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Plant Physiol. (1999) 121: 291-300
The Relationship between Ethylene Binding and Dominant
Insensitivity Conferred by Mutant Forms of the ETR1 Ethylene
Receptor1
Anne E. Hall,
Qianhong Grace Chen,
Jennifer L. Findell,
G. Eric
Schaller, and
Anthony B. Bleecker*
Department of Botany, University of Wisconsin, Madison, Wisconsin
53706 (A.E.H., Q.G.C., A.B.B.); and Department of Biochemistry and
Molecular Biology, University of New Hampshire, Durham, New
Hampshire 03824 (J.L.F., G.E.S.)
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ABSTRACT |
Ethylene responses in Arabidopsis are
mediated by a small family of receptors, including the
ETR1 gene product. Specific mutations in the N-terminal
ethylene-binding domain of any family member lead to dominant ethylene
insensitivity. To investigate the mechanism of ethylene insensitivity,
we examined the effects of mutations on the ethylene-binding activity
of the ETR1 protein expressed in yeast. The
etr1-1 and
etr1-4 mutations completely eliminated ethylene binding, while the etr1-3
mutation severely reduced binding. Additional site-directed mutations
that disrupted ethylene binding in yeast also conferred dominant
ethylene insensitivity when the mutated genes were transferred into
wild-type Arabidopsis plants. By contrast, the
etr1-2 mutation did not disrupt ethylene
binding in yeast. These results indicate that dominant ethylene
insensitivity may be conferred by mutations that disrupt ethylene
binding or that uncouple ethylene binding from signal output by the
receptor. Increased dosage of wild-type alleles in triploid lines led
to the partial recovery of ethylene sensitivity, indicating that dominant ethylene insensitivity may involve either interactions between
wild-type and mutant receptors or competition between mutant and
wild-type receptors for downstream effectors.
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INTRODUCTION |
Genetic studies in Arabidopsis have provided evidence that
ethylene perception in plants is mediated by a family of receptors, including the ETR1 gene product. The ETR1 gene
encodes a protein with homology to the two-component His kinase
regulators that control a variety of signaling cascades in prokaryotic
systems and some eukaryotic systems (Chang et al., 1993 ). While ETR1
was the first ethylene receptor to be identified in plants (Bleecker et
al., 1988), additional screens for ethylene-insensitive seedlings and
cloning by sequence similarity indicate that additional genes mediate
ethylene sensitivity in Arabidopsis (Hua et al., 1995, 1998; Sakai et
al., 1998). The ERS1, ETR2, EIN4, and
ERS2 genes all show a high degree of sequence similarity to
ETR1 and appear to comprise a small family of ethylene
receptors. Dominant point mutations that confer ethylene insensitivity
in planta have been isolated in the ETR1, ETR2,
and EIN4 genes, and all of these mutations are located
within the putative transmembrane domains in the N-termini of these
genes. Similar mutations introduced into ERS1 and
ERS2 also confer dominant insensitivity when transformed
into Arabidopsis plants (Hua et al., 1995, 1998). These studies
indicate that a single mutation in any one of these five genes is
sufficient to render plants insensitive to ethylene throughout the
plant.
Subsequent biochemical experiments have confirmed that the
ETR1 gene encodes an ethylene receptor. The N-terminal
hydrophobic domain of the ETR1 protein binds ethylene with high
affinity when expressed in yeast (Schaller and Bleecker, 1995). The
ethylene-binding (sensor) domain of ETR1 consists of three putative
membrane-spanning subdomains that are modeled as alpha helices
(Rodriguez et al., 1999). Notably, the etr1-1
mutation in subdomain 2 abolishes ethylene binding by the
yeast-expressed protein (Schaller and Bleecker, 1995). Biochemical
studies demonstrated that a copper ion in the N-terminal hydrophobic
domain of ETR1 is required for ethylene binding, and that the
etr1-1 mutation abolishes the capacity of the
receptor to coordinate this ion (Rodriguez et al., 1999).
Genetic evidence indicates that the ETR1 receptor family signals
through the Raf-like kinase CTR1. Loss-of-function mutations in
CTR1 show a constitutive triple-response phenotype,
indicating that CTR1 acts as a negative regulator of ethylene-response
pathways (Kieber et al., 1993). Recently, Hua and Meyerowitz (1998)
demonstrated that combining loss-of-function mutants in three or more
members of the ETR1 family also results in plants with a
constitutive ethylene-response phenotype. These results favor a model
for receptor signaling in which the ETR1 receptor family acts in
conjunction with CTR1 to suppress response pathways in the absence of
ethylene. Ethylene binding would convert receptors to a non-signaling
state, resulting in derepression of the response pathway.
Based on the concept of ethylene as a negative regulator of the ETR1
receptor family, we hypothesized that dominant insensitivity to
ethylene could result from mutations that disrupt ethylene-binding activity and lock receptors in a signaling state. The finding that the
etr1-1 mutation disrupts ethylene binding is
consistent with this hypothesis (Schaller and Bleecker, 1995). We
investigated whether this relationship could be extended to the other
mutant alleles, some of which do not completely abolish ethylene
sensitivity in planta (Guzman and Ecker, 1990; Chen and Bleecker,
1995). To further explore the relationship between ethylene binding and dominant insensitivity, we tested whether novel mutations in
ETR1 that abolished ethylene binding in yeast could confer
ethylene insensitivity to plants transformed with these mutant genes.
