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Plant Physiol. (1999) 119: 521-530
Two Arabidopsis Mutants That Overproduce Ethylene Are
Affected in the Posttranscriptional Regulation of
1-Aminocyclopropane-1-Carboxylic Acid Synthase1
Keith E. Woeste,
Chen Ye, and
Joseph J. Kieber*
Department of Biological Sciences, Laboratory for Molecular
Biology, University of Illinois at Chicago, Chicago, Illinois 60607
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ABSTRACT |
The Arabidopsis
mutants eto1 (ethylene
overproducer) and eto3 produce elevated
levels of ethylene as etiolated seedlings. Ethylene production in these
seedlings peaks at 60 to 96 h, and then declines back to almost
wild-type levels. Ethylene overproduction in eto1 and
eto3 is limited mainly to etiolated seedlings;
light-grown seedlings and various adult tissues produce close to
wild-type amounts of ethylene. Several compounds that induce ethylene
biosynthesis in wild-type, etiolated seedlings through distinct
1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) isoforms
were found to act synergistically with eto1 and
eto3, as did the ethylene-insensitive mutation
etr1 (ethylene resistant), which
blocks feedback inhibition of biosynthesis. ACS activity, the
rate-limiting step of ethylene biosynthesis, was highly elevated in
both eto1 and eto3 mutant seedlings, even though RNA gel-blot analysis demonstrated that the steady-state level
of ACS mRNA was not increased, including that of a novel Arabidopsis ACS gene that was identified. Measurements
of the conversion of ACC to ethylene by intact seedlings indicated that the mutations did not affect conjugation of ACC or the activity of ACC
oxidase, the final step of ethylene biosynthesis. Taken together, these
data suggest that the eto1 and eto3
mutations elevate ethylene biosynthesis by affecting the
posttranscriptional regulation of ACS.
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INTRODUCTION |
The gaseous hormone ethylene has been shown to influence numerous
plant growth and developmental processes, including germination, root-hair initiation, leaf and flower senescence and abscission, fruit
ripening, nodulation, and the response to a wide variety of stresses
(Mattoo and Suttle, 1991 ; Abeles et al., 1992). Much progress has been
made in elucidating the mechanisms of ethylene perception and signal
transduction (Ecker, 1995; Kieber, 1997a, 1997b), as well as the
ethylene-biosynthetic pathway (Kende, 1993). However, to fully
understand the mechanism of ethylene action, it is important to
delineate how its biosynthesis is regulated. We have chosen 3-d-old
etiolated Arabidopsis seedlings as a model system to unravel
this circuitry. Here we describe the characterization of two mutants
that affect the regulation of ethylene biosynthesis in etiolated
seedlings.
Almost all plant tissues have the ability to make ethylene, although in
most cases the amount made is very low. Ethylene production increases
dramatically during a number of developmental events such as
germination, leaf and flower senescence and abscission, and fruit
ripening (Yang and Hoffman, 1984; Mattoo and Suttle, 1991; Abeles et
al., 1992). A diverse group of factors modify the level of ethylene
biosynthesis, and a major effect of most of these is to increase the
steady-state level of ACS mRNA (see Olson et al., 1991,
1995; Rottmann et al., 1991; Botella et al., 1993, 1995), although
there is accumulating evidence to suggest that this enzyme is also
posttranslationally regulated (Nakajima et al., 1990; Spanu et al.,
1994; Oetiker et al., 1997; Vogel et al., 1998b).
The ethylene-biosynthetic pathway (for review, see Yang and Hoffman,
1984; Kende, 1993) starts with the conversion of Met to AdoMet by the
enzyme Met adenosyltransferase. ACS, which converts AdoMet to ACC
(Adams and Yang, 1979), is the first committed and generally
rate-limiting step in ethylene biosynthesis. ACS is encoded by a small
gene family comprising at least three to six members in the species
that have been closely examined. Distinct subsets of ACS
genes are expressed in response to various developmental, environmental, and hormonal factors.
In Arabidopsis six ACS genes have been identified
(ACS1-ACS6), two of which are nonfunctional (Liang et al.,
1992, 1995; Van der Straeten et al., 1992; Vahala et al., 1998).
Inhibition of protein synthesis by cycloheximide treatment induces
expression of the functional genes, suggesting that they are under
negative control (Liang et al., 1992). Wounding, auxin, LiCl, and
anaerobiosis differentially induce these genes (Liang et al., 1992,
1996; Van der Straeten et al., 1992). ACS2
expression is higher in young, developing leaves and flowers compared
with more mature tissues from these organs, and its expression is also
correlated with the initial stages of lateral root formation
(Rodrigues-Pousada et al., 1993). ACS5 is the major isoform
involved in the production of ethylene in response to low doses of
cytokinin in etiolated seedlings, and this regulation is primarily via
a posttranscriptional mechanism (Vogel et al., 1998b). ACS4
transcription is induced by auxin, and several auxin-responsive
elements have been identified upstream of the ACS4 coding
region (Abel et al., 1995). The steady-state level of ACS6
transcript is increased by treatment with ozone (Vahala et al., 1998).
