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P. W. MORGAN, E. BEYER, JR. and H. W. GAUSMAN'
Texas A (:, M University, College Station, Texas, U.S.A.
Ethylene effects on auxin physiology',2
SUMMARY - The effect of ethylene on auxin transport and destruction
was investigated using isotopically labelled 3-indoleacetic acid (IAA) and
I-naphthaleneacetic acid (NAA). Inhibition of basipetal auxin transport
was demonstrated in stem sections from cotton plants fumigated for only
3 hr and inhibition increased with time. 80th uptake and acropetal transport of auxin by cotton stem sections were inhibited by ethylene. but the
latter effect was not as pronounced as the former. When tested simultaneously. ethylene inhibited ha,ipetal tramport more than acropetal transport.
The proportion of "C in a homogenized sample which remained in the
supernatant after centrifugation was the same for ethylene-treated and
control stem sections subjected to auxin transport. Reduced auxin transport capacity after fumigation with ethylene was shown in a wide variety
of species and the degree of response varied widely. Cotton and okra were
highly susceptible to this effect of ethylene. cowpea and English pea were
intermediate in sensitivity while tomato and sunflower showed little or no
response in 15 hr. Cotton cotyledonary node explants decarboxylated
IAA-I-"C about 100.times faster than NAA-I-"c' but there was no
stimulation of decarboxylation when explants treated with auxin in lanolin
were subsequently fumigated. When previously fumigated stem sections
were allowed to transport IAA-I-"C supplied in agar blocks. there was a
significant increase in decarboxylation in cotton. okra. cowpea and English
pea. but the decarboxylation apparently did not account for the inhibition
of transport. The significance of this effect will be studied further. The
results are compatible with the hypothesis that exogenous ethylene modifies auxin transport and thereby contributes to abscission. reversal of
apical dominance and release of buds from dormancy. A role for native
ethylene in controlling auxin distribution and thus development seems an
additional possibility.
The possibility that ethylene and auxins interact to influence plant growth
has been of interest for many years (Laan. 1934; Zimmerman and Wilcoxon,
1935; Michener, 1938; and Borgstrom, 1939). It is within recent times
that this idea has received significant support. Researchers at Texas A & M
University have studied auxin-ethylene interactions since the early 1950's
'A contribution of the Texas Agricultural Experiment Station. Research was supported
by grants from the National Science Foundation.
"Some of the data in this paper will be part of a Ph.D. thesis by Elmo Beyer. Harold W.
Gausman initiated some studies reported here while a Post-Doctorate Research Associate
at Texas A & M University on leave from the University of Maine.
"Present address, USDA. Soil and Water Conservation Research Service, Weslaco, Texas.
1255
I ~_'h
Morgan, Beyer ami Gal/sman
(Hall. 1952; Hall et al., 1957), and they have sought to encourage the interest of others in this relationship. To this end, Hall and Morgan (1964)
presented evidence for two auxin-ethylene interactions at the 5th International
Conference on Plant Growth Substances in 1963. They reported: (1)
stimulation of ethylene synthesis by auxin, and (2) increase of in vitro IAAoxidase activity by ethylene. Recently, Morgan and Gausman (1966) have
published evidence for a third auxin-ethylene interaction, namely, an ethylenemediated reduction of auxin transport capacity.
Proof of auxin-mediated stimulation of ethylene synthesis by vegetative
tissue (Morgan and Hall, 1962; 1964; Hall and Morgan, 1964) verified
earlier, indirect observations of Zimmerman and Wilcoxon (1935). The
hypothesis, first put forward by Zimmerman and Wilcoxon (1935), that auxin
might produce some of its effects by stimulating the synthesis of ethylene,
was reproposed by Morgan and Hall, 1962, 1964; Hall and Morgan, 1964.
Since the 1963 conference, the auxin-mediated stimulation of ethylene
synthesis has been repeated by other workers and extended to other species
and auxins. This auxin-ethylene interaction has been shown to be associated
with or implicated in abscission, geotropism and phototropism (Abeles and
Rubinstein, 1964; Rubinstein and Abeles, 1965) as well as growth inhibition,
tissue proliferation, flowering, root growth inhibition and geotropism (Burg
and Burg, 1966, 1966a; Chadwick and Burg, 1967) and fruit maturation,
epinasty and leaf senescence (Maxie and Crane, 1967). It is likely that the
auxin-ethylene synthesis interaction is important in other plant responses
affected similarly by exogenous auxin and ethylene (Hall and Morgan, 1964;
Morgan, 1967b).
This paper reports additional studies on the inhibition of auxin transport
by ethylene and some results on the relationship between auxin destruction
and ethylene.
METHODS AND MATERIALS
General: The experiments were conducted with plants grown in a greenhouse (one exception noted ) in inert medium supplemented with a modified
Hoagland's nutrient solution. All cotton plants were Stoneville 213. The
details which varied between experiments are given in the text and tables.
IAA-1-"C. IAA-2-"C and NAA-1- C were obtained from Nuclear-Chicago
Corporation. Purity of these materials was verified by chromatography. All
counts were corrected for background and where specifically noted for
counter efficiency.
