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Plant Physiol. (1999) 120: 165-172
Differential Expression of Two Novel Members of the Tomato
Ethylene-Receptor Family
Denise M. Tieman and
Harry J. Klee*
Horticultural Sciences Department, P.O. Box 110690, University of
Florida, Gainesville, Florida 32611-0690
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ABSTRACT |
The phytohormone ethylene regulates
many aspects of plant growth, development, and environmental responses.
Much of the developmental regulation of ethylene responses in tomato
(Lycopersicon esculentum) occurs at the level of
hormone sensitivity. In an effort to understand the regulation of
ethylene responses, we isolated and characterized tomato genes with
sequence similarity to the Arabidopsis ETR1 (ethylene
response 1) ethylene receptor. Previously, we
isolated three genes that exhibit high similarity to ETR1 and to each
other. Here we report the isolation of two additional genes,
LeETR4 and LeETR5, that are only 42% and
40% identical to ETR1, respectively. Although the amino
acids known to be involved in ethylene binding are conserved, LeETR5
lacks the histidine within the kinase domain that is predicted to be
phosphorylated. This suggests that histidine kinase activity is not
necessary for an ethylene response, because mutated forms of both
LeETR4 and LeETR5 confer dominant ethylene insensitivity in transgenic
Arabidopsis plants. Expression analysis indicates that
LeETR4 accounts for most of the putative
ethylene-receptor mRNA present in reproductive tissues, but, like
LeETR5, it is less abundant in vegetative tissues. Taken
together, ethylene perception in tomato is potentially quite complex,
with at least five structurally divergent, putative receptor family
members exhibiting significant variation in expression levels
throughout development.
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INTRODUCTION |
Ethylene, a gaseous phytohormone, plays an important regulatory
role in such diverse plant developmental processes as fruit ripening,
abscission, seed germination, stem elongation, and leaf and flower
senescence (Abeles et al., 1992 ). Ethylene action can be regulated at
the level of both hormone synthesis and sensitivity. The regulation of
ethylene biosynthesis is especially well characterized (McKeon and
Yang, 1987). Ethylene is synthesized from
S-adenosyl-L-Met, which is converted
to ACC by ACC synthase. This is followed by the conversion of ACC to
ethylene by ACC oxidase. The biosynthetic pathway appears to be
regulated at the level of ACC synthase gene transcription (Olson et
al., 1991; Rottmann et al., 1991; Oetiker et al., 1997), and ACC
synthase genes are differentially regulated in response to various
developmental and environmental stimuli (Rottmann et al., 1991; Kieber
and Ecker, 1993; Theologis, 1993). ACC oxidase activity, although
present constitutively, is also regulated and may be responsible for
the fine regulation of ethylene levels present in plant tissues (Kende,
1993).
