dinitrophenol inhibits both ethylene produc- 55, 262-269. Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Ethylene in Plant Growth 597

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