11
Auxin Transport: General technique used have been described (Morgan
and Gausman, 1966). and they wiiI be reviewed only briefly along with a
description of modifications employed. Stem sections were cut and placed
apex down (base down in acropetal transport experiments) in small plastic
ETHYLENE EFFECTS ON AUXIN PHYSIOLOGY
1257
\ i,tis or beakers containing the HC-labelled auxin in a volume to limit immerTransport was allowed to proceed at
3() C. When the transport period was completed sections were divided into
sc~mcnts, segments were homogenized and aliquots of the homogenate were
dried and counted. In some cases, agar receiver blocks were placed on the
section base during transport and these were counted separately from the
tissue segments. Agar blocks were either counted with a Geiger-Muller
tube or, for the experiments in Tables V and VI, in a liquid scintillation
counter by the method of Lagerstedt and Langston, 1966.
For the experiment represented by Table II, a stem section holder
was constructed of plexiglass strips fitted with rubber septa with holes to
accommodate stem sections. Sections were secured downward in the holder,
lowered to touch the liquid IAA-I-"C source and then raised slightly. This
manipulation lifted small columns of liquid from the surface to the cut ends
of the sections. Entry of IAA-1-"C from a liquid source was thereby limited
to the cut surface.
In acropetal transport experiments, verification that transported He
represented auxin depended upon: (I) extraction and identification of IAA2_n C by chromatography as the only soluble "c containing material in cotton
tissue following basipetal transport (Morgan and Gausman, 1966), and (2)
similar patterns of transport of both IAA-2-"C and NAA-I-11C in either control or ethylene-treated sections (NAA being much less subject to metabolism
in cotton).
.
In the species survey, the transport technique developed for cotton and
cowpeas (Morgan and Gausman, 1966) was applied to several species as a
routine technique. The species and varieties tested were; Gossypium hirsutum L., Cotton, var. Stoneville 213; Hibiscus esculentus L., Okra, var.
Clemson Spineless; Pisum sativum L., English Pea, var. Alaska; Oryza sativa
L., Rice, var. Nato; Phaseolus vulgaris L., Black Valentine Bean, var. Asgrow
-Valentine; Zea mays L., Corn, var.' I 27CX441; Sorghum vulgare Pers.,
Grain Sorghum, var. RS 610; Raphanus sativus L., Radish, var. Scarlet Globe;
Vicia faba L., Broad Bean, var. Long Pod Flava; Cucurbita maximua
Duchesne., Squash, var. Early White Bush; Vigna sinensis Endl., Cowpea,
var. Blackeye; Helianthus annuus L., Sunflower, var. Red; Triticum aestivum
L., Wheat, var. Tascosa; Lycopersicon esculentum Mill., Tomato, var.
Summertime. Cotton, okra, rice, corn, grain sorghum, radish and wheat
were fumigated 19 days after planting. Broad bean, cowpea and sunflower
were fumigated 14 days after planting, while English pea, squash and tomato
were treated 9, 11 and 21 days after planting, respectively. The tissue used
was a 15 mm long stem section cut from directly below the petiole of the
first true leaf. Exceptions were wheat, corn, grain sorghum and rice where
a section was cut from the entire shoot starting immediately below the first
leaf blade. English pea and broad bean sections were cut from immediately
~ll1l1 of sections to less than 5 mm.
1258
Morgan, Beyer
lind Glil/Smllll
below the third node while squash was cut to yield a section from just below
the cotyledonary node.
The experiment described by Tables VI and VU was conducted with
plants from a growth room with 30 C days, 24 C nights and a 15 hr photoperiod. Plant age at fumigation was 16 days for cowpea, sunflower and okra,
20 days for cotton and tomatoes and 9 days for English pea. Stem sections
10 mm long were cut from the same reference point for each species used
in the previous survey experiments, except that cotton and okra sections
were cut from directly below the cotyledonary node. During transport,
stem sections in a plexiglass holder were in their natural orientation to
gravity. IAA-l-"C was applied to the apex of sections in 2 % agar blocks, 6.4
mm in diameter by 2.18 mm.
Ethylene Fumigation: Except where otherwise noted, intact plants were
exposed to 100 ,I{I ethylene per liter air at 30 C in the dark in a sealed, gastight container for 15 hr. Control plants from the same population were
enclosed in similar containers without ethylene. Stem sections were cut and
used for transport immediately after the containers were opened in the morning. For the experiments in Table V and VI, auxin transport was conducted
in desiccators which, for the previously fumigated sections, contained 100
ppm ethylene.
CO, Collectzon and Counting:
"CO, was recovered in 20% KOH or NaOH
in petri dishes in static systems or by scrubbing exhaust air in experiments
involving continuous air flow. "CO, aliquots were transferred by diffusion
to hyamine hydroxide and counted in a liquid scintillation system (Schwerner and Morgan, 1966), The system of diffusion, transfer and counting was
shown to be 50% etlicient.
RESUL TS ,AND DISCUSSION
Time sequence of eth)'lene inhibition of auxin transport.