Although the regulation of ethylene biosynthesis has been studied
extensively, much less is known about the regulation of ethylene
perception. Because ethylene is diffusible from the site of synthesis,
ethylene biosynthesized in one part of the plant can affect other
tissues as well. The ability of plants to regulate the diverse
developmental and environmental responses to ethylene in a
tissue-specific manner indicates that the perception of ethylene is a
complex process and that ethylene perception must be regulated. For
example, tomato (Lycopersicon esculentum) fruit undergoes a
well-defined transition in ethylene sensitivity at the onset of
ripening (Liu et al., 1985). Recent research has focused on the
isolation of genes involved in the ethylene signal transduction pathway
in both Arabidopsis and tomato. Several Arabidopsis mutants deficient
in the classic triple response of etiolated seedlings to ethylene have
been identified (Bleecker et al., 1988; Guzman and Ecker, 1990; Ecker,
1995; Roman et al., 1995; Kieber, 1997). Isolation of the mutant genes
responsible for these phenotypes has enhanced our understanding of the
mechanisms of ethylene perception and signal transduction (Chang et
al., 1993; Kieber and Ecker, 1993; Lehman et al., 1996; Chao et al.,
1997). Multiple mutations in one gene of Arabidopsis, ETR1
(ethylene
response 1), result in
dominant ethylene insensitivity (Bleecker et al., 1988). The ETR1
protein exhibits saturable, high-affinity binding of ethylene, and
mutations in the hydrophobic amino-terminal domain reduce or abolish
that binding (Schaller and Bleecker, 1995). Based on these results, it
has been concluded that ETR1 acts as an ethylene receptor in
Arabidopsis. Recently, it was shown that ETR1 is a member of
a gene family consisting of five members: ETR1,
ERS1, ETR2, EIN4, and ERS2
(Hua et al., 1995, 1998; Sakai et al., 1998). Mutations in the
membrane-spanning domains of each of these genes result in dominant
ethylene insensitivity (Hua et al., 1998; Sakai et al., 1998),
indicating a likely role for all of these proteins in ethylene signal
transduction. Loss-of-function mutants of the Arabidopsis
ETR1, ERS1, ETR2, EIN4, and
ERS2 genes did not have defects in ethylene responses;
however, mutants in three or four of these genes have constitutive
ethylene phenotypes in the absence of ethylene, indicating that these
genes negatively regulate ethylene responses (Hua and Meyerowitz,
1998).
ETR1 exhibits significant sequence homology to a class of His kinases
known as two-component regulators (Chang et al., 1993). In bacteria,
these signal transduction systems consist of two proteins, a sensor and
a response regulator, and mediate responses to a range of environmental
stimuli (Parkinson, 1993). The Arabidopsis ETR1 protein exists as a
membrane-associated dimer and can be divided into three domains (Chang
et al., 1993; Schaller et al., 1995). The amino-terminal sensor domain
contains three putative transmembrane segments within which all known
mutations resulting in loss of ethylene sensitivity are located. This
domain has been shown to bind ethylene when expressed in yeast, and the
etr1-1 mutation abolishes ethylene binding (Schaller and
Bleecker, 1995). The second domain exhibits homology to His kinases
that, in bacterial two-component sensing systems, are
autophosphorylated. This portion of the ETR1 protein has been shown to
exhibit His kinase activity in vitro (Gamble et al., 1998). The third
domain, the response regulator, may receive the phosphate from the His
of the His kinase domain at an Asp residue (Chang et al., 1993).
Nr (Never
ripe) is a semidominant mutant of
tomato originally identified by the inability of its fruit to undergo
ripening (Rick and Butler, 1956). Nr is now known to be ethylene insensitive in all tissues (Lanahan et al., 1994). For example, dark-grown seedlings do not show the "triple response" of
shortened, thickened hypocotyls and roots and a pronounced apical hook
when grown in the presence of ethylene. The mutant also exhibits
defects in ethylene-regulated processes, such as greatly impaired
pedicel abscission and significant delays in the onset of leaf and
flower petal senescence. The mutation responsible for the Nr
phenotype is a single-base change resulting in a Pro-to-Leu substitution in a protein, designated NR, that exhibits significant sequence similarity to ETR1 of Arabidopsis (Wilkinson et al., 1995). As
in Arabidopsis, tomato contains a family of putative ethylene-receptor
genes (Yen et al., 1995). Previously, we and others characterized three
tomato gene family members, LeETR1, LeETR2, and
NR, the first two of which contain response-regulator domains (Zhou et al., 1996a, 1996b; Lashbrook et al., 1998). In this
paper, we describe the isolation of two additional tomato genes,
designated LeETR4 and LeETR5, with sequence
similarity to Arabidopsis ETR1 and tomato NR,
bringing the number of tomato ETR1 homologs to five. LeETR4
and LeETR5 are divergent from the other tomato ETR1 homologs and
exhibit structural features not seen in these proteins. The presence of
multiple structurally divergent, putative ethylene receptors suggests
that the regulation of ethylene responses may be more complex than
previously recognized.