The ACS3 gene is most likely a pseudogene and
ACS1 encodes a nonfunctional ACS (Liang et al., 1995).
The final step of ethylene biosynthesis, the conversion of ACC to
ethylene, is catalyzed by the enzyme ACC oxidase, which can also play
an important role in regulating ethylene biosynthesis, especially under
conditions of high ethylene production (Nadeau et al., 1993; Kim and
Yang, 1994; Tang et al., 1994; Barry et al., 1996; Lasserre, 1996;
Mekhedov and Kende, 1996). ACC oxidase, like ACS, appears to be encoded
by a gene family whose members are differentially regulated in a number
of plants. There are several ACO genes in Arabidopsis
(Newman et al., 1994), at least one of which is induced by ethylene
itself (Gomez-Lim et al., 1993).
ACC can be conjugated to an inactive form, malonyl-ACC, by the enzyme
ACC malonyltransferase (Amrhein et al., 1981; Hoffman et al.,
1982; Kionka and Amrhein, 1984). A second ACC conjugate, 1-( -L-glutamylamino)cyclopropane-1-carboxylic acid,
has also been identified (Martin et al., 1995), although recent
evidence suggests that this is a much less abundant conjugate (Peiser
and Yang, 1998). There is some evidence that the level of ACC
conjugation is regulated, which may contribute to control of ethylene
production (Jiao et al., 1986).
Treatment of etiolated seedlings with ethylene results in a morphology
known as the triple response. In Arabidopsis the triple response
consists of shortening and radial swelling of the hypocotyl, inhibition
of root elongation, and exaggeration of the curvature of the apical
hook (see Fig. 1). This response has been used to identify mutants
disrupted in ethylene perception and signaling (Bleecker et al., 1988;
Guzman and Ecker, 1990; Kieber et al., 1993), as well as mutants
affected in the regulation of ethylene biosynthesis. Mutants in the
latter class fall into two categories: (a) those that fail to induce
ethylene in response to a particular inducer
(cytokinin-insensitive mutants, Cin; Vogel et
al., 1998a, 1998b); and (b) those that overproduce ethylene
(ethylene overproducer, Eto; Guzman and Ecker,
1990; Kieber et al., 1993). Three Eto loci have been identified:
eto1 is inherited as a recessive mutation, and
eto2 and eto3 are dominant. eto2 was
recently found to be the result of a disruption of the carboxy-terminal
11 amino acids of ACS5 (Vogel et al., 1998b). Here we
describe the physiological characterization of the eto1 and
eto3 mutants. This analysis suggests that these mutations
are affected in the posttranscriptional regulation of ACS.
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| Figure 1.
Phenotypes of 3-d-old etiolated wild-type (wt),
single-mutant, and double-mutant Arabidopsis seedlings (as indicated)
grown in air (top panels) or 10 µL L 1 ethylene (bottom
panels). Representative seedlings were picked and photographed.
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MATERIALS AND METHODS |
Plant Lines and Growth Conditions
The Columbia ecotype of Arabidopsis was used in this study. Seeds
were surface-sterilized as described previously (Vogel et al., 1998b),
resuspended in a suitable volume of top agar (0.8% low-melt agarose),
and spread onto Murashige and Skoog agar (Murashige and Skoog salts
[GIBCO-BRL], 2% Suc, and 0.8% agar, pH 5.7). Seeds were cold
treated for 4 d (4°C), exposed to light for 2 h, and then
moved to a dark incubator at 23°C. The time at which the vials were
moved to 23°C was designated as time 0. Adult plants were grown in
potting soil (Metro Mix 250, Grace-Sierra, Boca Raton, FL) under
continuous illumination at 23°C. The eto1-1 and eto3 mutants and the eto1-1/etr1-3 double mutant
were identified previously (Guzman and Ecker, 1990; Kieber et al.,
1993; Roman et al., 1995). The eto1-3 allele was isolated
from an x-ray-mutagenized population (ecotype Columbia) (Kieber et al.,
1993). This allele was used for all of the experiments, with the
exception of the eto1/etr1 double-mutant analysis, which
used the eto1-1 allele. The eto1-3/ein2 double
mutant was obtained by crossing an eto1-3 homozygote to a
ein2-1 homozygote. The F1 population
was allowed to self, and seedlings that were phenotypically
Eto were then selected in the
F2 line. Tall progeny from these individuals were
then selected and self-set seed was collected. A line that retested as
being ethylene insensitive and that overproduced ethylene was
identified and used in further experiments.