We have investigated the length of fumigation period necessary for ethylene to reduce the basipetal (polar) auxin transport capacity of stem sections
from cotton plants (Beyer and Morgan, 1967), Results indicated that reduction of transport capacity appeared after a few hours (between 6 and 9) and
increased with duration of ethylene fumigation (Table I). The phenomenon was studied further lIsing an apparatus which exposed the apical end
of stem sections to small columns of liquid lifted by surface tension from a
liquid donor source. Results of two experiments (Table II) show that
ethylene significantly reduced auxin transport capacity in stem sections after
plants were fumigated for only 3 hr. Generally, there was an inverse
relationship between the amount of "C recovered and the distance from the
absorbing segment. The ethylene-treated segments contained significantly
1259
ETHYLENE EFFECTS ON AUXIN PHYSIOLOGY
Effect of the duration of ethylene fumigation on the distribution of I':\.\-l-l'C
in stem sections of cotton following 2 hr of basipetal transport.·
-
Fumigation
Time,
Hours
3
6
9
Stem Segmen ts,
mm from Apical End
Treatment
10-20
Control
Ethylene
72
77
I )ifference
-s
Control
Ethylene
[ )ifference
Control
Ethylene
[ )itference
12
Control
Ethylene
I lifference
15
Control
Ethylene
[ )itference
I
20-30
I
CPM
43
41
30-40
I
Total
Transport
S~ Total
Reduction
0
40-50
37
29
30
33
181
181
+2
-1
+-l
()
61
69
37
,)7
27
26
24
18
149
150
-8
0
+1
+6
-1
72
61
38
34
29
22
29
23
168
140
+11
+4
+7
+6
+27
75
55
44
31
30
23
28
21
177
130
+20
+13
+7
+7
+47
28
18
15
9
13
8
14
8
70
43
+10
+6
+5
+6
+27
I
-1
+17
+27
+39
*Each datum is the average of observed cpm of 4 replications, each containing 5 stem
segments from 24 day old plants grown during January, 1965, Fumigation was at 26°C
and auxin supplied at 2 X 10-'[\1.
less He than the control segments in the three most basal segments. There
was a statistically significant increase in He accumulation in the absorbing
segment (0-1 mm) of ethylene-treated sections in all cases except one 3 hr
fumigation. This accumulation appears to be due to the inability of ethylenefumigated sections to move lie out of the absorbing segment into basal tissue,
an effect which may be due to binding. Differences in total uptake were
not statistically significant, but there was a trend toward a reduction of total
uptake in ethylene-treated sections. This trend increased with time as did the
accumulation of auxin in the absorbing tissue. Possibly, accumulation of
auxin in the absorbing tissue of ethylene-treated sections reduced the auxin
concentration gradient between tissue and source and thus eventually reduced
auxin uptake. The magnitude of the reduction of basipetal transport by
ethylene after 3 hr, apparent in experiment I where there was no difference in
total uptake, increased with time in agreement with the trend seen in Table I.
Differences in the timing of the auxin transport response in Tables I and II
Morgan, Beyer and Gal/sman
1260
Table II.
Effect of the duration of ethylene fUllligation on the distribution of IAA-l-"C
in cotton stem sections following 2 hr of basipetal transport.
EJ(periment,
Time
.**.
Stem Segments, Illm from ;\pictl End
Treatment
0-1
1-6
I
6-11
I
11-16
I
Tot<l.t
16-21
CI':\!
1,3 hr
83
!OO
116
61
51
46
103
29
26
15
315
314
-20'
-16
+10'
+ 17'··
+ 11'··
+1
114
125
110
96
40
26
24
12
5
2
293
261
-11
+14
+14"
+12'··
+3'"
+32
80
ISO
112
104
69
16
53
6
30
9
343
286
-70'·
+8
+53'"
+47'"
+21'"
+57
Control
Eth\ lene
170
227
130
101
65
13
41
7
9
6
415
354
[ lilferen(e
-57"
+29"
Control
Ethylene
I litference
II, 3 hr
Control
Ethylene
I )ilTerenee
I, 15 hr
Control
Ethylene
I lilTerence
II, 15 hr
I
+52"'1 +.H'··
+3"
+61
·Stati~tically sil!;niflc<lnt
··Stati~tically ~il!;nilicant
at 0.10 level.
at (LOS level.
···Statistical'" ,il!;nilicant at ()'(11 level.
····Experi"'eni~ "'ere conducted with 27 da\' old plant>; grown during February and June
1967 re'penivel\'. Each datulll i~ the average of ob,erved cpm of 5 replications each cuntaining oJ qenl sq;lIIent,. Fumigation II';" at .l()OC and auxin ,upplied at 1 X IU-'J!.
are probably due to plant variability or differences in fumigation temperature
and experimental techniques.
There is no indication from our data that the inhibition of auxin transport does not occur in vivo. One might argue that the effect of ethylene
on auxin transport occurs exclusively in broken cells at the absorbing surface
and is thus dependent upon a cut surface. However, in Table II when the
counts in the terminal segment (16-21 mm) were expressed as a percent of
the counts in the 1-16 mm region (thus excluding from consideration all HC
in the 0-1 mm segment) there was a smaller percent of transport in the
ethylene than control (8 So average for control versus 5 % for ethylene).
Cell counts indicate that there are 35 to 40 parenchyma cells per mm of
stem in cotton of this age; thus, some of the HC in the 0-1 mm segments
was not in cut cells and was transported auxin.
