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-
thesis, and growth in the meristems of roots, shoots, and
axillary buds, without influencing RNA synthesis. Apical
dominance 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 just
as if the apex had been removed. A similar mechanism
may underly ethylene-induced release from dormancy of
buds, 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 to
enormous size. These cells grow isodiametrically rather
than longitudinally because their newly deposited cellu-
lose microfibrils are laid down longitudinally rather than
radially. Tropistic responses are inhibited when ethylene
reversibly and rapidly prevents lateral auxin transport. In
most of these cases, as well as certain other instances,
ethylene action is mimicked by application of an auxin,
since auxins induce ethylene formation. Regulation by
ethylene extends to abscission, to flower formation and
fading, and to fruit growth and ripening. Production of
ethylene is controlled by auxin and by red light, auxin
acting 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 an
intervening step dependent upon inhibition of ethylene
production have been identified. Ethylene action requires
noncovalent binding of the gas to a metal-containing
receptor having limited access, and produces no lasting
product. The action is competitively inhibited by C02, and
requires 02. Ethylene is biosynthesized from carbons 3 and
4 of methionine, apparently by a copper-containing en-
zyme in a reaction dependent upon an oxygen-requiring
step with a Km = 0.2% 02. The oxidative step appears to be
preceded by an energy-requiring step subsequent to me-
thionine formation.
Early observations on the effects of ethylene on plant growth
are contained in a literature, dating to 1858 (1), that describes
the behavior of plants exposed to illuminating gas. In 1901,
the study of a strange growth habit of etiolated pea seedlings
raised in laboratory air contaminated with illuminated gas
revealed that the biologically active component of the gas is
ethylene (2). In the presence of ethylene the seedlings undergo
a "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 numerous
other changes in the growth and development of seedlings
might have received immediate attention had not it been
learned soon thereafter that ethylene ripens fruits (3). Almost
all effort was diverted to this economically important aspect
of ethylene action, and by the mid-1930s it was established
that ethylene is produced autocatalytically just in advance
of fruit ripening (4). The gas became known as the fruit-
ripening hormone, and not until the early 1960s was the role
Abbreviation: 2,4-D, 2,4-dichlorophenoxyacetic acid.
591
of ethylene in plant growth destined to be studied intensively
again.
Effects of ethylene on cell division
When etiolated pea seedlings are grown continuously in the
presence of a trace of ethylene, the stem hardly elongates and
root growth is inhibited about 60% (refs. 2, 5, 6; Fig. 3). A
swollen zone develops behind the root tip, root hairs prolifer-
ate, and the root deflects plageotropically in the gravitational
field (5-7); similar changes occur in the stem (2, 5, 6). The
major cause of the overall growth inhibition is cessation or
retardation of the mitotic process in the meristems of the root,
shoot, and axillary buds (5, 6). Within a few hours after
ethylene is applied, the number of mitotic figures in the stem
apex begins to decline, and within about 10 hr mitosis almost
stops. Auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D)
cause the same effect, at least in part by stimulating ethylene
production in the apex. Ethylene inhibits mitosis in the root
apex by about 60% and 2,4-D has a similar effect, but very
high concentrations of 2,4-D stimulate mitosis in the elon-
gating zone of the root just as in the elongating zone of the
stem. These divisions give rise to root initials in both tissues,
and ethylene does not interfere with their formation, although
it 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 hours
after 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 of
the stem where no cell divisions occur (Fig. 2; ref. 5). RNA
synthesis from [3H]uridine or [14C]ATP is not affected in
either tissue (Figs. 1 and 2; ref. 9). DNA synthesis is inhibited
because DNA polymerase activity is reduced (10). In roots,
shoots, and lateral buds of the etiolated pea plant there is a
quantitative relationship between the inhibitions of DNA
synthesis, 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 ethylene
production (11, 12). Inclusion of a cytokinin overcomes the
inhibitory action of ethylene or auxin (11-13), but whether
this is the manner in which auxin and cytokinin normally
control apical dominance is not resolved. A puzzling thing
about ethylene and bud growth is the fact that often apical
dominance is broken after an ethylene treatment even though
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
buds in the subapical zone of the shoot are released from cor-
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
FIG. 1. Effect of ethylene (100 nl/liter) applied to intact
etiolated pea seedlings (7-days old) on the rate of incorporation
of ['H]thymidine into DNA and [3Hjuridine into RNA. After
the seedlings were exposed to ethylene for the indicated number of
hours, either the hook elbow or the apical tip and plumular leaves
were excised and pulse-labeled with a solution containing 1
,ACi/ml of thymidine or uridine and 50 mM potassium phosphate
buffer (pH 7). DNA and RNA were extracted, and isolated; radio-
activity was determined. The results were the same regardless of
whether ethylene was present or absent during the pulse-labeling
period (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 then
removed (Table 1). When ethylene is removed after an 8- to
12-hr treatment essentially all buds are released from apical
TABLE 1. Ethylene-induced release of apical dominance in
5-week-old Petunia x hybrid Grandiflora calypso seedlings
(Ramos and Burg, 1972)
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
dormancy in root initials, buds, seeds, and tubors (14-16)
after brief ethylene treatment.