Ethylene Measurement
Hormones and CuSO4 were added to about 20 2-d-old etiolated seedlings that were grown in 22-mL GC vials
containing 3 mL of Murashige and Skoog agar by pipetting 200 µL of
solution on top of the seedlings. An equal volume of water plus solvent
was added to the control treatments. These vials were then flushed with hydrocarbon-free air and capped, and the accumulated ethylene measured
24 to 48 h later, as described previously (Vogel et al., 1998b).
Ethylene production was normalized to the number of seedlings in each
vial and the time between capping and sampling. All observations are
from at least three replicates, and each experiment was repeated at
least once with comparable results. To measure ethylene from adult
plants, tissues were detached, weighed, and then placed in 22-mL vials
containing 3 mL of Murashige and Skoog agar. The vials were flushed
with hydrocarbon-free air, sealed, and incubated in the light for the
indicated times. The amount of ethylene produced by light-grown
seedlings was determined by putting capped vials in a lighted growth
chamber for 72 h.
ACS Assays
ACS was assayed from 3-d-old etiolated seedlings as described
previously (Peck and Kende, 1995), with some modifications. Sterilized
seeds were plated on filter paper on Murashige and Skoog agar (10,000 seeds per 150-mm plate), cold incubated (4°C) for 4 d, and then
moved to a dark chamber for 3 d at 23°C. Ten grams of tissue was
added to 15 mL of buffer A (250 mM phosphate buffer, pH
8.0, 10 µM pyridoxal phosphate, 1 mM EDTA, 2 mM PMSF, and 5 mM DTT) and the sample was
homogenized on ice for 4 min (maximum speed with a PowerGen 700 homogenizer [Fisher Scientific]). The sample was centrifuged at
15,000g for 15 min and the supernatant was respun at
15,000g for 15 min. One milliliter of the supernatant was
placed into a 22-mL GC tube and 100 µL of 5 mM
AdoMet was added. This was incubated for 1 h at 22°C. The ACC
formed was converted to ethylene by addition of 100 µL of 20 mM HgCl, followed by 100 µL of a 1:1 mixture of
saturated NaOH:bleach (Lizada and Yang, 1979). The tubes were capped
immediately after addition of the NaOH/bleach and incubated on ice for
10 min. Five milliliters of headspace was removed with a syringe and
injected into a new vial, and the ethylene was measured as described
previously (Vogel et al., 1998b). All reactions were done in triplicate
and compared with controls, to which AdoMet was not added. Protein
concentration was determined using the Bradford assay as described by
the manufacturer (Bio-Rad).
RNA-Blot Analysis
Total RNA was prepared as described previously (Ausubel et al.,
1994) and poly(A+) RNA was isolated using
Oligotex-dT resin, as described by the manufacturer (Qiagen,
Chatsworth, CA). Five micrograms of poly(A+) mRNA
was separated on an agarose gel, blotted to a nylon membrane, and
hybridized to radiolabeled probes as described previously (Ausubel et
al., 1994). Fragments corresponding to each ACS gene and
 -tubulin were obtained by amplifying each from
Arabidopsis genomic DNA using PCR with oligonucleotide primers specific
for each gene, or in the case of ACS6, the insert from the
expressed sequence tag clone FAI88 (see below) was used. The signals
were quantified with a phosphor imager and normalized to the level of
the -tubulin control. This analysis was repeated once
with comparable results.
Isolation of ACS7
We searched the Arabidopsis expressed sequence tag database
(Newman et al., 1994) for sequences similar to those of ACS1
to ACS6. Three clones (FAI88, 288D2T7, and 240L12T7),
corresponding to an identical gene, were identified that were similar
to the previously identified Arabidopsis ACS genes, but
encoded a novel ACS isoform. Sequence analysis revealed that these
ACS7 cDNA clones were missing the 5 portion of the coding
region (see ``Results'').
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RESULTS |
Ethylene Biosynthesis in the eto1 and
eto3 Mutants Is Developmentally Regulated
The eto1 and eto3 mutants display a
constitutive triple-response phenotype as etiolated seedlings (Fig.
