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Plant Physiol. (1999) 120: 897-906
Ethylene Plays Multiple Nonprimary Roles in Modulating the
Gravitropic Response in Tomato1
Andreas Madlung,
Friedrich J. Behringer, and
Terri L. Lomax*
Department of Botany and Plant Pathology and Center for Gene
Research and Biotechnology, Oregon State University, Corvallis,
Oregon 97331-2902
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ABSTRACT |
Ethylene is known to interact with
auxin in regulating stem growth, and yet evidence for the role of
ethylene in tropic responses is contradictory. Our analysis of four
mutants of tomato (Lycopersicon esculentum) altered in
their response to gravity, auxin, and/or ethylene revealed
concentration-dependent modulation of shoot gravitropism by ethylene.
Ethylene inhibitors reduce wild-type gravicurvature, and extremely low
(0.0005-0.001 µL L 1) ethylene concentrations can
restore the reduced gravitropic response of the auxin-resistant
dgt
(diageotropica) mutant to wild-type levels. Slightly higher concentrations of ethylene
inhibit the gravitropic response of all but the ethylene-insensitive nr
(never-ripe)
mutant. The gravitropic responses of nr and the
constitutive-response mutant epi
(epinastic) are slightly and
significantly delayed, respectively, but otherwise normal. The reversal
of shoot gravicurvature by red light in the lz-2(lazy-2)
mutant is not affected by ethylene. Taken together, these data indicate
that, although ethylene does not play a primary role in the gravitropic
response of tomato, low levels of ethylene are necessary for a full
gravitropic response, and moderate levels of the hormone specifically
inhibit gravicurvature in a manner different from ethylene inhibition of overall growth.
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INTRODUCTION |
Gravity and light are important environmental cues that aid plants
in orienting themselves optimally to access life-supporting resources
such as water and light. The process by which plants orient their roots
and shoots with respect to gravity, gravitropism, has been studied
intensively for more than 100 years (Darwin, 1888 ). The Cholodny-Went
theory (Went and Thimann, 1937), widely regarded as the leading
hypothesis explaining gravitropism, postulates that the plant hormone
auxin, which is synthesized in the shoot apex and transported
basipetally down the shoot, is redistributed asymmetrically in response
to gravistimulation. This lateral redistribution of auxin leads to
higher concentrations of the hormone in the lower half of the stem,
which triggers an increased growth response in that region and results
in upward curvature of the plant.
The role of ethylene in the gravitropic response has been discussed
extensively in the literature, with research results split between two
opposing groups: those indicating that ethylene plays a role in the
gravitropic response (Zobel, 1973; Kang and Burg, 1974; Wheeler and
Salisbury, 1980, 1981; Clifford and Oxlade, 1989; Philosoph-Hadas et
al., 1996) and those supporting the opposing view (Clifford et al.,
1983; Kaufman et al., 1985; Harrison and Pickard, 1986; Woltering,
1991). It has also been reported that ethylene, at concentrations of
100 µL L1, can redirect 7-d-old etiolated pea
plants to grow downward rather than upward when gravistimulated (Burg
and Kang, 1993).
Most auxin-resistant mutants exhibit an altered gravitropic
phenotype, such as the Arabidopsis mutants aux1 (Bennett et
al., 1996), axr1 (Lincoln et al., 1990), axr2
(Wilson et al., 1990), and axr3 (Leyser et al., 1996), as
well as the tomato (Lycopersicon esculentum) mutant
dgt
(diageotropica; Kelly and Bradford, 1986; Hicks et al., 1989). These auxin-resistant mutants are also resistant to ethylene. However, small amounts of
ethylene have been reported to restore a normal gravitropic response in
dgt (Zobel, 1974), suggesting that ethylene may act downstream of auxin in gravitropic signal transduction. Elongation of dgt roots is less sensitive to application of ethylene
than its isogenic parent cv VFN8 (Muday et al., 1995), whereas the sensitivity of dgt shoot elongation to ethylene is greatly
increased when compared with the wild-type response (Shi and Cline,
1992). In contrast, it has been reported that the gravitropic behavior of light-grown wild-type tomato seedlings treated with ethylene and
ethylene inhibitors is not altered (Harrison and Pickard, 1986).
Although a transitory burst of ethylene has been observed in tomato
seedlings within 2 min of horizontal placement (Harrison and Pickard,
1984), a subsequent study concluded that the lack of measurable changes
in ethylene production during the first 3 h of the gravitropic
response was evidence that ethylene does not play a role in the signal
transduction cascade of graviresponses (Harrison and Pickard, 1986).
Kaufman et al. (1985) reported a sharp increase in ethylene production
between 6 and 24 h after gravistimulation in oats but concluded
that this increase occurred too late to be a cause for gravitropism. An
increase in the production of ethylene on the lower half of
gravistimulated dandelion plants has been observed; however, this
increase also occurred hours after the gravitropic response had been
initiated (Clifford et al., 1983), which led to the conclusion that
ethylene may modulate but not initiate the gravitropic response.