The results in Table II would be predicted from the model for auxin
transport proposed by Leopold and Hall (1966). In the model an increase
ETHYLENE EFFECTS ON AUXIN PHYSIOLOGY
1261
in fixation in undisturbed cells markedly increases the steepness of the curve
of auxin concentration versus distance from the donor. When the data in
Table II were averaged and plotted as cpm per mm versus distance from
the apex, the slope of the curve for the ethylene-treated stem section was
steeper than the one for the control. The accumulation of HC in the 0-1 mm
portion of the ethylene-treated stem sections could indicate such an increase
in some type of binding or destruction of the transport system. If the
distribution of auxin observed in ethylene-treated stem sections (Table II)
was due to binding in cut cells alone, one would expect saturation of this
layer of cells and a reversal' of the ethylene effect with time or auxin concentrations. In the study by Morgan and Gausman (1966) however, stem
segments were exposed to higher concentrations of IAA-2-"C and N AA-lHC for up to 23 hr; yet, a distribution pattern similar to that in Table II
persisted.
We conclude that ethylene-mediation of the basipetal auxin transport
system begins rather promptly after exposure to ethylene and increases with
time. While the ability to detect the change in the polar auxin transport system
appears to vary with the sophistication of the experimental approach, the
modification demonstrated here occurs before such relatively slow responses
of plants to ethylene as abscission, loss of apical dominance and reversal of bud
dormancy (Hall et al., 1957 and Heck et al., 1961). We observed little leaf
abscission with ethylene-treated cotton before 15 hr, and usually most appearance of small changes in auxin flow might serve as an inductive signal for
processes leading to abscission.
The results here provide an explanation for recent reports that ethylene
does not inhibit polar auxin transpo~t. Experiments by Burg and Burg (1965,
1965a, 1966) and Abeles (1966) involved exposure of stem sections to ethylene and auxin transport simultaneously for relatively short periods of time
(3 hr or less). After becoming aware of the paper by Morgan and Gausman
( 1966), Burg and Burg. (1966) verified that longer fumigation periods before
transport reduce auxin transport capacity in etiolated pea stems, and this observation was repeated more recently (Burg and Burg, 1966b). Based on the
statements of Burg and Burg (1966b) and the data in Tables I and II, we
conclude that there was no effect of ethylene on auxin transport in the earlier
experiments (Burg and Burg, 1965, 1965a, 1966 and Abeles, 1966) because
the length of fumigation was inadequate to demonstrate an effect with the techniques and species used (see later). Thus ethylene does reduce the capacity
of stem and petiole tissue to transport auxin basi pet ally. We call this effect of
ethylene an inhibition of auxin transport (Morgan and Gausman, 1966). In
1162
Morgan, Beyer and Gausman
our opinion, the expression that ethylene is not an inhibitor of auxin transport
but does destroy the auxin transport system (Burg and Burg, 1966, 1966b) is
undesirable because: (1) it is initially negative and therefore tends to obscure
the possible physiological significance of the interaction, and (2) it is essentially an unproven mechanistic theory based on timing information which is
modified by the present results. The distinction between occurrence of the
response in fumigated plants and its absence in sections (Burg and Burg,
1966b) may not be valid since Morgan and Gausman (1966) found the
response in both, provided fumigation preceeded testing of the rapidly exhausted transport system. The time sequence of the ethylene effect on auxin
transport shown here refutes the objections raised by Abeles (1966) to auxin
transport inhibition being involved in abscission.
Acropetal auxin transport.
After our first experiments indicated that ethylene reduced the polar
auxin transport capacity of tissues, we extended our study to determine the
effect of ethylene on acropetal (apolar) auxin transport (Morgan and Gausman, 1965).
Ethylene fumigation caused a statistically significant reduction in the
amount of "C from IAA-2-"C present in stem segments (Table III). With
one exception the reduction in "C with ethylene treatment occurred in all
Table III.
Effect "f eth\ Iene on uptake and di"triiJutioll of radioactivit\, from L\.-\-
2-"C in "tem secti,,", of ('ottOIl following' acropetal transport. :\verages of 1 and 2 hr
incubations with auxin ,at 2 X lll-":-'1.
Stem Segments, mm from Base
Experiment'"
Treatment
0-10
I
I
Total
10-20
I
20-30
I
30--W
C 1':\1
II
il20
Control
Ethylene
.t320
Si-!
.tOl
.+77
104
.+52
S9
8623
.+884
I lilfercllce
+28()O**
+1 i3*
+3i3**
+393**
+3739'
S8i.'i
3936
70S
325
427
169
.t65
131
7472
.t561
+.l80'
+258'
'Control
Eth,lene
I lifierence
! + 19,,9'
+334
+2911*
'Statisticalh' ,i~nit'lC;lnt at the (),OS len'l.
"Statisticall" ,ignilicant at the (),Ol level.
"'Experiments I and II \\'ere conducted with 21 and 25 da\' old planh rbpectivel,'. One
and 2 hr trial..; analYzed ,'eparately for each experiment. Each datum repre,ents the average
of.t replications, each containing .t stem sections. Data corrected for counter efficienc)'.