Several hours after ethylene application, the capacity of the
polar auxin transport system begins to decline (17, 18), and
within 10 hr it is inhibited almost 90% in pea subapical stem
tissue. As a result, the auxin content of the stem is lowered
markedly (19-21), possibly in part because auxin synthesis
also may be curtailed by ethylene (22). The cause of the block-
age of auxin transport is not known, but is not enhanced
auxin destruction (2, 23) or conjugation (24). In this manner
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
some plants when an ethylene treatment is terminated. Other
symptoms of auxin deficiency would be expected and have
been observed when ethylene is applied. This explains in part
how the gas causes the abscission of leaves, flowers, and fruits
(17, 25, 26) for auxin has the opposite effect. Both auxin and
ethylene have been implicated as natural regulators of the
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)
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-
anism by which ethylene promotes growth in these cases is
unknown, although an auxin assymetry in opposition to the
gravity 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-induced
ethylene production can be demonstrated by growing plants
under hypobaric conditions (6). The diffusivity of gases pass-
ing through lenticles or stomata is increased as the absolute
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
equivalent 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 the
inhibition of elongation caused by a 2,4-D spray and reveals
the fact that, when all auxin-induced volatile substances are
removed, 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 orientation
in 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 are
agents that cause cellular swelling (9, 12, 40, 41). Normally
the cellulose microfibrils are deposited in a transverse direc-
tion, restricting lateral expansion, but when ethylene (9) or
excess auxin (41, 42) is applied they are deposited instead in a
longitudinal direction so that longitudinal expansion may be
restricted and radial expansion promoted. As a result of this
changed pattern of cellulose deposition, the epidermal cells,
which are not restrained in outward expansion by neighboring
cells, 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 an
auxin and other factors. It is a relatively easy matter with
etiolated pea and certain other light and dark grown tissue
to demonstrate under these conditions that the classical bi-
phasic growth-response curve to applied auxin is due to a
promotive phase caused by induced growth at a low auxin
concentration, 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, especially
a synthetic nondegradable type, causes an additional inhibi-
tion, the herbicidal effect, which occurs independent of and
in addition to ethylene action (5, 7, 12, 43). Auxin-induced
inhibition of growth in pea roots also is explained in terms of
induced ethylene production, and under certain conditions
an additional, direct, herbicidal auxin effect (7, 43). This has
relevance to the geotropic curving of roots that, according to
the Cholodny-Went theory, is caused by accumulation of a
Ethylene in Plant Growth 593
EE
-2A
/
PEA SHOOT CABSBAGE SHOOT
3
2
+
C2
H;- PEA ROOT
I /l .
60 120 180 240
TIME (Minutes)
FIG. 3. Time course of the effect of 100 nl/liter of ethylene on
the elongation of etiolated pea and cabbage shoots, and pea roots.