Applied ethylene is known to inhibit hypocotyl elongation growth in
etiolated plants as part of the "triple response" phenomenon (Goeschl and Kays, 1975; Ecker, 1995). Growth inhibition due to high
ethylene leads to decreased tropic responses, which are by definition
dependent on growth. There is evidence that the reduction in elongation
caused by ethylene occurs via an interaction between ethylene and
auxin. Whereas ethylene production is stimulated by auxin, ethylene can
suppress polar (basipetal) transport of the auxin IAA (Schwark and
Schierle, 1992) and can also influence asymmetric distribution of auxin
(Schwark and Bopp, 1993). Ethylene mediates the formation and
maintenance of the seedling apical hook via an unknown component
downstream of CTR1, a protein kinase that is part of the ethylene
signal transduction pathway (Peck et al., 1998), and it has been
suggested that the Arabidopsis HLS1 gene controls
differential cell growth during hook formation by regulating auxin
activity via its N-acetyltransferase activity (Lehman et
al., 1996). The acetylation process itself may also be modulated by
ethylene. Recently, Luschnig et al. (1998) isolated the EIR1
gene from Arabidopsis. This gene shows homology to a bacterial membrane
transporter and, if mutated, confers reduced sensitivity to ethylene
and agravitropism to roots. Experimental evidence in yeast suggests
that EIR1 may play a role in auxin transport (Luschnig et
al., 1998).
Although growth inhibition by ethylene has been described mostly for
etiolated plants, it has been reported that ethylene promotes cell
growth and elongation in light-grown Arabidopsis seedlings maintained
on nutrient-deficient medium (Smalle et al., 1997). Ethylene promotion
of cell growth and elongation in the stem has also been reported for
the aquatic plant Ranunculus sceleratus when submerged in
water (Abeles et al., 1992), as well as for ethylene-treated etiolated
rice coleoptiles (Satler and Kende, 1985). In the meadow grass
Poa pratensis and oat, the ethylene-releasing compound ethephon has been implicated in the increase of tiller internode length (Abeles et al., 1992). Thus, it appears that ethylene
can both inhibit and promote stem growth. Although it is intriguing to
compare the multitude of different effects that ethylene has been
reported to exert on the growth process, it is important to note that
this information has been gathered from a large number of different
species. Different species may respond to the same level of ethylene in
different ways, as the literature clearly demonstrates.
Ethylene may also play a role in the integration of signals from light
and gravity. In soybean, red light was found to reduce ethylene
production by as much as 45% while promoting hypocotyl elongation
(Samimy, 1978). Both effects were found to be reversible by FR,
suggesting that phytochrome regulates hypocotyl growth via ethylene
(Samimy, 1978). Arabidopsis seedlings, when grown under red light, lose
their ability to reorient themselves to the gravity vector. This loss
is also reversible by FR and has been shown to be controlled by both
phytochrome A and phytochrome B (Poppe et al., 1996). In Arabidopsis,
the plant hormone cytokinin, acting via ethylene, can restore
gravitropism in seedlings that were rendered agravitropic by red light
(Golan et al., 1996).
One approach to elucidating how auxin, ethylene, and light interact in
shoot gravitropism is to study the response of mutants that are altered
in their response to one or more of these factors. To this end, we used
two tomato mutants with altered gravitropic responses: the
auxin-resistant dgt mutant (Kelly and Bradford, 1986; Hicks
et al., 1989), which exhibits a reduced gravitropic response (Lomax et
al., 1993), and the lz-2
(lazy-2) mutant in which the direction of shoot gravitropism is reversed in a
phytochrome-dependent manner (Gaiser and Lomax, 1993). We also
investigated whether two tomato mutants altered in their ethylene
physiology, the ethylene-overproducing epi
(epinastic) mutant of tomato (Fujino
et al., 1988) and the ethylene-insensitive nr
(never-ripe)
mutant (Wilkinson et al., 1995; Yen et al., 1995) exhibit alterations
in their gravitropic response mechanism. Taken together, the results
from these experiments lead us to propose that ethylene plays multiple,
but not primary, roles in modulating the gravitropic response in
tomato.
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MATERIALS AND METHODS |
Plant Material
Wild-type tomato (Lycopersicon esculentum Mill.)
varieties Ailsa Craig (AC) and Pearson (P), as well as four mutants,
epi, dgt, nr, and lz-2 were
used. The dgt and lz-2 mutants were maintained in
the AC background, whereas epi was in the VFN8 background. nr was used in AC, as well as the P isogenic parent line for
curvature experiments. Seeds of lz-2, epi, and
dgt, nr in AC, and AC were originally obtained from C.M.
Rick (University of California, Davis). Seeds of nr in the P
background, as well as wild-type P seeds, were kindly provided by Dr.
Harry Klee (Univerisity of Florida, Gainesville). All lines were
propagated by selfing at the Oregon State University Botany Farm
(Corvallis).
Gravicurvature Experiments
For the gravicurvature measurements (Figs. 1 and 2), plants were
grown in 10- × 10-cm pots filled with vermiculite and kept in darkness
at 29°C for 4 to 5 d (epi plants were grown for 6-7 d). Seedlings were gravistimulated in their pots in red light (General
Electric F40/Pl fluorescent lights filtered through a Roscolux Filter
no. 27, 2.51 µmol m2
s1 measured from 640-680 nm, transmission
maximum  = 660 nm; Rosco, Hollywood, CA) in a plant incubation
chamber (Hoffman, Albany, OR) at 29°C. At the indicated times,
individual representative plants were excised at the vermiculite level
and photocopied, and the curvature was determined with a protractor.