ETHYLENE EFFECTS ON AUXIN PHYSIOLOGY
T bl
1263
IV. Effect of ethylene on uptake and distribution of radioactivity from NAAstem sections of cotton following basipetal and acropetal transport for 4 hr.· .. •
1.~'Ce in
Direction
of
Transport
Basipetal
Acropetal
=0
Stem Segments, mm, Absorbing End
Treatment
Total
0-10
Control
Ethylene
9181
7931
Difference
+1250*
Control
Ethylene
4975
41M
Difference
+811**
I
10-20
CPl\!
1673
101
I
20-30
I
30-40
I
40-50
220
34
59
60
34
9
11167
8135
+1572**
+186**
-1
+25*
+3032**
119
34
26
9
26
9
5163
4225
+85
+17
17
9
+8
+17
+938**
*Statistically significant at 0.05 level.
**Statistically signilicant at 0.01 level.
. .
***NAA-I·IlC supplied at 2 X 10-'1\1 to 20 day old plants, 4 replIcatIOns of each treatment,
each containing 5 plants. Data corrected for counter efficiency.
segments and in the totals. The ethylene-treated sections in experiment I
contained a statistically significant lower percentage of their total He in the
20-30 and 30-40 mm segments than did the control; however, there were no
significant differences in the percent of total activity appearing in the various
segments of ethylene and control treatments in experiment II. In two other
experiments with cotton stem and petiole sections we found a significant reduction in IAA-2-"C in ethylene-treated sections, but no significant effect on
levels of activity in segments above the basal segment.
Ethylene was also shown to influence the distribution of N AA-1-"C.
Under conditions where ethylene significantly lowered both uptake and basipetal transport of the synthetic auxin (Table IV), ethylene reduced "C in the
absorbing segment during acropetal transport.
Evidently, when ethylene-treated tissues were exposed to acropetal auxin
transport, both the total amount of auxin present and the amount of auxin
transported were reduced. The inhibition of transport (as indicated by the
proportion of "C moved out of the absorbing segment) was only inhibited to
a statistically significant degree in experiment I, Table III, however, the trend
toward such an inhibition appeared in all but our first experiment. Auxin
uptake and transport have been shown to be separate processes (Goldsmith,
1967; Keitt and Baker, 1967), and irrespective of the mechanism involved, the
data here indicate that the effect of ethylene on acropetal uptake is more
evident.
1264
Morgan, Beyer and Gausman
Several of the effects of ethylene on acropetal auxin transport agree with
Leopold and Hall's (1966) model of auxin transport. Since ethylene inhibits
basipetal transport (Morgan and Gausman, 1966), a smilar effect on the
acropetal process would be expected because the model assumes that cells
transport auxin in both directions. In the model an increase in fixation
causes an increasing predominance of basipetal over acropetal transport with
time (Fig. 3, Leopold and Hall, 1966), which agrees with our data. In Table
IV, the ratio of total uptake in apical versus basal tissue was 2.2 to 1, while
the ratio of transported "C (activity not in the 0-10 mm segment) in basipetal
versus acropetal direction was 10.6 to I. Ethylene reduced both of these
ratios - to 1.9 to 1 and 3.3 to I respectively. The largest change was the
ethylene-mediated reduction of the ratio of basipetal to acropetal transport.
This ratio would not change, between control and ethylene plants, if
ethylene affected basipetal and acropetal auxin transport to an equal degree.
Several studies show that basipetal auxin transport predominates in young
tissue and declines with age; consequently acropetal transport eventually
increases in magnitude relative to basipetal transport (Jacobs, 1950';
McCready and Jacobs, 1963). Thus, the predominance of the ethylene
effect on basipetal transport may also be viewed as an acceleration of senescence in stem sections.
To the extent that there are apices and similar cells producing auxin and
transporting it basipetally, the effect of ethylene on polar auxin transport infers
a resultant decrease in auxin arriving at a given internode, petiole, bud or
abscission lone. This concluded result would not be changed by a simultaneous inhibition of acropetal auxin transport. Thus, the finding that ethylene
inhibits acropetal as well as basipetal auxin transport appears to add to the
over all significance of this auxin-ethylene interaction. If, as Jacobs (1962)
proposes, the transport capacity of the internode limits the amount of auxin
traveling through the stem. ethylene could modify overall auxin distribution
independently of synthesis.
Effect of ethylene on solubility of radioactivity from lie-labelled auxins.
In several of our auxin transport experiments, tissue homogenates from
ethylene and control segments were centrifuged at 20,000 x g for 30 min and
counts of the crude homogenates and the supernatant of the centrifuged aliquots compared. In 2 replicated experiments with IAA-2-"C in which basipetal transport was reduced by ethylene, there was no effect of the gas on the
percent of "C not precipitated, which was 38 : : ': : 2%. With NAA-l-"C (Table
IV) there was also no effect of ethylene on the proportion of "C not precipitated.
These tests indicate that ethylene does not reduce the uptake or transport
of either NAA-l-"C or IAA-2-"C by increasing binding of the auxins to a
ETHYLENE EFFECTS ON AUXIN PHYSIOLOGY
1265
particle which can be precipit-:.ted by centrifugation at 20,000 x g; however,
binding to a soluble material may be involved.
Survey of the response of auxin transport to ethylene in several species.