The accession numbers for the sequences reported in this article are
AF118843 (LeETR4) and AF118844 (LeETR5).
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MATERIALS AND METHODS |
Plant Material
Flowers from field-grown tomato (Lycopersicon
esculentum cv Pearson and the Nr mutant) plants were
tagged at anthesis, and the fruit was harvested at the reported days
after anthesis. Wild-type fruit at 30, 40, 50, and 58 d after
anthesis were immature, mature green, turning, and red ripe,
respectively. Leaf, epicotyl, and hypocotyl tissues were harvested from
greenhouse-grown cv Pearson and Nr seedlings. Flowers were
harvested from greenhouse-grown plants. Arabidopsis cv Columbia plants
were grown in a growth chamber under a 16-h daylength at 22°C.
Nucleic Acid Analysis
The Arabidopsis ETR2
(ethylene
response 2) cDNA (Sakai
et al., 1998) was kindly provided by Dr. Tony Bleecker (University of
Wisconsin, Madison). The ETR2 insert was used to screen
under low stringency a phosphate-stressed tomato root cDNA library in
 ZAPII (Stratagene) (kindly provided by Dr. K.G. Raghothama, Purdue
University, West Lafayette, IN). The resulting clones were sequenced
using synthetic oligonucleotide primers on a sequence analyzer (ABI,
Columbia, MD). Sequences of LeETR4 (accession no. AF118843)
and LeETR5 (accession no. AF118844) have been deposited in
GenBank. Amino acid identities and similarities were calculated using
the Gap program, and sequence alignments were performed using the
PileUp program with sequence-analysis software (Genetics Computer
Group, Madison, WI). Predictions of membrane-spanning domains were
performed using the EMBL PredictProtein e-mail server (Rost et al.,
1995).
RNase Protection Assays
Total RNAs were extracted from tissues frozen in liquid
N2 and stored at  80°C, as described
previously (Lashbrook et al., 1994). RNase protection assays were
performed using the RPA II kit (Ambion, Austin, TX) with modifications
(Lashbrook et al., 1998) using pBluescript vectors harboring a
PstI fragment containing nucleotides 2266 to 2565 of the
LeETR4 cDNA and a HindIII/ApaI fragment containing nucleotides 2718 to 3106 of the LeETR5
cDNA as the probes. Levels of mRNA were quantified using sense RNAs generated from a construct containing the 3 1697 bp or the 3 2010 bp
of the LeETR4 and LeETR5 cDNAs, respectively.
DNA Mutagenesis
Mutations in each of the tomato genes were introduced with the
QuikChange site-directed mutagenesis kit from Stratagene using oligonucleotides with the Cys TGT codon analogous to Cys-65 of ETR1
mutated to Ser TCT (NR, Cys-65; LeETR4, Cys-68; and LeETR5, Cys-90).
Mutations were confirmed by sequencing. The full-length mutated or
wild-type versions of LeETR4 and LeETR5 were
cloned into a vector under the control of the figwort mosaic virus
promoter (Richins et al., 1987) and followed by the Agrobacterium
tumefaciens nopaline synthase (nos) 3 terminator. The
promoter/mutated or wild-type ETR1 homolog/nos 3 cassettes
were cloned into a vector containing the neomycin phosphotransferase II
(nptII) gene under the control of the A. tumefaciens nos promoter and followed by the A. tumefaciens nos 3 terminator for selection of
transgenic plants by kanamycin resistance. This plasmid was introduced
into A. tumefaciens, and Arabidopsis plants were transformed
according to the method of Bechtold et al. (1993). Introduction of the
transgene was confirmed by PCR using oligonucleotides specific for the
nptII gene and by seedling germination on
kanamycin-containing plates.