1) caused by an overproduction of
ethylene. We analyzed ethylene biosynthesis from wild-type and Eto
mutant etiolated seedlings at various intervals during the first
several days of growth (Fig. 2). There
was almost no detectable ethylene produced by wild-type seedlings
during the early stages of germination (<24 h), followed by a low,
stable level during the next 6 d. Ethylene biosynthesis in
eto1-3 seedlings rose quickly between 48 and 60 h, and
remained at a steady, elevated level for the next 60 h, after
which it gradually declined to close to wild-type levels.
eto3 mutant seedlings displayed a similar pattern, although
their peak production was about 2.5-fold higher and the rate of decline
of production was increased compared with that observed in
eto1-3 seedlings. By d 9, both mutants produced close to
wild-type levels of ethylene.
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| Figure 2.
Ethylene biosynthesis by wild-type, single-mutant,
and double-mutant etiolated Arabidopsis seedlings. Seedlings were grown
for various times on Murashige and Skoog agar in GC vials and the
accumulated ethylene was measured. A, Time course of ethylene
production by etiolated wild-type, eto1-3, and
eto3 mutant seedlings. B, Time course of ethylene
production by etiolated eto1-3, ein2, and
eto1-3/ein2 double-mutant seedlings. The
data points in A and B represent the ethylene accumulated during the
time intervals 0 to 24, 24 to 48, 48 to 60, 60 to 72, 72 to 84, 84 to
96, 120 to 144, 144 to 168, and 168 to 216 h. The second time
point in each interval was plotted as the value for the
x axis. C, Ethylene accumulated by etiolated wild-type
eto1-1, etr1, and
eto1-1/etr1 double-mutant seedlings from
72 to 96 h. All values are means (±SD) of three
replicates.
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To determine if the decline in ethylene biosynthesis was caused by
feedback regulation, we examined the time course of ethylene biosynthesis in the double-mutant seedlings of eto1-3 and
the mutation ein2 ethylene
insensitive (Guzman and Ecker, 1990). The initial rate of
increase in ethylene biosynthesis was the same for both
eto1-3 and eto1-3/ein2 seedlings (Fig. 2B).
However, the double mutant continued to increase its rate of ethylene
biosynthesis beyond 60 h, the point at which the rate in
eto1-3 seedlings peaked. The peak of biosynthesis in the
double mutants was close to what one would predict from an additive
interaction, suggesting that these mutations act independently to
regulate ethylene biosynthesis. The rate of ethylene biosynthesis in
double-mutant seedlings also returned to close to that observed in
wild-type seedlings, although the rate of decline was somewhat slower
than in the eto1-3 single mutant. This suggests that the
decline in biosynthesis is not the result of negative-feedback
regulation from the elevated ethylene levels but, rather, may reflect a
developmental change in the regulation of ethylene biosynthesis
(although feedback regulation may alter the rate at which this occurs).
We examined ethylene production from various adult tissues to determine
if any were affected by the Eto mutations. Both eto1-3 and
eto3 affected ethylene biosynthesis almost exclusively in etiolated seedlings: light-grown seedlings (not shown), adult leaves,
flowers, and siliques from eto1-3 and eto3
mutants produced close to wild-type levels of ethylene (Fig.
3). Thus, these mutants are either
specific for etiolated seedlings or are involved in regulating ethylene
biosynthesis only in the dark.
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| Figure 3.
Ethylene produced by adult tissues of wild-type,
eto1-3, and eto3 mutant plants growing in
soil at 23°C under continuous illumination. Ethylene measurements
were as described in ``Materials and Methods''. Values are means
(±SD) of three replicates.
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Interaction of the Eto Mutants and Various Inducers of Ethylene
Biosynthesis
Ethylene biosynthesis in etiolated Arabidopsis seedlings is
strongly induced by a number of plant hormones, as well as by the
cupric ion. In some cases the target of these factors has been
demonstrated to be distinct ACS genes (Liang et al., 1992; Abel et al., 1995; Vogel et al., 1998b). We examined the interaction of
these factors and the eto1-3 and eto3 mutations
to begin to address how these signaling pathways are related. The
concentration of each inducer was chosen as the concentration that gave
the peak of induction in wild-type etiolated seedlings (Woeste et al.,
1999).
Both eto1-3 and eto3 displayed a synergistic
interaction with auxin, cytokinin, cupric ion, and 24-epibrassinolide
(Fig. 4). The level of ethylene produced
was generally close to multiplicative. For example, the
eto1-3 mutation elevated ethylene biosynthesis approximately
15-fold above wild-type levels, 5 µM BA
increased ethylene approximately 4-fold in etiolated, wild-type
seedlings, and BA-treated eto1-3 seedlings made close to
60-fold more ethylene than untreated, wild-type seedlings. In general,
the synergism of these inducers with the eto3 mutation was
slightly less than that observed with eto1-3. This synergism
indicates that the eto1-3 and eto3 mutations may
act interdependently with these factors in regulating ethylene
biosynthesis. Interestingly, exogenous ABA strikingly dampened the
ethylene overproduction observed in the Eto mutants, although it did
not appear to affect ethylene production in wild-type seedlings.