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MATERIALS AND METHODS |
Plant Materials
The etr1-1, etr1-2
(Bleecker et al., 1988; Chen and Bleecker, 1995),
etr1-3 (Guzman and Ecker, 1990),
etr1-4 (Chang et al., 1993),
etr2-1 (Sakai et al., 1998), and
ein4-3 mutants (Hua et al., 1998) have been
previously described. To reduce the probability of mutations at
additional loci, mutant lines were back-crossed to Arabidopsis ecotype
Columbia (wild type) at least three times before physiological
experiments. Heterozygous diploid plants were obtained by crossing
homozygous mutants and wild-type plants (ecotype Columbia). Triploid
plants were made by crossing homozygous mutant or wild-type diploid
plants to wild-type tetraploid plants (ecotype Bensheim), and the
resulting F1 seeds were used in the studies
described.
Growth Conditions and Ethylene Treatments
Seedling growth-response assays were carried out as described by
Chen and Bleecker (1995). For the experiment shown in Figure 4,
flow-through chambers were used with an ethylene concentration of 35 µL L 1. Ethylene concentrations were
determined by GC using a column of Carboxen 1000 (45/60-mesh size,
Supelco, Bellefonte, PA), with ethylene as the calibration standard.
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| Figure 4.
Analysis of ethylene binding and ethylene
responses in C65S and H69A mutants. A, Ethylene binding by C65S and
H69A proteins expressed in yeast. Dark bars represent the amount of
[14C]ethylene bound (disintegrations per minute per gram
of yeast) by transgenic yeast incubated with 0.07 µL L1
[14C]ethylene. Samples were run in triplicate and
SD bars are shown. White bars represent the amount of
[14C]ethylene bound (disintegrations per minute per gram
of yeast) by single samples of transgenic yeast incubated with 0.07 µL L1 [14C]ethylene and 1,000 µL
L1 [12C]ethylene (background binding). WT,
Wild type. B, Analysis of the triple response in dark-grown seedlings.
Transgenic lines are shown for plants transformed with the wild-type
ETR1 gene (WT), the ETR1 gene containing
a C65S mutation (C65S), and the ETR1 gene containing an
H69A mutation (H69A). For comparison, wild-type (ethylene-sensitive)
and etr1-1 mutant (ethylene-insensitive)
seedlings were grown under the same conditions. Seeds were grown for
4 d in 35 µL L1 ethylene, and two independent
transgenic lines are shown for each construct used. C, Expression level
of ETR1 protein. Expression levels of ETR1 protein were determined for
the transgenic lines from B. Membranes were isolated from etiolated
seedlings grown in air for 4 d. Membrane protein (15 µg) was
subjected to SDS-PAGE, and ETR1 was visualized by western blot.
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Measurement of Seedling-Growth Response
To measure root and hypocotyl length, seedlings were grown on
vertical plates and photographed with slide film or scanned directly
using a Vista-S6E scanner (UMAX, Fremont, CA). Slides were scanned and
saved as TIFF files, and measurements were made using the NIH Image
program (version 1.62, National Institutes of Health, Bethesda, MD).
Ethylene dose-response data were fitted to the Hill equation, used to
describe relationships of plant hormone concentrations and responses
(Weyers et al., 1987). Dose-response curves were generated using COPLOT
(CoHort, Berkeley, CA) and MacCurve Fit (version 1.3, Macintosh) and
were based on non-linear least squares regression.
Construction of Site-Directed Mutants for Yeast and Transgenic
Plant Expression
Site-directed mutations for yeast expression were introduced into
a full-length ETR1 cDNA clone (cETR1-5.2)
subcloned into the pALTERII vector. Mutations were introduced using the
Altered Sites mutagenesis system (Promega) according to the
manufacturer's instructions, and mutations were confirmed by dideoxy
sequencing (Sanger et al., 1977). Constructs were transformed using the
lithium acetate method (Schiestl and Gietz, 1989) into
Saccharomyces cerevisiae strain LRB520 (MAT his3leu2trp1
ura3-52yck2-1::HIS3) and expressed in yeast as previously
described (Schaller and Bleecker, 1995). For wild-type ETR1
expression in Arabidopsis, a 7.3-kb genomic fragment subcloned into the
EcoRI site of pBlueScript (Chang et al., 1993) was removed
by digestion with BamHI and SalI and cloned into
pBIN19 (Bevan, 1984). For mutant gene expression in Arabidopsis the
ETR1 genomic clone was subcloned into pALTERII.
Site-directed mutants of the genomic ETR1 fragment were
confirmed by dideoxy sequencing, excised from the PALTERII vector, and
subcloned into pBIN19 using BamHI and SalI.
Ethylene Binding to Transgenic Yeast
Ethylene-binding experiments were performed as previously
described (Schaller and Bleecker, 1995) using a modification of the
method originally described by Sisler (1979). Yeast cells were grown to
mid-log phase at 30°C, harvested by centrifugation at
1,500g for 5 min, washed with water, and collected in 1-g
aliquots by vacuum filtration on glass fiber discs (Whatman). Yeast
samples (1 g) were sealed in jelly jars containing an injection port in the lid. Samples were incubated in the presence of
[14C]ethylene with or without 1,000 µL
L1 [12C]ethylene.