Ethylene-Insensitivity Assay
Seeds from transgenic Arabidopsis plants were plated on medium
containing Murashige and Skoog salts, 1% Suc, 0.5 mM
ACC, 1 mM GA3, and 1% agar and then
stored at 4°C for 3 d and at room temperature for 6 d in
the dark.
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RESULTS |
Cloning of Tomato Ethylene-Receptor Homologs
To identify genes that may be involved in ethylene perception in
tomato, the Arabidopsis ETR2 cDNA (Sakai et al., 1998) was used to screen a tomato-root cDNA library at low stringency. Sequencing of four ETR2-hybridizing clones resulted in the
identification of two classes of tomato cDNAs. The longest clones
within each class were designated LeETR4 and
LeETR5. LeETR4 is a cDNA of 3337 bp that contains
a single open reading frame encoding a protein of 761 amino acids with
a predicted molecular mass of 85.1 kD. The LeETR5 cDNA
consists of 3370 bp and encodes a deduced protein of 767 amino acids
with a predicted molecular mass of 85.9 kD. The predicted LeETR4 and
LeETR5 proteins are somewhat larger than the other members of the
tomato ETR1 family (LeETR1, 84.2 kD; LeETR2, 81.7 kD; and NR, 71.0 kD).
Each of these cDNAs has an unusually long 5-untranslated sequence: 576 nucleotides in LeETR4 and 680 nucleotides in
LeETR5. The 3-untranslated region consists of 389 nucleotides in LeETR5 and 477 nucleotides in
LeETR4. LeETR4 is 78% similar and 60% identical to the
Arabidopsis ETR2 protein, whereas LeETR5 is 76% similar and 54%
identical.
Structural Analysis of the Tomato Ethylene-Receptor Homologs
The Arabidopsis ETR1 protein has been divided into three domains:
the sensor, the His kinase, and the response regulator (Chang et al.,
1993). Figure 1 shows the amino acid
identities and similarities of the tomato ETR1 homologs compared with
the Arabidopsis ETR1 ethylene receptor in each of the three domains.
The similarity among these genes is most evident in the sensor domain,
whereas the His kinase and response-regulator domains are more
divergent. NR is the only member of the tomato putative
ethylene-receptor family that lacks the response-regulator domain. An
alignment of the tomato and Arabidopsis ETR1 and ETR2 deduced amino
acid sequences is shown in Figure 2. The
three membrane-spanning regions critical for ethylene binding are well
conserved; however, LeETR5 is predicted to have a fourth
membrane-spanning domain within an amino-terminal extension of the
protein (Fig. 2). An alternative prediction of membrane-spanning
domains using the method of Hofman and Stoffel (1993) posits a fourth
membrane-spanning domain in both LeETR4 and LeETR5.
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| Figure 1.
Schematic representation of the structure of the
tomato LeETR proteins. By analogy to the Arabidopsis ETR1 protein, the
three domains of the proteins include the sensor domain, with three or
four hydrophobic regions (represented by black boxes) capable of
spanning a membrane (amino acids 1-325 of ETR1); the signaling domain
(amino acids 326-609 of ETR1), with the conserved domains of bacterial
His kinases represented by black boxes and the conserved His
represented by a star; and the response-regulator domain (amino acids
610-738 of ETR1), with the conserved domain containing an Asp that is
capable of receiving the phosphate from the His kinase represented by a
black box (Chang et al., 1993). The percentage of amino acid identity
and similarity between the Arabidopsis ETR1 protein and each of the
tomato ETR1 homologs within each of the three domains is shown below
the schematic of each protein.
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| Figure 2.