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| Figure 4.
Ethylene produced by wild-type and mutant
etiolated Arabidopsis seedlings in response to treatment with ABA (75 µM), 2,4-D (160 µM), BA (5 µM), CuSO4 (20 mM), and 1 µM 24-epibrassinolide (24-epi). For all treatments except
BA, seedlings were grown in GC vials, and 200 µL of the solutions of
the indicated concentrations was added at 48 h. The vials were
flushed with hydrocarbon-free air, sealed, and returned to the dark at
23°C. The ethylene that accumulated during the next 24 h was
measured. For BA treatment, the seedlings were germinated on Murashige
and Skoog medium supplemented with 5 µM BA and the
ethylene production during the first 72 h of germination was
measured. Values are means (±SD) of three replicates.
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Interaction with Mutants Affected in Ethylene Biosynthesis
Ethylene-insensitive mutations produce elevated levels of
ethylene, most likely because they block the feedback inhibition of
ethylene biosynthesis in vegetative Arabidopsis tissue (Guzman and
Ecker, 1990). To determine the effect of ethylene feedback on the Eto
mutants, we constructed double mutants of eto1 and the
ethylene-insensitive mutants ein2 and etr1-3
(ethylene resistant) (Fig. 1). The average
length of hypocotyls from the double mutants was not significantly
different from that of each ethylene-insensitive parent (not shown),
which indicates that the etr1-3 and ein2
mutations are epistatic to eto1. This confirms the
expectation that mutations defective in the perception of ethylene act
downstream of those affecting ethylene biosynthesis (Roman et al.,
1995). However, under the conditions that we used, the hypocotyl
lengths of ein2 etiolated seedlings were much more variable
than those of either the wild-type or eto1-3/ein2 etiolated
seedlings (not shown). It is interesting that the apical hooks of the
eto1-3/ein2 double-mutant etiolated seedlings generally
appeared to be more closed that those of the ein2 single
parents (Fig. 1), which have a hook that is less angled than that of
wild-type etiolated seedlings.
When combined with the eto1-1 mutation, the
etr1-3 mutation displayed a synergistic interaction in terms
of the amount of ethylene produced (Fig. 2C). However, the amount of
ethylene produced by the eto1-3/ein2 double mutant appeared
to be close to additive relative to the parental seedlings (Fig. 2B).
This difference may reflect a branch in the ethylene-response pathway
or perhaps subtle differences between the eto1-1 and
eto1-3 alleles.
eto1 and eto3 Etiolated Seedlings
Have Elevated Levels of ACS Activity
One likely target of these ethylene-overproducing mutants is ACS,
the rate-limiting step of ethylene biosynthesis. Previous work
demonstrated that ACS is the target of various inducers of ethylene
biosynthesis in etiolated Arabidopsis seedlings by both transcriptional
and posttranscriptional mechanisms (Liang et al., 1992, 1996; Van der
Straeten et al., 1992; Abel et al., 1995; Vogel et al., 1998b). We
assayed the level of ACS in crude extracts from wild-type and
eto1-3 and eto3 etiolated seedlings (Fig.
5). Both mutants showed high elevated
levels of ACS activity compared with wild-type etiolated seedlings,
which had barely detectable levels of ACS activity. This indicates that
increases in ACS activity may be responsible for the elevated ethylene
biosynthesis observed in the mutant seedlings.
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| Figure 5.
ACS activity in crude extracts from 3-d-old,
etiolated wild-type (WT), eto1, and eto3
mutant seedlings. The enzyme was assayed by incubating crude extracts
in buffer with or without AdoMet, and then measuring the amount of ACC
formed by converting it to ethylene (see ``Materials and Methods'').
The activity was calculated by subtracting the amount of ethylene
produced in the absence of added AdoMet and then normalizing to the
protein concentration of each sample. Values are means
(±SD) of three replicates.
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The formation of ACC is generally the rate-limiting step of ethylene
biosynthesis, but regulation may also be mediated by changes in ACC
oxidase levels or by changes in the amount of ACC that is conjugated.
To address this issue, we examined the ability of intact wild-type and
mutant seedlings to convert exogenous ACC to ethylene, which should
reflect the level of ACC oxidase activity minus any ACC that becomes
conjugated (Fig. 6). Both eto1-3 and eto3 mutants were indistinguishable
from wild-type seedlings in their conversion of ACC to ethylene over a
wide range of exogenous ACC concentrations. Consistent with this,
eto1-3 and eto3 seedlings contained wild-type or
slightly elevated levels of ACC N-malonyltransferase and
-glutamyltranspeptidase activity (not shown). These data
suggest that these mutants elevate ethylene biosynthesis primarily by
increasing ACS activity.