The data presented were pooled from three experiments.
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| Figure 1.
Phenotype of wild type (WT, cv AC),
dgt, lz-2, epi, and
nr tomato seedlings. Seedlings were germinated in the
dark for 4.5 d (epi for 6.5 d) and then
oriented vertically in darkness (left), vertically in red light (R,
center), or horizontally in red light (right) for 20 h.
Representative seedlings were selected and photographed. Note that the
lz-2 mutant in the upright or horizontal positions bends
downward when exposed to red light.
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| Figure 2.
The kinetics of gravitropic responses of etiolated
auxin- and ethylene-response mutants of tomato in red light (R) at
29°C compared with wild type (WT). A, Short-term kinetics. B,
Kinetics over 20 h from the same experiments.  , Wild type cv
AC;  , dgt;  , lz-2;  ,
nr in AC background; and  , epi. Plants
were grown in darkness for 4.5 d (epi for 6.5 d) and subsequently transferred to an incubator equipped with
fluorescent lights filtered through red Roscolux filters. At the times
indicated, representative plants were cut at the vermiculite level and
photocopied. The angle of gravicurvature was determined from the
photocopies using a protractor. Data were pooled from three independent
experiments and are mean values. Error bars reflect ±SE,
n = 6 to 21 (with n = 9.3 on
average per time point).
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Ethylene Evolution Measurements
A gas chromatograph (model GC-8A, Shimadzu, Kyoto, Japan) equipped
with a flame-ionization detector and a 122-cm Poropak Q column (Waters)
was used for all measurements of ethylene evolution. Approximately 20 seeds were germinated on 1 mL of 1% agar in 10-mL vials (Fisher
Scientific) and grown in the dark for 4.5 d at 29°C. Prior to
gravistimulation, the vials were capped with an airtight serum stopper
(Fisher Scientific). Care was taken to use plants that were short
enough not to reach the top of the vial or the serum stopper to prevent
artifactual ethylene production resulting from seedling damage or
stress response (Lehman et al., 1996). Upright controls or
gravistimulated seedlings, which had been reoriented 90°, were
either exposed to red light or kept in the dark. At the indicated
times, a 1-mL headspace sample was withdrawn from the vial using a 1-mL
tuberculin syringe with a 25-gauge needle (Becton Dickinson) and
injected into the gas chromatograph. Ethylene concentrations were
determined from a standard curve, and total ethylene evolution was
normalized to the fresh weight of the seedlings.
Gravicurvature Response to Applied Ethylene
Plants used for measurements of gravicurvature in response to
various ethylene concentrations (Fig. 5) were grown in 3-mL scintillation vials filled with vermiculite and kept in darkness at
29°C for 4 to 5 d (epi plants were grown for 6-7 d).
Plants measuring approximately 1.5 to 2 cm from the root/shoot node to the hook were selected (epi plants were generally shorter
and measured only 1-1.5 cm), and an interval 1 cm down the hypocotyl from the top of the hook was marked with black ink (Steig Products, Lakewood, NJ) to monitor elongation growth during gravistimulation. Subsequently, six to seven vials containing one seedling each were set
in a holder with the cotyledons pointing up. The holders were
transferred into 1-L Mason jars lined with Whatman 3MM paper, and the
jars were sealed airtight. Prior to gravistimulation, the jars were
injected with ethylene at various concentrations and then reoriented
90°. All plants were kept under red light (General Electric 40R red
fluorescent tubes filtered through red acrylic, Shinkolite 102 [Argo
Plastics Co., Los Angeles, CA] 0.95 µmol m2
s1 measured from 640-680 nm, transmission
maximum = 642 nm) during gravistimulation. Fluence
measurements were made with an LI-1800 spectroradiometer (LI-COR,
Lincoln, NE). All manipulations of the plants were done under dim-green
safelight. After gravistimulation, the plants were excised at the
vermiculite level and photocopied. The length of the marked interval
was measured, and curvature was determined with a protractor. Data were
pooled from three to six independent experiments, and the
SE was calculated.
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| Figure 5.
Ethylene stimulation and inhibition of
gravicurvature at concentrations that do not inhibit hypocotyl growth.
Wild type (WT, ), lzy-2 (), dgt
(), nr in AC (), and epi ().
Gravicurvature (A) and elongation (total length of marked interval
after 20 h, C) were measured after 20 h of gravistimulation
in red light. The bending capacity (B) was derived from the
gravicurvature data in A as the percentage change to show differences
of sensitivity in the mutants.
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Ethylene Inhibitor Studies
For studies with ethylene inhibitors (Fig. 4), plants were grown
and gravistimulated in sealed jars as described above for the
gravicurvature experiments. NBD (Aldrich) was pipetted onto the filter
paper just prior to gravistimulation and allowed to evaporate after the
jars were sealed airtight. AVG (Sigma) was applied by watering upright
plants with AVG solutions 5 h before gravistimulation to allow
time for uptake of AVG through the root system.