Amino acid sequence alignments of the five tomato
ETR1 homologs and Arabidopsis ETR1 and ETR2. Tomato sequences are
designated LeETR1, LeETR2 (Lashbrook et al., 1998), LeNR (Wilkinson et
al., 1995), LeETR4, and LeETR5 (this paper). Arabidopsis ETR1 and ETR2
are designated AtETR1 and AtETR2, respectively (Chang et al.,1993;
Sakai et al., 1998). Shaded areas represent putative membrane-spanning
domains. Cons, Consensus sequence;  , Cys residues involved
in dimerization (Schaller et al., 1995);  , amino acids in which
mutations result in dominant ethylene insensitivity in Arabidopsis
(Ala-31 to Val, Ile-62 to Phe, Cys-65 to Tyr, and Ala-102 to Thr) or
tomato (Pro-36 to Leu in NR) (Wilkinson et al., 1995); * at nucleotide
391, the autophosphorylated His (Gamble et al., 1998); * at nucleotide
718, Asp suggested to act as a receiver of the phosphate; boxed areas
are conserved regions in bacterial His kinases and response regulators
(Parkinson and Kofoid, 1992).
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The amino acids mutated in the known dominant ethylene-insensitive
mutants of Arabidopsis and tomato are conserved in the tomato ETR1
homologs (Fig. 2, arrows). Other invariant amino acids include the two
Cys residues (at positions 4 and 6 of ETR1), which were demonstrated to
be involved in covalent dimerization of the ETR1 protein, although the
intervening amino acid is absent in LeETR5 (Schaller et al., 1995). The
five sequence motifs characteristic of bacterial His kinases (Fig. 2,
boxed regions) are well conserved in LeETR1, LeETR2, and NR; however,
the third and fourth motifs are highly divergent in LeETR4 and LeETR5.
The putative autophosphorylated His (Fig. 2, asterisk at nucleotide
391) in the His kinase domain is present in all of the tomato homologs
except LeETR5. This His is also missing in the Arabidopsis ETR2 and
ERS2 proteins (Hua et al., 1998; Sakai et al., 1998), and when it is
mutated in the ETR1 protein, phosphorylation activity is abolished
(Gamble et al., 1998). The three domains conserved in bacterial
response regulators are also conserved in the tomato proteins
containing a response-regulator domain (Fig. 2, boxed regions). The Asp
that has been suggested to act as a phosphate receiver is present in all of the proteins containing the response-regulator domain.
Phylogenetic analysis of the Arabidopsis and tomato ETR1 homologs (Fig.
3) indicates that LeETR1 and LeETR2 are
most closely related to ETR1 of Arabidopsis, whereas NR is most closely
related to the ERS protein of Arabidopsis. LeETR4 and LeETR5 are more closely related to Arabidopsis ETR2 and EIN4. Arabidopsis ERS2 is
divergent from all other known tomato and Arabidopsis ETR1 homologs.
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| Figure 3.
Phylogenetic analysis of the Arabidopsis and
tomato ETR1 homologs aligned by the Clustal program. Tomato sequences
are designated LeETR1, LeETR2 (Lashbrook et al., 1998), LeNR (Wilkinson
et al., 1995), LeETR4, and LeETR5 (this paper). Arabidopsis sequences
are designated AtETR1 (Chang et al., 1993), AtERS1 (Hua et al., 1995),
AtEIN4 (Hua et al., 1998), AtETR2 (Sakai et al., 1998), and AtERS2 (Hua
et al., 1998). Numbers represent the percentages of amino acids that
differ between two proteins (Saitou and Nei, 1987).