Seedlings were about 3-cm high when they were treated; roots
were about 3-cm long. All tissue was preadapted to dim green
light 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 of
the root as a result of geostimulation. Several studies have
suggested that it is not auxin per se that causes the growth
inhibition, but rather some product of auxin action that can
diffuse across the root (44). That ethylene may be this sub-
stance is indicated by the fact that the geotropic curving of
roots 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 from
curving geotropically (43), and it has this same effect on the
stem of pea and certain other seedlings (46, 47). The gas also
prevents phototropism in mustard and radish seedlings, as
well as the development of a spontaneous curvature in pea-
stem segments (23). The latter curvature develops during the
first few hours of incubation, and is perceptible within 15 min
after the tissue is cut, but is stopped completely before that
time by applied ethylene. Ethylene does not retard elongation
of these same stem segments for 2-3 hr (9, 23), so some other
action of the gas underlies its efficacy in preventing tropistic
curvature. This function of ethylene has been examined with
[I4C]indole-3-acetic acid, and has been found to be based on
the ability of the gas to completely, rapidly and reversibly
inhibit the auxin lateral transport system that is sensitive to
gravity (23). As soon as ethylene is removed, normal curvature
again develops. In this way ethylene may exert feedback
control 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
594 Burg
ripening. Flowering of all Bromeliads, including the commer-
cially important pineapple, is stimulated by very brief exposure
to 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 that
ensues after pollination of certain flowers. The pollen is a rich
source of auxin, and releases sufficient growth hormone to
stimulate ethylene production in the stigma. [14C]Indole-3-
acetic acid remains localized there, but because ethylene
production 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-fading
spreads to the outer appendages (50).
Factors controlling ethylene synthesis
Two natural factors controlling the rate of ethylene synthesis
have been identified, auxin and red light. Induction of ethyl-
ene synthesis by an auxin occurs after a lag of 30-60 min, and
according to inhibitor studies must involve de novo synthesis
of a requisite enzyme (52, 53). The enzyme is labile, so that if
cycloheximide is added after ethylene production has been
stimulated by an auxin, the production stops within a few
hours. Auxin must be continuously present in vegetative
tissue for ethylene to be produced, and the rate of production
is proportional to the endogenous content of indole-3-acetic
acid (24, 43, 52, 53). If auxin is removed from the bathing
solution, or if the tissue is induced to develop an auxin-
conjugating system, whereby the endogenous auxin content
is lowered, ethylene production is diminished proportionately.
In etiolated seedlings, ethylene is produced primarily in the
apex (11, 12, 54), the site of auxin production and therefore
the tissue richest in auxin content. After a seedling is exposed
to red light, ethylene production in the apex declines pro-
gressively, at least in part because the ability of auxin to
stimulate 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 inhibitor
of ethylene activity, C02, or by removal of ethylene in a
hypobaric atmosphere. In this manner it has been demon-
strated that endogenous ethylene production is responsible
for the formation of the seedling hook, which protects the
young leaves or cotyledons from mechanical damage during
their emergence from the soil (12, 57). When the seedling
reaches the light, ethylene production is suppressed, the hook
opens, and the leaves expand. In darkness the hook opens if
the seedlings are placed in a hypobaric chamber or exposed to
C02 (5, 54, 55, 57). In fact, the hook never forms in darkness
if the seedling is continuously grown under either of these
conditions (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 repressed
ethylene production are stimulation of carotinoid and antho-
cyanin synthesis (55, 57), and enhancement of geotropic
sensitivity (48). It takes only 0.1 nl/liter of ethylene to half-
inhibit hook opening in the light, and the same amount of
gas to half-inhibit cell division in the dark, so if there is nor-
mally enough ethylene present in the etiolated plant to cause
hook formation, there must also be enough present to regu-
late cell division. This can be directly demonstrated by re-
Proc. Nat. Acad. Sci. USA 70 (1973)
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 carbon
adjacent 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 biological
activity, and a known metal binder, carbon monoxide, will
replace ethylene in all its actions at a concentration of several
hundred nl/liter. It has been concluded that ethylene binds to
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
deuterated ethylene is applied to responsive plants (58, 59).
The transient nature of the binding is also revealed by the
fact 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 product
of ethylene action is produced as a result of binding to its
receptor.