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| Figure 4.
The ethylene action inhibitor NBD inhibits wild
type (WT) gravicurvature but not elongation. The 4.5-d-old etiolated
wild-type (AC) seedlings were marked with ink at the top and bottom of
a 1cm interval extending from the top of the hook down the hypocotyl.
Total hypocotyl length was approximately 1.5 cm. Seedlings in air-tight
jars were treated with the indicated concentrations of NBD and
reoriented with respect to gravity. After 20 h of
gravistimulation, the increase in length of the marked hypocotyl region
(% elongation increase of marked interval at 0 h; ) and
hypocotyl curvature ( ) were measured. Data are representative of
three experiments, n = 6 to 8 for each point.
Results are means ± SE.
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Statistical Analysis
All statistical analyses were performed using Microsoft Excel
software. P values reflect those of two-sided Student's t
test analyses.
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RESULTS |
The dgt, lz-2, epi, and nr
Mutants of Tomato Exhibit Altered Gravitropic Responses
If ethylene plays an important role in the gravitropic response of
plants, then mutants that are altered in ethylene perception or
response should exhibit altered gravitropism. Alternatively, analysis
of the ethylene physiology of known gravitropic mutants may provide
insight into the role of ethylene in plant responses to gravity. In
Figure 1, the morphology of four tomato
mutants, epi (an ethylene-overproducing mutant),
nr (an ethylene-insensitive mutant that has been
demonstrated to lack an ethylene receptor), dgt (an
auxin-resistant mutant with a retarded gravitropic response), and
lz-2 (a mutant that exhibits a phytochrome-regulated
reversal of the shoot gravitropic response), is compared with that of
wild-type seedlings. All seedlings were grown in ambient air and kept
either upright in darkness (Fig. 1, left), upright in red light (Fig. 1, center), or gravistimulated in red light (Fig. 1, right). When vertically oriented, all of the seedlings maintained correct
orientation away from the gravity vector with the exception of
lz-2 seedlings which bent downward in red light.
Gravistimulation, achieved by placing the plants horizontally in the
presence of red light results in complete upward reorientation of
wild-type, epi, and nr seedlings, incomplete
upward curvature of dgt seedlings, and reversed (positive) curvature of lz-2 seedlings. Under all of the conditions,
seedlings carrying the epi lesion display characteristics of
wild-type seedlings treated with high ethylene concentrations,
including shortened and thickened hypocotyls. It is interesting that
epi seedlings achieved correct reorientation even with
severely stunted hypocotyl growth.
Kinetic analysis of gravitropic curvature revealed additional
alterations in the gravitropic responses of all four mutants (Fig.
2). The normal upward gravitropic
response of dark-grown wild-type tomato seedlings was initiated within
15 to 30 min of gravistimulation and was completed by 4 h, whereas
the auxin-resistant dgt mutant exhibited a slower and
incomplete response to gravity, reaching only approximately 50°
curvature after 20 h. The gravitropic response of the ethylene
overproducer epi was also reduced during the first 12 h
after gravistimulation but reached wild-type levels (75°-80°
curvature) by 20 h. Under these experimental conditions, the
lz-2 mutant curved upward during the first 3 h of
gravistimulation before it reoriented and by 20 h had curved about
80° downward. Whereas the nr mutant in the AC background
exhibited a nearly normal gravitropic response, the initiation of
curvature was slightly delayed in nr plants when compared
with the wild-type response. This delay was detected during the first
30 to 60 min after reorientation (Fig. 2A). However, curvature of
nr plants reached wild-type levels within 90 min of
gravistimulation. Similar results were observed with nr in
the P background (data not shown).
Ethylene Evolution during the Gravitropic Response
Because phytochrome mediates the reversal of gravicurvature in the
lz-2 mutant and red light has been shown to regulate
ethylene concentrations and hypocotyl elongation, we hypothesized that the lz-2 lesion altered ethylene synthesis or action. We
tested this hypothesis by comparing the evolution of ethylene by
wild-type, dgt, lz-2, and epi plants,
which had either been gravistimulated or maintained upright in either
darkness or red light. During a 20 h time course, we observed
similar rates of ethylene evolution for wild-type, lz-2, and
epi plants in both red light and darkness and dgt
plants in darkness (Fig. 3). The
exception was a sharp decrease at 20 h in ethylene evolution by
dgt seedlings that were gravistimulated in red light.
However, the kinetics of this reduction did not correlate with the
reduction in the dgt gravitropic response observed as early
as 30 min after gravistimulation (compare Fig. 2A and Fig. 3B). It was
interesting to note that at 10 h gravistimulated wild-type and
lz-2 plants, as well as both stimulated and unstimulated dgt plants, showed a plateau in their ethylene production.
The rate of ethylene production was increased between 10 and 20 h in wild type, lz-2, and the ungravistimulated
dgt mutant, which was in sharp contrast to the
gravistimulated dgt mutant. It is interesting that with the
epi mutant a linear increase in ethylene production was
observed for ungravistimulated plants, whereas gravistimulated
plants evolved approximately twice as much ethylene as the vertically
oriented plants at 10 h. By 20 h, this difference in ethylene
production between gravistimulated and vertical epi plants
had disappeared (Fig. 3D).