[14C]ethylene (specific activity = 56.9 mCi/mmol) was used as the mercuric perchlorate complex, and trapped
ethylene was released by the addition of 1 mL of saturated LiCl. After
incubation for 4 h at 22°C, samples were removed from the jars,
aired for 6 min, then transferred to individual jars with 0.3 mL of
mercuric perchlorate in a scintillation vial. These jars were heated to
65°C for 90 min, and then allowed to stand for 24 h to trap the
ethylene released from the samples. Trapped
[14C]ethylene was quantified by liquid
scintillation counting.
Protein Isolation
For membrane isolation, yeast cells were grown according to
standard procedures (Ausubel et al., 1994) and isolated by
centrifugation for 5 min at 5,000 rpm in a centrifuge (model RC-5B
Superspeed, Sorvall), washed with water, and respun at 3,100 rpm for 5 min (AccuSpin FR, Beckman). Yeast cells (25 g) were resuspended in 45 mL of membrane extraction buffer (100 mM NaCl, 50 mM Tris-HCl, pH 6.4, 1% [v/v] DMSO, and 1 mM PMSF), and cells were disrupted by two 1-min treatments
with a bead beater (BioSpec Products, Bartlesville, OK) separated by a
30-s interval. Cell debris were pelleted at 10,000 rpm for 10 min and
discarded. Membranes were isolated from the supernatant by
ultracentrifugation at 30,000 rpm (SW41 Ti rotor, Beckman) for 30 min
(model L8-70, Beckman) and resuspended in membrane resuspension buffer
(10 mM MES [2-(N-morpholino)-ethanesulfonic acid], pH 6.0, 10% [w/v] Suc, and 1% [v/v] DMSO)
at a concentration of 5 g/mL (membranes from 5 g of yeast cells/mL
of buffer), and frozen at  80°C.
The TCA precipitation method was used for the isolation of total yeast
proteins. Yeast cultures (25 mg) used in the ethylene-binding assays
were incubated with cold TCA for 15 min on ice. Yeast were spun down
for 5 min at top speed in a microcentrifuge, and the acid supernatant
was removed. The cells were then resuspended in 100 µL of SDS-PAGE
loading buffer (125 mM Tris [pH 6.8], 20% [v/v]
glycerol, 4% [w/v] SDS, and 0.01% [w/v] bromphenol
blue), and incubated for 1 h at 37°C before SDS-PAGE analysis
(Laemmli, 1970).
For isolation of Arabidopsis membranes, etiolated seedlings (1 g) were
homogenized at 4°C in extraction buffer (50 mM Tris, pH
8.0, 150 mM NaCl, 10 mM EDTA, and 20%
[v/v] glycerol) containing the protease inhibitors PMSF (1 mM), leupeptin (10 µg/mL), and pepstatin (1 µg/mL). The
homogenate was strained through Miracloth (Calbiochem) and centrifuged
at 8,000g for 15 min. The supernatant was centrifuged at
100,000g for 30 min, and the membrane pellet was resuspended
in 10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, and
10% (v/v) glycerol with protease inhibitors.
Protein concentration was determined by the Lowry method (Lowry et al.,
1951) after addition of samples to 0.4% (w/v) deoxycholate to
extract protein from membranes. BSA was used as a standard.
Western Blotting and Protein Quantification
For western blotting, proteins were fractionated via SDS-PAGE
using 8% (w/v) gels, transferred to PVDF membranes (Millipore), and incubated for 1 h with the HRR antibody (Schaller et al., 1995) at a 1:2,500 dilution. Immunodecorated proteins were visualized with a chemiluminescent system according to the manufacturer's instructions (Kirkegaard and Perry, Gaithersburg, MD). Exposed film was
then scanned, saved as TIFF files in Photoshop (version 4.0, Adobe
Systems, Mountain View, CA), and the immunodetectable protein bands
were quantified densitometrically using imaging software.
Arabidopsis Transformation and Growth
Constructs in the pBIN19 vector were introduced into
Agrobacterium tumefaciens (strain GV3101) and used to
transform Arabidopsis ecotype Columbia by vacuum infiltration (Bechtold
et al., 1993). Seeds were plated onto agar plates, and transformed
plants were selected based on resistance to kanamycin (50 µg/mL)
and/or insensitivity to ethylene. Plants were selfed and homozygous
lines identified in subsequent generations. For growth of Arabidopsis
plants in pots, a 3:1 mixture of Metromix 360 (Scotts-Sierra
Horticultural Products, Marysville, OH) to perlite was used. Plants
were maintained in an environmental growth chamber at 22°C with a
16-h daylength.
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RESULTS |
Effects of Mutations in the ETR1 Gene on
Seedling-Growth Response
There are four known dominant mutations in the ETR1
gene: etr1-1 (Bleecker et al., 1988),
etr1-2 (Chen and Bleecker, 1995), etr1-3 (formerly ein1-1;
Guzman and Ecker, 1990), and etr1-4 (Chang et
al., 1993). All were isolated using screens for seedlings that were
insensitive to ethylene-mediated growth inhibition (Bleecker et al.,
1988). The mutations are clustered in the three predicted transmembrane
domains of the protein, and all are point mutations that result in the
following amino acid substitutions: Ala-31 to Val
(etr1-3), Ile-62 to Phe
(etr1-4), Cys-65 to Tyr
(etr1-1), and Ala-102 to Thr
(etr1-2) (Fig. 1).