C)nsiderable difference was noted in the effect of ethylene on auxin
transpori in cowpea and cotton (Morgan and Gausman, 1966). Since wide
variations in species sensitivity to ethylene exist, a survey was conducted to
determine the degree of ethylene-mediated reduction of auxin transport in
divergent species (Morgan, 1967a).
Auxin uptake varied, but since our primary interest was in auxin transport, the amount of lie transported to a given segment or agar block was
expressed as a percent of the total auxin absorbed or transported for that
particular species and treatment. Next, the percentage of activity in a given
segment or agar block in ethylene plants was divided by the same value for
the control and the result expressed in percent. If the proportion of total
activity reaching agar receiver blocks had been the same in ethylene-treated
and control plants, a value of 100 would be obtained and the value would
decrease as the ethylene effect increased.
Results of the survey are presented in Table V as the averages of all
experiments. These values are not absolute, but several observations are
Table V. Effect of ethylene on the percent of absorbed I:\A-l-'jC which was transported
beyond the apical 5 mm of a 15 mm stem segment and on the percent of the transported
IAA-I-IlC which was moved into agar receiver blocks. Species listed from most to least
sensitive based on averages of the two criteria.
Total
Transported**
E/C',;
Species*
Cotton (4)
Okra (1)
English Pea (2)
Rice (1)
Black \'alentine nean (2)
Corn (2)
Sorghum (2)
Radish (2)
nroad Bean (2)
Squash (2)
Cowpea (2)
Sunflower (1 )
\\'heat (2)
Tomato (2)
,
44
68
56
47
75
82
80
50
70
81
88
73
96
95
Agar Blocks***
E/C(~,
28
17
38
51
35
38
B
75
57
63
60
77
66
102
Average
36
42
48
49
55
60
62
63
64
72
74
75
81
99
*:\umber of experiments indicated in parenthesis after common name. Effect of ethylene
larger in one of two experiments for Engli:.h peas, corn, radish, "quash and wheat. J..\.-\-I-I4C
supplied at 1 X 10-':\1.
**1 'C in basal stem segments and agar block,; divided b\· total I'C in ethdene treatment
divided by the same vallie for the control treatment and 'the results expres~ed as percent.
***I·C in agar blocks divided by the I'C in basal stem segments in eth\·lene treatment
divided by the same value for the control treatment and the result expressed in percent.
1266
Morgan, Beyer and Gausman
warranted. Ethylene reduced auxin transport capacity to some degree in a
wide variety of species. Some species, cotton being the best example,
exhibited a large decline in auxin transport due to ethylene by both of the
indices used. Some species showed very little effect of ethylene on auxin
transport capacity - the obvious example is tomato. The monocots were
distributed from more sensitive to more insensitive groups, and cowpea
showed relatively little ethylene effect on auxin transport. Based on extensive
abscission and inability to recover after fumigation, cowpea was classified as
one of the most sensitive species to ethylene (Heck and Pires, 1962). Both
of these symptoms could indicate serious disruption of auxin physiology, but
this disruption may not reach its maximum within the 15 hr treatment period
employed in these tests.
The above studies were extended with 6 species representing both extreme and intermediate rankings in Table V. Agar blocks were used as donor
sources of IAA-l-"C to preclude the possibility of surface creeping on
hirsute stems. The relative effect of ethylene on auxin transport between
species (Table VI) was quite similar to that indicated in survey experiments
(Table V). Ethylene caused a statistically significant reduction of lAA-I-HC
transported into agar receiver blocks in cotton, okra, cowpea and English
pea. In cotton. okra. and cowpea there was a characteristic accumulation of
HC in the apical segment and decrease in activity in the basal segment, a
distribution pattern which can indicate a disruption of transport. However,
the differences between "c activity in apical and basal segments of ethylene
and control tissues for these species were only statistically significant for
cotton. The existence of the trends plus real differences in the agar blocks
indicates that ethylene modified the auxin transport system in okra and
cowpea. The reduction in transported IAA-l-" C in okra is more impressive
in view of the significantly higher level of "C taken up by the ethylenetreated okra in this experiment. In this and the survey experiments, auxin
transport in the English pea was inhibited to a lesser degree than several
other species. There wa's apparently little or no inhibition of auxin transport
in tomato or sunflower under conditions of this experiment. In both species
total uptake and transport were significantly higher in ethylene sections.
When the indices in Table V were calculated for the data in Table VI,
the ranking of the species was similar to that in Table V. This result further
suggests a modification of transport in cotton. okra. cowpea and English
pea and a very small effect with tomato and sunflower.
While there is no direct proof that the interaction of ethylene and auxin
transport occurs in the plant. its in vivo occurrence is supported by Guttenberg
and Steinmetz (1947). They found that the auxin recovered from A vena
coleoptile tips by diffusion through A vena coleoptile sections is reduced if
the sections arc cut from ethylene treated plants. They obtained similar results
with sunflower and beans. Their technique did not distinguish between
1267
ETHYLENE EFFECTS ON AUXIN PHYSIOLOGY
Table VI. Effect of ethylene on L\.~-~-I4C transport in 6 species. Transl?ort conduct~d
for!' hr with agar donor blocks cont,lInrng L\.-\-I-I4C at 1 X 1O~6!\I. SpeCl~s ~~range~ In
order from most sensitive to least based on averages of two CrItena used In I able \ .