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| Figure 3.
Ethylene evolution is not altered by
gravistimulation and/or red light. Approximately 20 seeds were planted
in each 10-mL vial containing 1 mL of 1% agar and incubated at 29°C
in the dark for 4.5 d. Vials were capped with serum stoppers prior
to treatment. A, Wild type (wt, AC); B, dgt; C,
lz-2; D, epi.  , Upright plants in red
light; , gravistimulated plants in darkness (dashed line); ,
gravistimulated plants in red light (dashed line);  , upright
plants in darkness. SE bars are shown where larger than the
symbol, n = 5 to 20 vials per point. Data were
pooled from six independent experiments. FW, Fresh weight.
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Ethylene Inhibitors Specifically Inhibit the Gravitropic
Response
If ethylene is required for a complete shoot gravitropic response,
then inhibition of ethylene action or synthesis should inhibit
gravitropism. Gravicurvature of wild-type plants exposed to varying
concentrations of the ethylene action inhibitor NBD was inhibited
approximately 50% at 0.87 mM (70 µL) NBD, with maximum inhibition (75%) achieved at 1.13 mM (90 µL; Fig.
4) NBD. The overall elongation of
the uppermost 1 cm of the hypocotyl was not significantly affected by
NBD at these concentrations (Fig. 4). Treatment with the ethylene
synthesis inhibitor AVG yielded results similar to those observed with
NBD. At the highest concentration tested (100 µM), AVG
inhibited curvature 18% (data not shown), which was a statistically
significant decrease (P = 0.01). As with NBD, elongation growth
was not affected by concentrations of AVG, which significantly affected
gravicurvature (data not shown).
Ethylene Has Concentration-Dependent Effects on Curvature
To further test the potential of ethylene to modify the response
of plants to gravity, we measured gravicurvature in the presence of
ethylene concentrations varying from 0.0001 to 0.1 µL
L1 (Fig. 5A). The
gravitropic response of the ethylene-insensitive nr mutant
was not altered at any ethylene concentrations tested. Seedlings of all
other genotypes showed a sharp decrease in curvature in response to low
concentrations of ethylene. Inhibition of the gravitropic response
of wild-type and epi seedlings was detected at 0.01 µL
L1 with 50% inhibition at 0.1 µL
L1, whereas curvature of lz-2 and
dgt seedlings was inhibited at even lower ethylene
concentrations (significant inhibition was observed at 0.001 and 0.005 µL L1, respectively; Fig. 5B). Treatment with
0.05 to 0.1 µL L1 ethylene resulted in nearly
complete inhibition of the dgt gravitropic response but only
50% inhibition for wild-type, epi, or lz-2
plants (Fig. 5B). Thus, all tomato genotypes that retain sensitivity to
ethylene exhibit dose-dependent inhibition of their bending behavior in
response to gravistimulation.
Those ethylene concentrations that significantly inhibited
gravicurvature (0.001-0.1 µL L1) produced no
significant change in overall elongation growth in the 1-cm interval
below the hook, which included the curvature zone (Fig. 5C).
Statistically significant reduction of elongation growth was observed
only for dgt and lz-2 at ethylene concentrations higher than 0.05 µL L1 (P = 0.01). Those
ethylene concentrations are 10- to 50-fold higher than those necessary
to significantly inhibit gravicurvature (compare Fig. 5, B and C).
Thus, inhibition of elongation growth and curvature apparently occur
independently at the exogenous ethylene concentrations used here.
The reduced gravitropic response of the dgt mutant is
restored by very low levels of ethylene. Fumigation of dgt
seedlings with ethylene concentrations as low as 0.0005 µL
L1 for 20 h resulted in gravicurvature
equal to that of wild-type plants (Fig. 5A). This is a 40% stimulation
of bending capacity over untreated dgt plants (Fig. 5B). To
determine whether ethylene restored the dgt mutant to a
full, wild-type gravitropic response, we compared the kinetics of
curvature of gravistimulated wild-type and dgt seedlings in
the presence and absence of the optimal ethylene concentration, 0.0005 µL L1 (Fig. 6).
Whereas the wild-type response was similar in either ambient air or
ethylene, the gravitropic response of the dgt mutant was
accelerated by ethylene at this very low concentration. The kinetics of gravicurvature for dgt seedlings in the presence
of ethylene were, however, not identical to the wild-type gravity response. Curvature of dgt seedlings in the presence of
ethylene is still slower than that of wild-type seedlings and the
acceleration of dgt bending by ethylene is not noticeable
until 8 h after gravistimulation. By this time, reorientation of
wild-type seedlings with respect to gravity was essentially complete.
In comparison, the complete reorientation of dgt seedlings
required 16 h even in the presence of optimal ethylene
concentrations. It appears that ethylene enhances and sustains the
long-term response of dgt hypocotyls to gravity but does not
phenocopy the wild-type gravitropic response (Fig. 6).
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| Figure 6.