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| Figure 1.
Ethylene dose-response curves of seedling growth
for the etr1 alleles. A schematic of ETR1 indicates the
protein's predicted domains. The three predicted transmembrane
subdomains within the N-terminal hydrophobic domain are shaded light
gray. The GAF domain (Aravind and Ponting, 1997) is shaded dark gray.
The His kinase domain is indicated by diagonal lines; black boxes
within this area represent the five consensus motifs (H, N, G1, F, and
G2) found in bacterial His kinases. The response regulator domain is
indicated by diagonal bars. The point mutations that constitute the
four dominant etr1 alleles are indicated. Single-letter
abbreviations for amino acids are designated. Ethylene dose-response
curves of seedling growth for the etr1 alleles are shown
for hypocotyl (A-D) and root (E-H). Dose-response curves are shown
for wild type ( , WT) and mutants ( ), including
etr1-1 (C and G),
etr1-2 (D and H),
etr1-3 (A and E), and
etr1-4 (B and F). Mutant and wild-type
dark-grown seedlings were incubated for 3 d in air or a range of
ethylene concentrations (0-1,000 µL L1) (log values
shown on the x axis). Hypocotyl and root length values
are reported as a percentage of the wild-type maximum (wt max) and
represent the means ± SE of 50 measurements. Error
bars are not shown if smaller than the smallest displayed. ND, No
detectable ethylene.
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To determine the effects of these four mutations on ethylene
sensitivity in planta, we analyzed the ethylene dose response for
hypocotyl and root growth in mutant and wild-type etiolated seedlings.
Seedlings were grown in the presence of a range of ethylene
concentrations (0-1,000 µL L1). Hypocotyl
and root lengths were measured after 3 d of growth in the dark,
and dose-response relationships were compared with isogenic wild-type
seedlings.
Plants homozygous for the etr1-1 (Fig. 1, C and
G) and etr1-4 alleles (Fig. 1, B and F) showed no
responsiveness to applied ethylene in either roots or hypocotyls. The
dose-response curves for these mutants appear as negative responses
compared with wild-type seedlings, because wild-type seedlings grown in
air showed a slight response to endogenous ethylene that the mutants
did not. In the etr1-3 line, the maximum ethylene
response of seedling growth was 10% of the wild type in the hypocotyl
(Fig. 1A) and 23% of wild type in the root (Fig. 1E). In the
etr1-2 line, the mutant seedlings had a
maximum response of 42% of wild type in the hypocotyl (Fig. 1D) and
60% in the root (Fig. 1H).
These results indicate that the etr1-1 and
etr1-4 alleles completely eliminate
seedling-growth responses over a range of ethylene concentrations, and
confirm previous observations that the etr1-2 and
etr1-3 alleles are only partially effective in
eliminating seedling-growth responses (Guzman and Ecker, 1990; Chen and
Bleecker, 1995). Furthermore, for the etr1-2 and
etr1-3 alleles, the partial ethylene sensitivity
was more apparent in the root than in the hypocotyl.
Effects of Mutations on the Ethylene-Binding Activity of the
ETR1 Receptor
Based on the result that the etr1-1 mutation
completely abolished ethylene binding by the yeast-expressed protein
(Schaller and Bleecker, 1995), we examined the effects of the other
existing mutations on the ethylene-binding capacity of mutant receptors expressed in yeast. Site-directed mutations were introduced into the
ETR1 cDNA, corresponding to the
etr1-2, etr1-3, and
etr1-4 alleles. Each mutant protein was expressed
in yeast and assayed for ethylene binding. Protein expression levels
for the mutants were quantified on western blots probed with an
anti-ETR1 antibody (HRR) (Schaller et al., 1995), and all strains
produced immunodetectable protein at levels of at least 60% relative
to yeast expressing the ETR1 protein.
Yeast expressing the etr1-4 protein showed no detectable ethylene
binding, even though the level of immunodetectable protein was slightly
higher in the etr1-4-expressing yeast strain than in the wild-type
ETR1-expressing strain (Fig. 2). A more
conservative substitution at this residue (I62A) also completely
eliminated ethylene binding, indicating that the disruption of binding
by the etr1-4 mutation does not result simply
from substituting a bulky side chain (I62F) at this position.
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| Figure 2.
Analysis of ethylene binding by mutant ETR1
proteins. A schematic of the amino acids 1 to 128 of the ETR1 protein
is shown, and relative positions of amino acid changes () in the
three hydrophobic domains are included. For site-directed mutations,
single-letter abbreviations are noted for amino acids. Dark bars
represent the amount of [14C]ethylene bound
(disintegrations per minute per gram of yeast) by transgenic yeast
incubated with 0.07 µL L1 [14C]ethylene.
Samples were run in triplicate and SD bars are shown. White
bars represent the amount of [14C]ethylene bound
(disintegrations per minute per gram of yeast) by single samples of
transgenic yeast incubated with 0.07 µL L1
[14C]ethylene and 1,000 µL L1
[12C]ethylene (background binding). Total yeast proteins
were isolated and analyzed by western-blot analysis. The expression
level of each of the mutants was determined by densitometric
quantification of western blots, and is reported as expression level
relative to wild-type ETR1 protein.