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| Figure 6.
Ethylene produced by etiolated wild-type (wt),
eto1, and eto3 Arabidopsis seedlings in
response to varying concentrations of supplemented ACC. Seedlings were
grown on 3 mL of Murashige and Skoog agar in GC vials. ACC of the
indicated concentrations, in a total volume of 200 µL, was added to
the seedlings 48 h after moving to 23°C. The vials were then
flushed with hydrocarbon-free air and sealed, and the ethylene produced
during the next 24 h was measured. Values are means
(±SD) of three replicates.
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eto1 and eto3 Etiolated Seedlings
Have Wild-Type Levels of ACS mRNA
Elevation of ethylene biosynthesis has often been correlated
with increases in the steady-state levels of ACS mRNAs,
although in a few cases posttranscriptional control has also been
demonstrated. To determine if elevated ACS mRNA contributes
to the increased ethylene biosynthesis observed in eto1 and
eto3 etiolated seedlings, we analyzed ACS mRNA
levels by northern blotting (Fig. 7). We analyzed the expression of ACS2, ACS4,
ACS5, and ACS6, the three previously identified,
active ACS genes in Arabidopsis. In addition, we analyzed a
novel ACS gene that we found by searching the Arabidopsis expressed sequence tag database (Newman et al., 1994), which we have
named ACS7 (Fig. 8).
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| Figure 7.
RNA-blot analysis of ACS mRNA
levels in etiolated seedlings. Five micrograms of poly(A+)
RNA from 3-d-old wild-type (open bars), eto1 (closed
bars), and eto3 (hatched bars) seedlings was separated
by agarose-gel electrophoresis, blotted to a nylon membrane, and
hybridized to an ACS2, ACS4,
ACS5, or -tubulin (TUB)
probe. A, The original images of the blots. B, The quantification of
the blots in A. The signal from each band was quantified using a
phosphor imager. The value for each ACS band was divided
by its -tubulin loading control. The highest level in
each set was assigned a value of 1, and the other values are expressed
relative to this. The ACS5 blot was from an independent
RNA blot and was normalized to its own -tubulin
loading control (not shown).
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| Figure 8.
A comparison of the protein sequences deduced from
the cDNA clones for the Arabidopsis ACS genes. Regions
of amino acid identity are shaded. The alignment was produced using
Clustal software. The sequences for ACS2, ACS4, and ACS5 were from
Liang et al. (1992). The sequence for ACS6 was from Vahala et al.
(1998). The ACS7 protein sequence was deduced from the DNA sequence of
the expressed sequence tag FAI88 (Newman et al., 1994). The amino acid
position of the first residue shown for each protein is indicated in
the first line.
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The ACS7 cDNA clones all appear to lack the 5 end of the
gene, because none was as large as the size predicted from RNA gel-blot analysis (approximately 1.6 kb) and the open reading frame continued to
the 5 end of the longest sequence. However, the 3 end of the gene is
intact and an in-frame stop codon is present, as is a short
poly(A+) tail. A comparison of the predicted
amino acid sequence of ACS7, derived from the sequence of a
non-full-length cDNA expressed sequence tag clone, with the other
Arabidopsis ACS genes reveals that this clone lacks the
variable carboxy-terminal extension present in other ACS proteins (Fig.
8). This carboxy-terminal domain has been demonstrated in at least two
cases to negatively regulate the function of the ACS protein (Li and
Mattoo, 1994; Vogel et al., 1998).
Overall, the level of expression of all five ACS genes was
very low, in most cases just above the level of detection using 10 µg
of poly(A+) mRNA. The steady-state level of mRNA
for ACS5 in eto1-3 and eto3 etiolated
seedlings was close to the level observed in wild-type seedlings. This
was also confirmed by quantitative reverse-transcriptase PCR analysis
(not shown). ACS2 and ACS4 steady-state levels
were actually lower in the Eto mutants, perhaps reflecting
negative feedback from the elevated ethylene levels. There was no
detectable ACS6 or ACS7 expression in either
wild-type or mutant etiolated seedlings, although treatment with the
protein-synthesis inhibitor cycloheximide resulted in high levels of
ACS7 expression (not shown), as is the case with the other
Arabidopsis ACS genes (Liang et al., 1992). These results
indicate that the increased ACS activity is not caused by elevated gene
expression, but is likely attributable to a posttranscriptional
mechanism.