Low concentrations of ethylene (0.0005 µL
L1) restore the dgt gravitropic response
but not with wild-type (WT) kinetics. Plants were grown and treated as
for Figure 5. Wild type with ethylene (), WT without ethylene (),
dgt with ethylene (), and dgt without
ethylene (). Results are means ± SE,
n = 10 to 25, pooled from three to six independent
experiments.
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DISCUSSION |
If ethylene plays a primary role in shoot gravitropism, as has
been proposed (Wheeler and Salisbury, 1980, 1981), then mutants that
are altered in ethylene responsiveness should exhibit profound alterations in their gravitropic response. We found that seedlings of
the tomato ethylene-response mutants nr and epi
do exhibit a gravitropic phenotype (Fig. 2). The epi mutant
was previously shown to overproduce ethylene (Fujino et al., 1988),
whereas nr is an ethylene-insensitive mutant (Wilkinson et
al., 1995). Curvature of both mutants in response to gravistimulation
is delayed in comparison with wild-type seedlings, with the
epi phenotype being much more severe than that of
nr (Fig. 2). Both mutants can, however, achieve full
reorientation with respect to gravity within 20 h. This relatively
minor reduction in the gravitropic response of these ethylene mutants
provides strong evidence that ethylene does not play an essential or
primary role in the gravitropic response of tomato.
Seedlings carrying the dgt lesion have a slower gravitropic
response and never achieve greater than 50o
curvature in ambient air (Fig. 2). The dgt lesion was
previously shown to confer greatly reduced auxin sensitivity in
hypocotyls (Kelly and Bradford, 1986) and increased sensitivity to
ethylene in shoots (Shi and Cline, 1992; Fig. 5), and it confers a
different gravitropic phenotype than either epi or
nr. The slow but complete gravitropic response of
nr and epi also did not resemble that of the
lz-2 mutant of tomato, which, under the red-light conditions used here, initially curved upward in a manner similar to wild-type seedlings and then reversed direction of growth, resulting in downward curvature (Figs. 1 and 2). The lz-2 mutant seems to
exhibit a biphasic gravitropic response. Similarly, epi
responds to gravistimulation initially only in an extremely delayed and
reduced fashion but curves up rather rapidly after 12 h, reaching
full curvature after 20 h of gravistimulation. This biphasic
response in epi correlates with an increase in ethylene
evolution at 10 h (Fig. 3D).
There are contradictory reports in the literature with regard to
ethylene synthesis in response to gravistimulation (Kaufman et al.,
1985; Harrison and Pickard, 1986). Treatment with ethylene was reported
to restore normal gravitropic orientation to mature dgt
plants (Zobel, 1973; Jackson, 1979), and application of ethylene can
reverse the direction of the shoot gravitropic response in etiolated
pea seedlings (Kang and Burg, 1974; Burg and Kang, 1993) and in mature
tomato petioles (Kang and Burg, 1974). However, neither red-light
treatment nor gravistimulation induced changes in the ethylene
synthesized by etiolated seedlings of wild-type or any of the mutants
tested (Fig. 3). These observations agree with previous reports
showing no measurable differences in ethylene production within 3 h of gravistimulation using light-grown wild-type tomato seedlings
(Harrison and Pickard, 1986). Overall levels of ethylene evolution by
the epi and lz-2 mutants were slightly lower than
those observed for wild-type or dgt plants. Red light, therefore, does not act through increased ethylene evolution to reverse
the lz-2 gravitropic response.
Although the epi mutant has been characterized as an
ethylene overproducer, ethylene synthesis by epi seedlings
was similar or even reduced compared to wild-type seedlings. This
finding is in agreement with observations that hypocotyls are the
tissue least affected by the epi lesion and the only organ
tested that does not overproduce ethylene (Fujino et al., 1988). The
morphology of epi seedlings is strikingly similar to a
constitutive ethylene response in many respects, including thicker and
shorter hypocotyls (Fig. 1; Ursin, 1987). However, it has not been
shown that epi cosegregates with the constitutive
triple-response gene (ctr) isolated from Arabidopsis (Kieber
et al., 1993). Although the reduced gravitropic phenotype of
nr and epi seedlings indicated that normal
ethylene responsiveness was necessary for a full gravitropic response,
large changes in ethylene synthesis were not observed (Fig. 3).
One exception to the inability of red light or gravistimulation to
produce changes in ethylene evolution measurable by GC was the
reduction in ethylene synthesis by dgt seedlings that had
been gravistimulated in the presence of red light (Fig. 3B). This
suggested that the sluggish gravitropic response exhibited by
dgt (Fig. 2) may be the result of diminished ethylene
production. However, the observed difference in ethylene evolution was
significant only after 20 h of treatment and thus cannot explain
the reduction in the dgt gravitropic response, which was
measurable as early as 30 min after gravistimulation (compare Fig. 2
with Fig. 3B). In addition, curvature of dgt seedlings
gravistimulated in darkness for 20 h was not significantly
different from that of dgt plants gravistimulated in red
light (data not shown). Whereas red light was previously reported to
cause a marked deceleration of ethylene production in pea (Goeschl et
al., 1967) and soybean (Samimy, 1978), we observed no effect of either
red light or gravistimulation alone on ethylene production in
dgt or any other tomato genotype tested. The dgt
lesion does, however, render ethylene synthesis sensitive to the
simultaneous application of gravity stimulus and red light (Fig. 3B).