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When the etr1-3 protein containing the A31V mutation was expressed in
yeast, a greatly reduced level of ethylene binding was detected. In
this case, the level of immunodetectable protein was also reduced to
60% of wild type. However, even when corrected for expression level,
the etr1-3 protein showed only 23% of the binding activity measured in
yeast expressing wild-type ETR1. By contrast, protein expressed from
the construct containing the etr1-2 mutation
displayed high levels of ethylene-binding activity. When corrected for
the level of immunodetectable protein, the etr1-2 protein showed 50%
more binding activity than the wild-type protein.
Western-blot analysis indicated that each of the yeast-expressed mutant
proteins were membrane associated and formed disulfide-linked dimers,
similar to the wild-type ETR1 protein (Schaller et al., 1995) (Fig.
3). Therefore, the effects of the
site-directed mutations on ethylene binding did not appear to result
from gross disruptions of the protein structure or membrane
association.
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| Figure 3.
Western-blot analysis indicates mutant ETR1
proteins form disulfide-linked dimers. Membranes isolated from yeast
expressing each of the mutant ETR1 proteins were incubated in the
presence (+) or absence () of 100 mM DTT for 1 h at
37°C, and separated by SDS-PAGE. Western-blot analysis comparing
wild-type ETR1 protein to the mutant proteins was carried out using an
anti-ETR1 antibody (HRR). In the presence of reducing agent (A), the
proteins migrate as a 79-kD monomer, while in the absence of reducing
agent (B), a 147-kD dimer is also detected.
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Taken together, these data indicate that not all of the dominant
mutations in ETR1 abolish ethylene binding, and the
ethylene-insensitive phenotype observed in the etr1 mutant
lines may be caused by defects in ethylene binding, signal
transduction, or both.
Two Novel Site-Directed Mutations in ETR1 Abolish Ethylene Binding
in Yeast and Confer Dominant Insensitivity to Transgenic Arabidopsis
Plants
The finding that the etr1-1,
etr1-3, and etr1-4
mutations reduce or eliminate the ability of ETR1 to bind ethylene
raises the possibility that the dominant ethylene insensitivity
exhibited by the mutant plants results from the disruption of the
receptor's ability to bind ethylene (Schaller and Bleecker, 1995). We
therefore hypothesized that any mutation in ETR1 that
eliminates ethylene binding by ETR1 expressed in yeast might also cause
dominant ethylene insensitivity when transformed back into Arabidopsis.
To test this, two site-directed mutations that have not been identified from screens for ethylene-insensitive seedlings (C65S and H69A) were
introduced in ETR1 (Schaller and Bleecker, 1995; Rodriguez et al., 1999). We then analyzed the effects of these mutations on
ethylene binding by the yeast-expressed protein (Rodriguez et al.,
1999) and on ethylene sensitivity in planta.
Cys-65 and His-69 are likely candidates to coordinate the copper ion
that mediates ethylene binding to the ETR1 receptor (Schaller and
Bleecker, 1995; Rodriguez et al., 1999). We hypothesized that if metal
coordination is required for ethylene binding, mutations in residues
critical for metal coordination would abolish ethylene binding.
Consistent with our hypothesis, the C65S and H69A mutations completely
eliminate ethylene binding by the yeast-expressed protein (Rodriguez et
al., 1999) (Fig. 4A).
To determine the effects of the C65S and H69A mutations on ethylene
sensitivity in planta, these mutations were also made in a 7.3-kb
genomic DNA fragment containing the ETR1 gene (Chang et al.,
1993), cloned into a plant transformation vector (Bevan, 1984), and
transformed into wild-type Arabidopsis (Bechtold et al., 1993).
Transgenic lines that segregated for kanamycin resistance as single
loci were identified and used for further analysis of ethylene
responses.
Seedlings transformed with the mutant ETR1 genomic clones
were clearly insensitive to ethylene (Fig. 4B). Dark-grown transgenic seedlings gassed with ethylene lacked the triple-response phenotype, which includes a shortened hypocotyl and root, radial thickening of the
hypocotyl, and an exaggerated apical hook. Instead, the transgenic
seedlings showed the etiolated morphology typical of air-grown plants,
and mimicked the appearance of ethylene-insensitive etr1-1 mutant plants. Transgenic plants grown in
the light for 2 weeks also showed no responsiveness to ethylene, and
lacked such morphological changes as a shortened hypocotyl and root and compressed leaves (data not shown). Because ethylene insensitivity in
these transgenic plants occurs in a genetic background containing wild-type ETR1, the ethylene insensitivity conferred by the
C65S and H69A mutations is dominant. In contrast, plants transformed with the wild-type ETR1 genomic fragment showed normal
ethylene responses (Fig. 4B).
The expression level of ETR1 protein in transgenic plants was
determined by performing western-blot analysis with membranes isolated
from etiolated seedlings (Fig. 4C). All of the transgenic lines had
higher levels of ETR1 protein compared with the level in wild-type
seedlings, which is consistent with effective transgene expression. The variability in protein levels may be due to positional effects upon transgene expression. The higher levels of
immunodetectable ETR1 present in the transgenic lines cannot account
for the ethylene insensitivity observed in the lines transformed with
the site-directed mutations, as the lines transformed with wild-type
ETR1 and the C65S construct show similar levels of
immunodetectable protein. These results indicate a strong correlation
between dominant ethylene insensitivity in planta and a lack of
ethylene binding by the ETR1 receptor.