Species
Treatment
Apical
Segments
Basal
Segments
Agar
Blocks
Total
105ol5
10368
CPM'"
Cotton
6850
98ol8
3580l
512
III
-2998'
+3072"
+103--
+177
olO-i7
6752
1902
1097
IOol
31
6053
7880
-2705
+805
+73-
-1827-
Control
Ethylene
3ol13
3826
1195
951
Sol
28
ol662
ol805
DifTerence
-ol13
+2H
+26"
-H3
6581
71ol2
902
1219
107
52
7509
8ol13
-561
-317
+55-
-823
Control
Ethylene
2970l
-1607
951
1024
55
79
3980
5710
Difference
-1633--
-73
-2-1
Control
Ethylene
1682
2580l
80S
1121
55
90
DitTerence
-902
-316-
Control
Ethylene
Difference
Okra
Control
Ethylene
I )ifterence
Cowpea
English Pea
Control
Ethylene
Difference
Tomato
Sunflower
8
-35
-1730-'
25-12
3795
-1253*
-Statistically significant at the (i.05 level.
-'Stati,tically sig-nificant at the ().(JI level.
-'-COllllts corrected for cOllnter efficiency. Each datum represents average cpm per rep
for -1 reps, 7 plallh per rep. Each segment 5 111111 long.
ethylene effects on synthesis,' destruction and transport, but their results
indicate that the supply of diffusable, native auxin in physiological amounts
is disrupted by ethylene. Another indirect indication that ethylene may
inhibit auxin transport in vivo is the observation by Goeschl and Pratt (1965,
1966) that most of the ethylene produced by the etiolated pea arises from
the plumular hook region. Exposure to light reduces production of ethylene
by pea seedlings (Goeschl, 1967).
Scott and Briggs (1960) found the apical bud was the sole source of
diffusable auxin in the epicotyl of the green pea, but they could not obtain
auxin by diffusion from etiolated peas even though they applied KeN or
chlorogenic acid to inhibit oxidation. This failure to obtain diffusable auxin
from etiolated pea seedlings may have been due to a reduction in the capacity
of the auxin transport system caused by abundant ethylene production
1268
Morgan. Beyer and Gausman
(which inhibits plumule opening of beans, Kang et al., 1967; and peas,
Goeschl, 1967).
There is now evidence that other plant hormones, gibberellins and
cytokinins, promote the transport of auxin (Jacobs and Case, 1965; Pilet,
1965; McCready et al., 1965). In view of the growing recognition of ethylene as a hormone, its association with senescent tissue (Hall et aI., 1957)
and the decline in polar auxin transport with age (Jacobs, 1950; McCready
and Jacobs, 1963), it seems possible that endogenous ethylene along with
other hormones may function to regulate auxin distribution and thus plant
development. Results here and earlier (Guttenberg and Steinmetz, 1947;
Morgan and Gausman. 1966) are compatible with a hypothetical role of
ethylene as a regulator of auxin distribution. The enhancement of ethylene
synthesis by both auxin (Morgan and Hall, 1964) and abscisic acid (dormin)
(Abeles and Holm, 1966) supports the hypothesis. We are now investigating this interesting idea.
Effect of ethylene on auxin destruction.
Ethylene increased both IAA-oxidase and peroxidase activity of extracts
of fumigated plants (Morgan and Hall, 1963; Hall and Morgan, 1964), and
these enzymes could destroy IAA, be directed to other functions or remain
nonfunctional. Morgan and Gausman (1966) concluded that the effect' of
ethylene on auxin transport was not just an acceleration of auxin breakdown,
but this conclusion docs not eliminate the possible importance of auxin destruction in plant responses to ethylene.
To clarify the effect of ethylene on auxin destruction, we are using
IAA-I-"C and measuring decarboxylation. Decarboxylation of IAA-I-"C by
petioles of cotton cotyledonary node explants was approximately 100 times
greater than for N AA-I-' 'C (Fig. I). The magnitude of the differences in
Fig. I were verified in 2 additional experiments with N AA-l-"C and IAAI_lie. These results indicate that cotton plants have an auxin destruction
system much more specific for native IAA than for synthetic NAA. The
cotton explant, shown to contain a functional IAA-oxidase system (Schwertner and Morgan, 1966), showed reduced decarboxylation of IAA-I- H C
applied in lanolin when fumigated with ethylene. Recovery of "CO" from
the ethylene-treated explants was only 83 % and 85 % of the control after
5 days in separate experiments. Similar results were obtained in 3 additional
experiments where ex plants were immersed in IAA-I-"C solution and then
exposed to ethylene or room air (control). Evidently under these conditions,
ethylene either: (I) reduced auxin transport to a degree which spared auxin
from destruction. (:2) depleted available energy by accelerating respiration
(Herrero and HaiL 1960). or (3) did not stimulate decarboxylation of IAA.