Amongst the active compounds substituting for ethylene is
allene, a close analogue of C02. Because C02 inhibits fruit
ripening, the possibility was investigated that it might act
as a competitive inhibitor of ethylene action through its
similarity to allene. Competition between ethylene and C02
was demonstrated by use of the Lineweaver-Burke plots, and
subsequently has been established for essentially all actions
of ethylene (7, 45). By the same approach it was shown that
ethylene action requires 02. As the 02 concentration is di-
minished, the amount of ethylene required for a half-maximal
response is increased (45). 02 also is needed for ethylene
production (Figs. 4 and 5; refs. 60, 61). These interactions ex-
plain why controlled atmospheres low in 02 and high in C02
extend the storage life of many fruits. A simpler and better
method 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 method
greatly 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 been
constructed and tested commercially, and the first storage
warehouses 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 carbons
3 and 4, carbon 1 is converted to C02, carbon 2 yields formate
but 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 dimethyl
mercaptan, into homoserine to form homocysteine, which is
recycled through several steps to methionine (65). Model
systems producing ethylene from methionine and other com-
pounds have been described including one that utilizes Cu+
and ascorbate or peroxide (66). A peroxidase system, requiring
Mn++ (or peroxide), S03--, and monophenol, degrades the
2-keto analogue of methionine, 2-keto4-methylthiobutyrate,
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
TABLE 3. Effect of arsenate and phosphate on ethylene
production (Imaseki and Burg, 1972)
Relative ethylene production
0-2 hr 2-4 hr
Control 100 100
AsO4 (30 mM) 102 75
AsO4 + P04 (100 mM) 98 91
AsO4 (50 mM) 68 39
As04 + P04 (100 mM) 98 82
As04 + Met (10 mM) 68 43
Conditions are similar to those described in the footnote in
Table 2, except that the apple discs were floated on 2 ml of the
rinsing solution contained in each Erlenmeyer flask. When
arsenate + 100 mM phosphate (pH 7) were added, the 100 mM
Tris.HCl buffer (pH 7) was omitted, and the result was com-
pared to that with a control having 100 mM phosphate buffer
but no arsenate.
these conditions the apparent Km is 20%02 and stimulation
by 100% 02 occurs for several hours (Figs. 4 and 5). It is
significant that in this case 100% 02 only stimulates ethylene
synthesis if the tissue was preincubated in N2 (Fig. 4) for it
is known that some substance accumulates under anaerobic
conditions that causes ethylene production to be accelerated
for several hours after air is readmitted (60). This substance,
probably methionine, can limit the rate of ethylene synthesis
so that in the presence of a liquid phase, 100% 02 is only
stimulatory when the other factor is present in sufficient
concentration. In the absence of a liquid phase, the Km for
the 02 dependency of ethylene production is about 0.2%
(Table 2). These data indicate that respiration and ethylene
production have the same high affinity for 02, and since there
are no known oxidases other then cytochrome oxidase with
this characteristic (72), the 02 dependency in both cases
must reflect involvement of the respiratory electron-transport
system rather then an oxidase specific to ethylene synthesis.
Oxidation must occur close to the terminal step in ethylene
biosynthesis, 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 it
occur from [U-'4C]methionine (71), so the decarboxylation is
an oxidative process. When air is readded, for several hours
each 14CO2 derived from [1-14C]methionine is accompanied by
one ['4C]ethylene derived from [U-14C]methionine, but after
several hours when the initially high rate of ethylene synthesis
characteristic of N2-treated tissue subsides to the normal
aerobic rate, stoichiometry is no longer maintained and some
of the decarboxylated methionine does not yield ethylene.
A close connection between oxidative decarboxylation of
methionine and evolution of ethylene also can be shown by
means of inhibitors. Ethylene production is inhibited by
diethyldithiocarbamate (66), suggesting that a copper-
containing enzyme may be involved, although other metals
cannot be excluded. With 500 ,uM diethyldithiocarbamate the
inhibition is fairly specific to ethylene formation, reducing
the rate by 90% without interfering with respiratory CO2
evolution 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-
Proc. Nat. Acad. Sci. USA 70 (1973)
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 insensitive
to cycloheximide even when slices are treated for 6 hr, so the
rate-limiting enzymes in fruit tissue are not labile as they are
in vegetative tissue induced to produce ethylene by an auxin.
N-acetyl methionine is a substrate for ethylene production in
the 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 the
inhibition is reversed by phosphate. These data and the fact
that dinitrophenol and respiratory poisens inhibit ethylene
biosynthesis (60, 66) suggest the existance of a high-energy
step in the conversion of methionine to ethylene. S-Adenosyl
methionine is a possible intermediate since it is formed in
good 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 (but
not the peroxidase system), perhaps because the adenosine
moeity coveys a positive charge to the sulfur just as Cu+ is
proposed to do.
The- substrate for ethylene production, methionine, is
formed 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 from
methionine have to be carried by a cofactor to the respiratory
electron-transport system. Presumably because of these
numerous interactions between the mitochondria and the
ethylene producing system, it has not yet been possible to
assemble a cell-free system capable of evolving the gas.
This work was supported by National Science Foundation
Grant GB-27424.
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