Effects of Gene Copy Number on Mutant Phenotypes of the
Dominant Ethylene-Insensitive Lines
To determine the degree of dominance of mutations in four
ethylene-insensitive lines, we examined the relationship between the
seedling-response phenotype and mutant gene copy number. Lines that
were heterozygous for the etr1-1 allele showed no
response to applied ethylene and were indistinguishable from homozygous mutants (Fig. 5, A and B). To further
increase wild-type over mutant gene copy number, we crossed a
homozygous etr1-1 diploid line to a wild-type
tetraploid line (ecotype Bensheim), providing us with triploid
F1 progeny with two wild-type and one mutant allele of ETR1. Interestingly, we found that when the ratio
of wild-type to mutant gene number was increased in a triploid
background, the effects of the etr1-1 mutation
were partially attenuated (Fig. 5C). The mutant/wild-type/wild-type
lines displayed 27% of the maximum seedling response shown by the
wild-type/wild-type/wild-type controls, indicating that partial
responsiveness to applied ethylene was restored by increasing the
wild-type allele copy number. The possibility that the partial recovery
of responsiveness in the triploid line was due to genetic background
effects of the ecotype Bensheim parent line was ruled out by additional
experiments with Bensheim/Columbia heterozygote diploid lines (Chen and
Bleecker, 1995, and Q.G. Chen and A.B. Bleecker, unpublished data).
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| Figure 5.
Effects of gene dosage on the
etr1, etr2, and ein4
dominant mutant phenotypes. Dose-response curves for seedling-growth
response in hypocotyl tissues are shown for wild-type () and mutants
() including etr1-1 (A-C),
etr1-2 (D-F),
etr2-1(G-I), and
ein4-3 (J-L). Genotypes of mutants are
indicated as mt/mt for homozygous diploid mutants, mt/wt for
heterozygous diploid mutants, and mt/wt/wt for heterozygous triploid
mutants. Values represent the means ± SE of 20 measurements. Error bars are not shown if smaller than the symbol. ND,
No detectable ethylene.
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Restoration of ethylene responsiveness by increasing the wild-type
allele dosage was even more apparent in the case of the weaker
etr1-2 allele (Fig. 5, D-F). The percent
wild-type response increased from 42% in the homozygous
etr1-2 mutant (Fig. 5D) to 55% in the
heterozygote (Fig. 5E). This result does demonstrate the dominance of
the mutant over the wild-type allele for even a weak etr1
mutant, but also shows that the dominance is incomplete. Increasing the
ratio of wild-type to mutant copies in the mutant/wild-type/wild-type triploid line increased responsiveness to 84% of that obtained in the
wild-type/wild-type/wild-type control, demonstrating that the effects
of this mutant allele can be almost completely overcome by increasing
the wild-type allele copy number (Fig. 5F).
We included two additional lines in our dose-response analysis that
contain dominant mutations in the putative ethylene receptors ETR2 and EIN4 to compare the extent of their
dominance to the etr1 lines. The
etr2-1 mutation results in the conversion of
Pro-66 to Leu (Sakai et al., 1998), while the
ein4-3 mutation results in the conversion of
Thr-117 to Met (Hua et al., 1998). Like etr1-2 mutants, both etr2-1 and
ein4-3 mutants showed some sensitivity to
ethylene, even in the homozygous mutant lines. The
ein4-3 mutation also showed a gene dosage effect
similar to etr1-2 in that dominance was
incomplete (Fig. 5, J-L). Interestingly, the
etr2-1 allele was more completely dominant than
the other alleles tested. Mutants showed about 35% of wild-type
responsiveness in the presence of zero, one, or two copies of the
wild-type allele (Fig. 5, G-I). Similar results were also obtained
with the more sensitive root-growth assay (Q.G. Chen and A.B.
Bleecker, unpublished results).
| |
DISCUSSION |
The genetic evidence from combined loss-of-function mutations in
members of the ETR1 receptor family supports an inverse-agonist model
for receptor signaling in which unbound receptors repress the
ethylene-response pathway (Hua and Meyerowitz, 1998). Consistent with
this model, we hypothesized that mutations in the ETR1-like genes that cause dominant ethylene insensitivity in planta could work
by disrupting the ethylene-binding activity of the ethylene sensor
domain. This in turn could lock the receptors in a constitutively active signaling state in both air and ethylene. The yeast expression system allowed us to examine the specific effects of mutations in the
ethylene sensor domain on ethylene binding.
Two of the four mutations tested in this study resulted in mutant
proteins unable to bind ethylene. Specifically, the
etr1-1 (Schaller and Bleecker, 1995) and
etr1-4 mutations, which result in strongly
ethylene-insensitive plants, completely abolished ethylene binding by
the receptor. This relationship may also hold for the other members of
the ETR1-like family, because the amino acid substitution
equivalent to etr1-4 causes dominant
insensitivity when it occurs in EIN4 or is introduced in
ERS1 (Hua et al., 1995) and ERS2 (Hua et al.,
1998).
We also determined that novel site-directed mutations that would
abolish ethylene binding and in turn confer dominant insensitivity to
transgenic plants could be introduced into ETR1. Both the
C65S and H69A mutations, located within the second hydrophobic
subdomain of ETR1, completely abolished ethylene binding in
yeast (Schaller and Bleecker, 1995; Rodriguez et al., 1999) and
conferred dominant ethylene insensitivity in planta. These two
mutations not only strengthen the link between ethylene binding and
dominant insensitivity, but illustrate the possibility of modulating
ethylene sensitivity in plants by selective mutation of the
ETR1 gene.