In another approach to the question of ethylene-auxin destruction interaction, decarboxylation of IAA-I-"C by previously fumigated plants was
ETHYLENE EFFECTS ON AUXIN PHYSIOLOGY
1269
30
(\J
o
u
~ 25
/AA-/-Y·
V>
ct
~
ffi
•
20
~
u
w
a:
~u
15
•
a
w
~
0-
'0
ct
~
!zw
5
u
a:
~
NAA-/- /4C
_ _ _ _ _ _ _ 0 _____ 0 _ _ _ _ 0
2
4
6
8
DAYS
Fig. 1. Accumulative decarboxylation of IAA-l-"C and NAA-l-"C. Auxins at 5 x
to-"M applied in lanolin to petioles of cotton cotyledonary explants. Data are averages
of 2 replications (observed cpm recovered divided by total applied dpm). Autoxidation
of same batch of IAA-l-"C in same collection system was less than 0.2 percent in 6
days.
measured in some of our trans'port studies. In Table VI, each species was
enclosed in a separate desiccator with KOH during auxin transport after which
stem section holders were removed rapidly, desiccator lids replaced and the
KOH collected 18 hr later. Ethylene greatly increased the decarboxylation of
IAA-I-"C by those species in which there was also an effect on auxin transport. In cotton and cowpea ethylene more than doubled decarboxylation, and
the effect was almost as large with English pea and okra (Table VII). There
was no detectable effect of ethylene on decarboxylation of IAA-l-"C in
tomato and sunflower. With cotton, English pea and cowpea but not okra
there was a 27% average reduction in the amount of IAA-l-"C that could
be detected in the agar donor blocks as well as a significant reduction in
the total recovery of "C from the ethylene treatment.
The results in Table VII, although not representing recovery of all lAC
released as "CO" indicate that ethylene fumigation increased the capacity of
stem sections from some species to decarboxylate IAA-I-HC. The response
was less apparent or absent in species not showing an effect of ethylene on
auxin transport under the conditions of the test. There are two apparent
1270
Morgan, Beyer and Gallsman
I'able VI l.
Effect of ethylene OJl decarboxylation of I.-\.-\-I-"C as indicated by recovery
of I'C0 2 . Data from transport experiment shown in Table VI.
Species
Cotton
Okra
Cowpea
English Pea
Tomato
Sunflower
Control
Ethylene
CPM per replication'
1MO
3889
1898
2981
2269
5982
~669
772~
130-1
1110
1.132
1089
[)ifference
-22~9
-108,)
-3713
-.lOSS
+128
-21
'Corrected for counter efficiency. Counts of duplicate aliquot,; from single samples frolll
each treatment.
explanations for the increased decarboxylation and the decreased HC present
in donor blocks: (I) ethylene may actually increase both auxin uptake and
in vivo destruction. or (2) ethylene may increase the oxidases (peroxidases)
present at the cut surface, some of which might be secreted into the agar
blocks. It seems unlikely that ethylene increases overall uptake of auxin
since the contrary has often been indicated in NAA-I-I'C and IAA-2-"C
experiments (Morgan and Gausman, 1966) in which decarboxylation is insignificant or would not affect measurements of total uptake. Thus. the second
possibility or a combination of it with some iijcrease of internal decarboxylation seems to be the most reasonable explanation.
One might conclude. as did Michener (1938), that ethylene simply
increases destruction of IAA thus decreasing auxin available to the transport
system. This interpretation is not compatible with the results in Table VI.
Ethylene did not significantly reduce the total amount of I'C recovered in the
stem segments and agar receiver blocks from either cotton, English pea,
cowpea or okra; yet, in all of these species there was some indication that
ethylene reduced auxin transport. Transport was reduced by ethylene in
Table II (3hr exposure, experiment I) but not total 'Ie present. Also, there
are several cases where uptake and transport of NAA-I-"C were inhibited by
ethylene (Table IV and Morgan and Gausman. 1966) while decarboxylation
of NAA-l-"C appears insignificant in cotton (Fig. I and Morgan and Gausman, 1966). Since the effect of ethylene on transport is not fully explained
by either induced changes in uptake or destruction, there may be independent
effects of ethylene on all 3 processes.
CONCLUSIONS
We conclude that the ethylene-mediated reduction of basipetal auxin
transport capacity in stem sections begins rather promptly after plants
are exposed to the gas and increases with time. The inhibition of both acropetal
ETHYLENE EFFECTS ON AUXIN PHYSIOLOGY
1271
and basipetal transport by ethylene and the predominance of the effect on
the later process indicated that ethylene would reduce the supply of auxin
reaching a given internode, petiole, bud or abscission zone. This reduction
of polar transport would be similar to the condition in senescent tissue and
could play" causative role in abscission and loss of apical dominance.
The effect of ethylene on auxin transport is not due to binding of auxin
to a centrifugable particle. The appearance of ethylene-mediated auxin
transport inhibition in many species suggests a functional sole for the phenomenon. Quantitative differences in auxin-transport modification by ethylene,
while of possible physiological significance, do not appear to be a general
explanation of species sensitivity to ethylene.
While the stimulation of decarboxylation of IAA by ethylene does occur
under some circumstances, we conclude that this process does not fully explain
the inhibition of basipetal transport of either auxin used in the studies here.
Possibly both inhibition of auxin transport in stems and petioles and stimulation of auxin destruction in abscission zones and buds could occur in the same
plant in response to ethylene.
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,I