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Proc. Nat. Acad. Sci.

USA
Vol. 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- 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 in- swollen zone develops behind the root tip, root hairs prolifer-
hibits cell expansion within 15 min, but delays differentia-
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 root,
radially. Tropistic responses are inhibited when ethylene
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 the same effect, at least in part by stimulating ethylene
fading, and to fruit growth and ripening. Production of
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 and auxins appear to function in this case through an inter-
4 of methionine, apparently by a copper-containing en-
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
592 Burg Proc. Nat. Acad. Sci. USA 70 (1978)

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


--RNA [3HIURIDINE

a 6

0
<

IJ
F 0
2 0 O

0-

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).

C2H4
dominance. In Petunia the effect of applied ethylene on apical
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

at indicated time (days) The overall elongation rate of several intact seedlings, includ-
Duration of ing etiolated pea, is strongly inhibited by ethylene within 15
C2H4 treatment 2 4 5 7
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
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 dura-
tion 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- EE
-2A
anism by which ethylene promotes growth in these cases is /
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 expan-
sion 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 +
C2
H;- PEA ROOT
pressure is decreased, and it follows from Fick's law and can
be directly demonstrated that, at equilibrium, the endog-
enous partial pressure of any vapor produced within a plant
is directly related to the absolute atmospheric pressure (39).
One-fifth of an atmosphere of water-saturated O2, which is
I /l .
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 micro-
fibrils. 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
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
a noncovalent bond for no exchange of deuterium occurs when
Factors controlling ethylene synthesis
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
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 im-
portant 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/' (%02)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).
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
AsO4 (50 mM) 68 39 100 mM it profoundly inhibits ethylene synthesis in vivo,
As04 + P04 (100 mM) 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.
treated tissue ethylene is produced at a linear rate (Fig. 4, 7. Chadwick, A. V. & Burg, S. P. (1967) Plant Physiol. 42,
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,
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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)
the rate by 90% without interfering with respiratory CO2 Plant Physiol. 42, 1803.
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evolution or 02 consumption. Under these conditions forma- 402.
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Similarly, 50 AM dinitrophenol inhibits both ethylene produc- 55, 262-269.
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Planta 90, 333. 61. Lieberman, M., Kunishi, A. T., Mapson, L. W. & Wardale,
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40. Probine, M. C. (1965) Proc. Royal Soc. Ser. B, 161, 526. 66. Lieberman, M., Kunishi, A. T., Mapson, L. W. & Wardale,
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