In addition, we have found that there may be a quantitative
relationship between ethylene binding and insensitivity:
etr1-3 plants retained some sensitivity to
ethylene, while the mutant form of the protein bound a very small but
detectable amount of ethylene. One possible explanation for the reduced
level of binding we detected in the etr1-3 protein is that the mutant
etr1-3 receptors may have a reduced affinity for ethylene. This could
result in an incomplete derepression of the ethylene transduction
pathway, producing plants that exhibit partial insensitivity to
ethylene.
A second hypothesis to account for how dominant mutations result in
receptors constitutively signaling even in the presence of ethylene is
that signal propagation, rather than signal perception, is altered. Our
studies with the etr1-2 protein are consistent with this hypothesis:
ethylene-binding assays carried out with yeast expressing the etr1-2
protein indicated that this mutant protein was able to bind ethylene in
excess of wild-type binding levels. While we cannot rule out the
possibility that the etr1-2 mutation disrupts
ethylene binding in planta (e.g. through forming heterodimer
complexes), our data suggest that the etr1-2 mutant protein is altered
in its ability to transduce, rather than to perceive, the ethylene
signal. Furthermore, we must also consider the possibility that the
dominant mutations that do affect ethylene binding may also affect
signal output. For example, if the mutations that disrupt ethylene
binding also lock the receptor in a hyperactive signaling state, an
explanation would be provided for the observation that similar
mutations in any one of five receptor isoforms leads to ethylene
insensitivity.
A more complete understanding of how mutations in the ETR1
family lead to dominant ethylene insensitivity requires more knowledge about the mechanism used by the receptors to transmit signals to
downstream effectors. A reasonable assumption is that ethylene binding
to the sensor domain of a receptor induces a conformational change that
is propagated to the transmitter domain. Transduction of the signal
could occur via modulation of His kinase activity in the transmitter
domain, as it does in many bacterial two-component regulators
(Parkinson, 1993). His kinase activity has been demonstrated for the
ETR1 kinase domain expressed in yeast (Gamble et al., 1998).
Alternatively, conformational changes resulting from ethylene binding
could be transmitted directly to downstream effectors via
protein/protein interactions. In support of this possibility, Clark et
al. (1998) found that the transmitter domains of ETR1 and ERS1 interact
with the regulatory domain of the CTR1 kinase in yeast two-hybrid and
in vitro pull-down assays.
With either of the above mechanisms, mutations anywhere in the receptor
that lock the transmitter domain in the appropriate signaling state
would lead to dominant insensitivity to ethylene. In this regard, the
etr1-2 mutation is located in the third
hydrophobic subdomain, between the residues that are known to be
required for ethylene binding (Schaller and Bleecker, 1995) and the
cytoplasmic transmitter domain. Ethylene-induced conformational changes
would likely be propagated through the third hydrophobic subdomain. This could make the third hydrophobic subdomain particularly
susceptible to mutations that lock the transmitter in a particular
signaling mode. It will be of interest to make additional mutations in
this domain of ETR1 and examine their effects on ethylene binding by the receptor and ethylene responses in transgenic plants. It will also
be interesting to examine the effects of the
ein4-3 mutation on ethylene binding, as this
mutation is also located in the third hydrophobic subdomain of
EIN4 (Hua et al., 1998).
While genetic studies are consistent with the hypothesis that mutations
isolated thus far in the ETR1 family achieve dominance through a gain-of-function mechanism (Hua and Meyerowitz, 1998), these
experiments did not clarify whether mutant receptor isoforms act
through interactions with other wild-type receptor isoforms or
independently. The insensitivity of the etr2-1
mutant allele to increased wild-type allele dosage in the triploid line
experiments (Fig. 5I) is consistent with the latter possibility
(assuming that the Bensheim background has a functional ETR2 allele).
However, in the case of the etr1-1,
etr1-2, and ein4-3 alleles,
increasing the ratio of wild-type allele dosage reduced the level of
ethylene insensitivity in the triploid lines. This recovery of
sensitivity by an increased ratio of wild-type to mutant alleles could
result if the dominant insensitivity caused by these alleles is
mediated through interactions of wild-type and mutant receptor
isoforms. These could take the form of receptor heterodimers and/or
higher order multimeric complexes of receptor clusters. Interestingly, the latter possibility has been suggested for the
ETR1-related bacterial chemoreceptors, based on models in
which alterations in one receptor can change the function of receptors
held in close proximity by non-covalent interactions (Liu et al.,
1997).
Alternatively, this gene dosage effect could result from increased
competition by wild-type receptor isoforms for downstream effectors
(e.g. CTR1). Either of these models could account for the reduction in
ethylene insensitivity with increased wild-type gene dosage.
| |
FOOTNOTES |
1
This work was supported by the National Science
Foundation (grant no. MCB-9603679 to G.E.S. and grant no. MCB-9513463
to A.B.B.), the HATCH project (grant no. 386 to G.E.S.), the Department
of Energy (grant no. DE-FG02-91ER20029 to A.B.B.), and the Department of Energy-National Science Foundation-U.S. Department of Agriculture Collaborative Research in Plant Biology Program (grant no. BIR92-20331 to support A.E.H.).