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DISCUSSION |
The eto1 and eto3 mutants were isolated as
seedlings that displayed a constitutive triple-response phenotype
caused by an elevation of ethylene biosynthesis. We demonstrate that
this ethylene overproduction is brought about by increased ACS
activity, and that this is most likely the result of
posttranscriptional regulation. This conclusion is based on
measurements of ACS activity and mRNA levels, as well as the
interactions with various inducers of ethylene biosynthesis. This is
consistent with previous reports of posttranscriptional regulation of
ACS in etiolated Arabidopsis seedlings (Vogel et al., 1998b), and
suggests that this may be a major mechanism for modulating the level of
ethylene biosynthesis in this tissue. Alternatively, it is possible
that an additional ACS gene(s) is present in Arabidopsis and
that the transcription of this gene is elevated in the Eto mutants.
However, if this were the case, one would predict an additive
interaction of these mutations with inducers that act through
independent ACS genes, which is clearly not the case with
either auxin or cytokinin. Thus, the model that is most consistent with
the data presented here is that the Eto mutations affect the
posttranscriptional regulation of ACS.
One possible mechanism for this posttranscriptional regulation is that
ACS protein could be modified to increase its activity or stability,
and the Eto mutations affect this modification. Protein
phosphorylation has been found to play a role in regulating the
function of ACS in tomato (Spanu et al., 1994), and perhaps eto1 and eto3 affect the phosphorylation state of
ACS. Alternatively, the translation of ACS mRNA could be
enhanced by the eto1 and eto3 mutations, leading
to increased levels of ACS enzyme.
Arabidopsis does not display a detectable burst of ethylene in the
first 24 h of germination, as is observed in some other plant
species (Yang and Hoffman, 1984; Abeles et al., 1992), even though ethylene can stimulate germination in Arabidopsis (Bleecker et
al., 1988). In contrast to the wild-type, eto1,
eto3, and ein2 mutant seedlings display a sharp
increase in ethylene production starting at about 48 h. This
increase is then followed by a gradual decline to close to wild-type
levels. The constitutive triple-response phenotype of eto1
and eto3 seedlings becomes decreasingly distinct as
etiolation continues beyond 4 d (K.E. Woeste and J.J. Kieber, unpublished data), which reflects this diminution of ethylene biosynthesis. One model for this decrease in ethylene biosynthesis in
the Eto mutants is that there is a developmental change in the regulation of ethylene biosynthesis. Alternatively, it may reflect
an exhaustion of some metabolite required for ethylene biosynthesis,
although the observation that eto1/ein2 double mutants decrease ethylene production more slowly suggests that this is not the
case, because they presumably have similar metabolic limitations. The
hypothesis that developmental changes affect the function of
ETO1 and ETO3 is also supported by the
observation that mutations in these genes affect only etiolated
seedlings. It is possible that the ETO1 and ETO3
gene products are only required in etiolated seedlings, or,
alternatively, that they affect the regulation of ethylene in multiple
tissues but only in the dark, perhaps playing a role in the circadian
regulation of ethylene biosynthesis (Finlayson et al., 1998).
In Arabidopsis vegetative tissue, ethylene biosynthesis appears to be
autoinhibitory (Guzman and Ecker, 1990), although the mechanism for
this negative-feedback regulation is unknown. Autoinhibition in other
systems has been linked to decreased ACS, decreased ACC oxidase, and/or
up-regulation of ACC conjugation (Abeles et al., 1992). The
ethylene-insensitive mutants etr1 and ein2 block
this negative-feedback regulation in etiolated seedlings, leading to an
increase in ethylene production (Guzman and Ecker, 1990). The additive
interaction of eto1 and ein2 suggests that these
mutations act in parallel to regulate ethylene biosynthesis.
etr1, unlike ein2, displays a synergistic
interaction with eto1, which suggests that eto1
and etr1 act in an interdependent manner to regulate ethylene production. For example, etr1 could elevate
ethylene biosynthesis by increased ACS transcription or
decreased ACC conjugation, either of which, when coupled with the
effect of eto1 on the posttranscriptional regulation of ACS,
would lead to a synergistic interaction. The observation that
etr1 and ein2 differ in their interaction with eto1 suggests that feedback regulation may occur via
multiple mechanisms, and that the feedback pathway may be complex.
We evaluated the effects of a number of inducers of ethylene
biosynthesis on eto1 and eto3 mutants to
determine how they interact. We found that both cytokinin, which in low
doses acts almost exclusively through the ACS5 isoform
(Vogel et al., 1998b), and auxin, which acts through ACS4
(Abel et al., 1995), appear to interact with the Eto mutations in a
synergistic fashion. In addition, cupric ion and 24-epibrassinolide
also act synergistically with eto1 and eto3. This
suggests that these factors, like etr1, act interdependently to regulate ethylene biosynthesis. This is readily explained in the
case of auxin: auxin elevates ACS4 mRNA levels, and
eto1 and eto3 increase ACS gene
function posttranscriptionally, which together would lead to a
synergistic effect. Likewise, cupric ion has also been shown to elevate
ACS mRNA levels in tobacco (Avni et al., 1994), and thus its
synergism with the Eto mutants could occur by a similar mechanism.