The basis for this sensitivity remains to be elucidated. It is
interesting to note that increased sensitivity occurred only in
dgt shoots, which exhibit increased sensitivity to ethylene
(Shi and Cline, 1992; Fig. 5) and resistance to auxin (Kelly and
Bradford, 1986; Muday et al., 1995; Coenen and Lomax, 1998).
The epi mutant was reported to overproduce ethylene in all
tissues except hypocotyls (Fujino et al., 1988). Our results confirm this finding for the most part. However, at 10 h of
gravistimulation we observed significantly higher ethylene evolution by
gravistimulated epi seedlings versus nongravistimulated
epi plants. This increased evolution of ethylene
approximately correlates with a somewhat increased rate of curvature at
10 h in Figure 2, indicating that ethylene may cause (or be the
result of) a second phase of curvature leading to full 90° curvature
after 20 h of gravistimulation. Although these correlations are
intriguing, it is important to note that measurements of ethylene
evolution by intact seedlings do not focus on the target tissue that is
affected during gravitropic elongation growth. Therefore,
graviresponsiveness or the lack thereof may not necessarily reflect the
necessity for ethylene in a normal response in this experiment.
Evidence that ethylene can modulate the gravitropic response is
provided by the ability of ethylene action and synthesis inhibitors to
reduce gravicurvature in wild-type seedlings. Inhibition of curvature
by NBD, an ethylene action inhibitor (Fig. 4), and AVG, an ethylene
synthesis inhibitor (data not shown), occurred at concentrations that
did not significantly inhibit the overall elongation of the marked
region of the hypocotyl. This confirmed results of experiments
conducted in cocklebur, which demonstrated a reduction in
gravicurvature by ethylene action and synthesis inhibitors (Wheeler and
Salisbury, 1980, 1981) and indicates that low levels of ethylene are
necessary for a full gravitropic response. However, a basal gravitropic
response still occurred even at high concentrations of NBD. AVG was
less effective in inhibiting the gravitropic response than NBD,
possibly because of inefficient uptake by the roots.
Further support for the ability of ethylene to stimulate the
gravitropic response of tomato seedlings was revealed by the restoration of curvature in dgt seedlings to wild-type
levels by treatment with extremely low levels of ethylene (Figs. 5 and 6). These ethylene concentrations are 5- to 10- fold lower that those
reported to restore the wild-type gravitropic phenotype of mature
dgt plants (Zobel, 1973, 1974; Jackson, 1979). However, even
in the presence of 0.0005 µL L1 ethylene, the
initial curvature of dgt seedlings was much slower than that
of wild-type seedlings. An increase in the curvature rate of
ethylene-treated dgt seedlings after 12 to 20 h (Fig. 6) subsequently restored the mutant hypocotyls to full curvature. This
divergence from wild-type kinetics suggests that low levels of ethylene
can stimulate gravicurvature but not via direct repair of the
dgt lesion (Fig. 6).
Ethylene was found to play additional concentration-dependent roles in
modulating the gravitropic response of etiolated tomato seedlings.
Whereas extremely low levels of ethylene appeared to be necessary for a
full gravitropic response, intermediate ethylene concentrations
(0.005-0.1 µL L1) inhibited wild-type
gravicurvature (Fig. 5, A and B). The gravitropic response of the
dgt and lz-2 mutants exhibited increased
sensitivity to inhibition by ethylene, whereas the nr
gravitropic response was not inhibited by ethylene concentrations as
high as 0.1 µL L1. Inhibition of the reversed
gravitropic curvature of lz-2 plants in red light occurred
at 0.001 to 0.1 µL L1 ethylene,
concentrations that are 1000-fold lower than those reported to induce
reversal of gravitropic orientation of etiolated pea seedlings (Burg
and Kang, 1993). In the etiolated seedlings used here, lz-2
and wild-type seedlings exhibited severe triple-response symptoms
(stunted growth, radial expansion, and exaggerated hook curvature) at
concentrations greater than 1.0 µL L1 (data
not shown). Since neither red light nor gravistimulation altered
ethylene evolution by lz-2 plants, and ethylene treatment inhibited gravitropism but did not repair the reversed-gravitropic lz-2 phenotype, we suggest that ethylene does not play a
role in red-light regulation of the direction of growth in tomato.
Ethylene inhibits hypocotyl elongation in a variety of species,
including etiolated Arabidopsis (Goeschl and Kays, 1975; Ecker, 1995;
Peck et al., 1998) and yet accelerates hypocotyl growth in light-grown
Arabidopsis (Smalle et al., 1997) and peanut (Goeschl and Kays, 1975).
In this study we found no significant inhibition or stimulation of
overall elongation within the marked region that included the
gravitropic bending zone by ethylene concentrations that inhibited
gravicurvature (Fig. 5C). The lack of correlation between inhibition of
gravicurvature and inhibition of growth rates suggests that the
differential growth involved in gravicurvature is not regulated in the
same manner as overall stem elongation.