*
Corresponding author; e-mail Bleecker{at}facstaff.wisc.edu; fax
608-262-7509.
Received March 30, 1999;
accepted June 6, 1999.
| |
ACKNOWLEDGMENTS |
We thank C. Chang for the tetraploid Bensheim lines. A.E.H.
thanks T.A. Richmond for computer assistance and K. Elliot for assistance with illustrations. We thank Anita Klein, Wayne Fagerberg, and members of the Bleecker lab for critical reading of the manuscript.
| |
LITERATURE CITED |
Aravind L,
Ponting CP
(1997)
The GAF domain: an evolutionary link between diverse phototransducing proteins.
Trends Biochem Sci
22:
458-459
[CrossRef][ISI][Medline]
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA,
Strohl K, eds (1994) Current Protocols in Molecular Biology. John
Wiley & Sons, New York
Bechtold N,
Ellis J,
Pelletier G
(1993)
In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants.
CR Acad Sci Ser III
316:
1194-1199
Bevan M
(1984)
Binary Agrobacterium vectors for plant transformation.
Nucleic Acids Res
12:
8711-8721
[Abstract/Free Full Text]
Bleecker AB,
Estelle MA,
Somerville C,
Kende H
(1988)
Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana.
Science
241:
1086-1089
[Abstract/Free Full Text]
Chang C,
Kwok SF,
Bleecker AB,
Meyerowitz EM
(1993)
Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators.
Science
262:
539-544
[Abstract/Free Full Text]
Chen GQ,
Bleecker AB
(1995)
Analysis of ethylene signal transduction kinetics associated with seedling-growth responses and chitinase induction in wild-type and mutant Arabidopsis.
Plant Physiol
108:
597-607
[Abstract]
Clark KL,
Larsen PB,
Wang X,
Chang C
(1998)
Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors.
Proc Natl Acad Sci USA
95:
5401-5406
[Abstract/Free Full Text]
Gamble RL,
Coonfield ML,
Schaller GE
(1998)
Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis.
Proc Natl Acad Sci USA
95:
7825-7829
[Abstract/Free Full Text]
Guzman P,
Ecker JR
(1990)
Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.
Plant Cell
2:
513-523
[Abstract/Free Full Text]
Hua J,
Chang C,
Sun Q,
Meyerowitz EM
(1995)
Ethylene insensitivity conferred by Arabidopsis ERS gene.
Science
269:
1712-1714
[Abstract/Free Full Text]
Hua J,
Meyerowitz EM
(1998)
Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana.
Cell
94:
261-271
[CrossRef][ISI][Medline]
Hua J,
Sakai H,
Nourizadeh S,
Chen QG,
Bleecker AB,
Ecker JR,
Meyerowitz EM
(1998)
EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis.
Plant Cell
10:
1321-1332
[Abstract/Free Full Text]
Kieber JJ,
Rothenberg M,
Roman G,
Feldmann KA,
Ecker JR
(1993)
CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases.
Cell
72:
427-441
[CrossRef][ISI][Medline]
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685
[CrossRef][Medline]
Liu Y,
Levit M,
Lurz R,
Surette MG,
Stock JB
(1997)
Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.
EMBO
16:
7231-7240
[CrossRef][ISI][Medline]
Lowry OH,
Rosebrough NJ,
Farr AL,
Randall RJ
(1951)
Protein measurement with the Folin phenol reagent.
J Biol Chem
193:
265-275
[Free Full Text]
Parkinson JS
(1993)
Signal transduction schemes of bacteria.
Cell
73:
857-871
[CrossRef][ISI][Medline]
Rodriguez FI,
Esch JJ,
Hall AE,
Binder BM,
Schaller GE,
Bleecker AB
(1999)
A copper cofactor for the ethylene receptor ETR1 from Arabidopsis.
Science
283:
996-998
[Abstract/Free Full Text]
Roman G,
Lubarsky B,
Kieber JJ,
Rothenberg M,
Ecker JR
(1995)
Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway.
Genetics
139:
1393-1409
[Abstract]
Sakai H,
Hua J,
Chen QG,
Chang C,
Bleecker AB,
Meyerowitz EM
(1998)
ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis.
Proc Natl Acad Sci USA
95:
5812-5817
[Abstract/Free Full Text]
Sanger F,
Nicklen S,
Coulson AR
(1977)
DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci USA
74:
5463-5467
[Abstract/Free Full Text]
Schaller GE,
Bleecker AB
(1995)
Ethylene binding sites generated in yeast expressing the Arabidopsis ETR1 gene.
Science
270:
1809-1811
[Abstract/Free Full Text]
Schaller GE,
Ladd AN,
Lanahan MB,
Spanbauer JM,
Bleecker AB
(1995)
The ethylene response mediator ETR1 from Arabidopsis forms a disulfide-linked dimer.
J Biol Chem
270:
12526-12530
[Abstract/Free Full Text]
Schiestl RH,
Geitz RD
(1989)
High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier.
Curr Genet
16:
339-346
[CrossRef][ISI][Medline]
Sisler EC
(1979)
Measurement of ethylene binding in plant tissue.
Plant Physiol
64:
538-542
[Abstract/Free Full Text]
Weyers JDB,
Paterson NW,
Brook A
(1987)
Towards a quantitative definition of plant hormone sensitivity.
Plant Cell Environ
14:
1-12
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Top
Abstract
Introduction