However, the synergism of cytokinin and the Eto mutations is somewhat
surprising, because both appear to affect the posttranscriptional
regulation of ACS. This interaction suggests that cytokinin and the
Eto mutants affect distinct posttranscriptional mechanisms,
which could include translational efficiency of ACS mRNA and
protein stability or activity.
To evaluate the possibility that the Eto mutations affect
the regulation of ACC oxidase, we provided wild-type and mutant plants
with an excess of substrate (ACC) and determined that Eto mutants and wild-type plants convert ACC to ethylene at the same rate
over a 3-log range of concentrations (Fig. 6). These results suggest
that ACC oxidase is not the rate-limiting step of ethylene biosynthesis
in etiolated Arabidopsis seedlings, as is also the case in many other
plant tissues, and that the Eto mutations do not affect the
metabolism of ACC. This, coupled with direct measurements of ACC
malonyltransferase and glutamyltranspeptidase activities, which showed
the Eto mutants did not decrease ACC conjugation, supports
the hypothesis that eto1 and eto3 primarily
affect ACS activity.
ABA treatment reduces ethylene production in eto1 and
eto3 mutant etiolated seedlings. ABA has also been shown to
reduce the induction of ethylene biosynthesis by a number of factors in
other plant tissues, including IAA-stimulated and drought-stressed
leaves (Wright, 1980; Yoshii and Imaseki, 1981; McKeon et al., 1982; Tan and Thimann, 1989). The reduction of ethylene biosynthesis by ABA
has been linked to both decreased ACC oxidase levels and increased ACC
conjugation (McKeon et al., 1982; Corbineau et al., 1989; Tan and
Thimann, 1989). The difference in the effect of ABA on Eto
mutants versus its effect on wild-type etiolated seedlings can be
explained if regulation by ABA is only required under conditions of
high ethylene production, conditions not normally found in germinating
wild-type Arabidopsis seedlings. The Eto mutations do
not display any other obvious defects in ABA responses. ABA may act to suppress excess ethylene production downstream of the wild-type gene product of both Eto mutations.
The emerging picture from these and other studies is that
posttranscriptional events play an important role in regulating ethylene biosynthesis. The interactions of the Eto mutations
with other regulators of ethylene biosynthesis suggest that the
pathways regulating this biosynthetic pathway are complex, reflecting
the multitude of regulatory inputs that affect ethylene biosynthesis. Cloning of the genes corresponding to the eto1 and
eto3 mutations should shed further light on the role that
these genes play in regulating ethylene biosynthesis.
| |
FOOTNOTES |
1
This work was supported by U.S. Department of
Agriculture grant nos. 95-37304-2294 and 97-01425 to J.J.K.
*
Corresponding author; e-mail jkieber{at}uic.edu; fax
1-312-413-2691.
Received August 20, 1998;
accepted October 22, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ACS, ACC synthase.
AdoMet, S-adenosyl-Met.
| |
ACKNOWLEDGMENT |
We thank Melinda Martin for assaying malonyltransferase and
glutamyltranspeptidase activities.
| |
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March 1, 2002;
53(368):
391 - 398.
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H.-P. Peng, C.-S. Chan, M.-C. Shih, and S. F. Yang
Signaling Events in the Hypoxic Induction of Alcohol Dehydrogenase Gene in Arabidopsis
Plant Physiology,
June 1, 2001;
126(2):
742 - 749.
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R. M. Harper, E. L. Stowe-Evans, D. R. Luesse, H. Muto, K. Tatematsu, M. K. Watahiki, K. Yamamoto, and E. Liscum
The NPH4 Locus Encodes the Auxin Response Factor ARF7, a Conditional Regulator of Differential Growth in Aerial Arabidopsis Tissue
PLANT CELL,
May 1, 2000;
12(5):
757 - 770.
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M. Tatsuki and H. Mori
Phosphorylation of Tomato 1-Aminocyclopropane-1-carboxylic Acid Synthase, LE-ACS2, at the C-terminal Region
J. Biol. Chem.,
July 20, 2001;
276(30):
28051 - 28057.
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A. J. Wright, H. Knight, and M. R. Knight
Mechanically Stimulated TCH3 Gene Expression in Arabidopsis Involves Protein Phosphorylation and EIN6 Downstream of Calcium
Plant Physiology,
April 1, 2002;
128(4):
1402 - 1409.
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Abstract
Introduction