If ethylene plays a primary role in gravitropism (Wheeler and
Salisbury, 1980, 1981; Clifford and Oxlade, 1989; Philosoph-Hadas et
al., 1996), then an ethylene-insensitive mutant should be agravitropic. Surprisingly, the nr mutant, which is insensitive to
ethylene in both seedling and mature stages (Fig. 5; Wilkinson et al., 1995), displayed only a slightly retarded gravitropic response (Fig.
2). Although it is possible that the nr mutation is leaky or
that other members of the ethylene receptor gene family can compensate
for the missing nr gene product, this does not seem likely
because nr is completely insensitive to ethylene with
respect to inhibition of the gravitropic response at the concentrations tested in Figure 5A. The fact that nr seedlings are
insensitive to ethylene inhibition of curvature and can attain full
reorientation with respect to gravity suggests that inhibition of
curvature by ethylene does not play a prominent role in the generation
of a normal gravitropic response. The ethylene-overproducing
epi mutant could be expected to be either severely inhibited
in its graviresponsiveness or enhanced, depending on which part of the ethylene dose-response bell curve is mimicked by the mutation. However,
although exhibiting striking similarities to the constitutively ethylene-responding Arabidopsis ctr mutant, epi
did not exhibit an opposite graviresponse to nr. It is
possible that higher ethylene concentrations within the tissue lead to
a condition that is supraoptimal for the gravitropic response and
therefore inhibit or retard the process. However, until the gene
products of dgt, epi, and lz-2 are
identified, these questions cannot be answered.
The gravitropic response of the auxin-resistant dgt mutant
is more severely delayed and reduced than that of nr.
Therefore, sensitivity to auxin appears to play a more important role
in the gravitropic response mechanism than ethylene sensitivity or synthesis. Application of very low levels of ethylene can, however, compensate for reduced auxin responsiveness by enhancing and sustaining the slower dgt response (Fig. 6). The Cholodny-Went
hypothesis suggests that tropic curvatures are the result of increased
auxin concentrations on one side of a stem (Went and Thimann, 1937). Other studies have provided evidence that gravistimulation results in
alterations in auxin sensitivity (MacDonald and Hart, 1987; Rorabaugh
and Salisbury, 1989). Our results suggest that ethylene may amplify a
signal that either stimulates the asymmetric redistribution of auxin or
increases the auxin sensitivity of the cells in the lower half of a
gravistimulated hypocotyl. It has been proposed that ethylene modulates
lateral auxin transport, especially in the apical hook of etiolated
seedlings (Schwark and Bopp, 1993; Lehman et al., 1996; Peck et al.,
1998). Ethylene may compensate for the reduced auxin responsiveness of
the dgt mutant by enhancing lateral transport of auxin to
target cells in the hypocotyl epidermis. In mutants that have reduced
ethylene sensitivity, such as nr, the stimulation of auxin
transport by ethylene may be attenuated, leading to a partial or
delayed gravitropic response but not eliminating the basal rate of
lateral transport of auxin. This possibility is also supported by the
recent finding that eir-1, a root-specific, agravitropic,
ethylene-insensitive mutant of Arabidopsis, likely owes its phenotype
to a dysfunctional auxin transporter (Luschnig et al., 1998). However,
it is possible that stimuli other than ethylene also enhance lateral
auxin transport and thus compensate for the inability of nr
to respond to ethylene normally. The same argument can also be applied
to ethylene stimulation of auxin responsiveness in the
dgt mutant.
Our genetic analysis has revealed that, although ethylene does not play
a primary role in the gravitropic response of etiolated seedlings, it
can act as a modulator of gravitropism by either stimulating or
inhibiting curvature. The mechanism by which ethylene influences
gravitropism remains to be elucidated. However, these studies indicate
that ethylene is part of a complex feedback mechanism in the
gravitropic response similar to that demonstrated for the role of
ethylene in elongation growth. Ethylene may play an important role in
the initiation or maintenance of differential growth responses that
help emerging seedlings detect obstructive objects and adjust growth
rates and direction accordingly. Alternatively, ethylene modulation of
the gravitropic response may be a residual interaction resulting from
the mechanisms governing hook formation and maintenance. These studies
provide testable hypotheses that can be used to elucidate the
interaction between auxin and ethylene not only in regulating
gravitropism but also in integrating that information with other
environmental cues.
| |
FOOTNOTES |
1
This research was supported by a doctoral
fellowship from the Deutsche Studienstiftung (to A.M.), a National
Aeronautics Space Administration (NASA) Space Biology Research
Associate award (to F.J.B.), and grants from the NASA Gravitational
Biology and Ecology Program and the National Science Foundation
Integrative Plant Biology Program to T.L.L.
*
Corresponding author; e-mail lomaxt{at}bcc.orst.edu; fax
1-541-737-3573.
Received December 18, 1998;
accepted April 7, 1999.
| |
ABBREVIATIONS |
Abbreviations:
AVG, aminovinylglycine.
FR, far red light.
NBD, norbornadiene.
| |
ACKNOWLEDGMENTS |
We thank Dr. Harry Klee for the generous gift of mutant
nr and P seeds, Dr. Daniel Arp for use of the gas
chromatograph, and TJ White for critical reading of the manuscript.
| |
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Top
Abstract
Introduction