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Mutagenesis of the Tomato Ethylene-Receptor Homologs
Introduction of mutations into the membrane-spanning domains of
the Arabidopsis ETR1, ERS, EIN4, ETR2, and ERS2 proteins results in
ethylene insensitivity in Arabidopsis (Chang et al., 1993; Hua et al.,
1995, 1998; Sakai et al., 1998). To assess the potential role of LeETR4
and LeETR5 in ethylene perception, we introduced mutations into the
membrane-spanning domain of these proteins. We chose to mutate the Cys
aligning with Cys-65 of ETR1 to Ser because this mutation has been
shown to be effective in conferring ethylene insensitivity in
Arabidopsis and resulted in a loss of ethylene binding when the mutated
etr1-1 was expressed in yeast (Chang et al., 1993; Schaller
and Bleecker, 1995). The mutated or wild-type tomato cDNAs under
control of the figwort mosaic virus 35S promoter (Richins et al., 1987)
were introduced into Arabidopsis. This cross-species approach has been
shown to confer ethylene insensitivity in petunia and tomato plants
using the Arabidopsis etr1-1 gene under the control of a
constitutively expressed promoter (Wilkinson et al., 1997). Progeny
from transgenic plants for each of the gene constructs were tested for
ethylene insensitivity, and the results are shown in Table
I and Figure 4. Introduction of each of the mutated
tomato genes (Cys-65 to Ser) conferred ethylene insensitivity, as
judged by elongation of the hypocotyl and the absence of an apical
hook, comparable to that of ein3-1 in approximately
two-thirds of the transgenic lines (Fig. 4). Introduction of the
wild-type LeETR4 gene did not result in increased ethylene
insensitivity of transgenic Arabidopsis; however, 36% of the
transgenic lines were significantly shorter than wild-type Columbia
when grown in the presence of ACC (data not shown). These results may
indicate an increased sensitivity to ethylene or an effect of the
transgene on seedling growth. Introduction of the wild-type
LeETR5 into Arabidopsis resulted in a somewhat longer
phenotype in 23% of transformed lines; however, the frequency of the
longer hypocotyl and root phenotype was much less than that exhibited
by plants transformed with the mutant LeETR5 gene construct
(data not shown). As shown in Figure 4, the phenotype of both LeETR4
and LeETR5 wild-type overexpressers is consistent with ethylene
sensitivity, as indicated by the shorter, thicker hypocotyl and the
exaggerated apical hook.
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Table I.
Ethylene insensitivity of Arabidopsis seedlings from
independent transgenic lines transformed with the mutated (mut) or
wild-type (WT) tomato ethylene-receptor homolog genes, LeETR4 and
LeETR5, and control, wild-type Columbia and ethylene-insensitive ein3-1
seedlings
Data shown indicate lengths (±SE) of etiolated seedlings
grown in the presence of ACC, as described in ``Materials and Methods''.
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| Figure 4.
Phenotype of etiolated Arabidopsis seedlings
transformed with the mutated or wild-type tomato putative
ethylene-receptor genes, LeETR4 or
LeETR5, under the control of a constitutive promoter
grown in the presence of ACC, as described in ``Materials and Methods''. A, Wild-type Columbia; B, ethylene-insensitive mutant
ein3-1; C, transgenic Arabidopsis expressing the mutated
LeETR4 gene; D, transgenic Arabidopsis expressing the
wild-type LeETR4 gene; E, transgenic Arabidopsis
expressing the mutated LeETR5 gene; and F, transgenic
Arabidopsis expressing the wild-type LeETR5 gene.
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Expression Patterns of the Tomato Ethylene-Receptor Homologs
RNA expression patterns of LeETR4 and LeETR5
in tomato tissues were determined by RNase protection assays (Fig.
5). Expression levels of
LeETR4 and LeETR5 mRNAs were highly regulated
among plant tissues, with high levels of expression in reproductive tissues but low levels in vegetative tissues. High levels of
LeETR4 and LeETR5 RNA expression were found in
flower buds and increased in mature flowers. LeETR5
constitutes approximately 0.025% of the total RNA in mature flowers,
the highest level of this mRNA in all tomato plant tissues examined.
Low levels of LeETR4 and LeETR5 RNA expression
were found in leaves, epicotyls, and hypocotyls. LeETR5 RNA
expression in roots was similar to that in ripe fruit and flower buds,
whereas low levels of LeETR4 were found in roots. In
wild-type cv Pearson fruit, expression of LeETR4 and
LeETR5 RNA did not change significantly from 30 to 58 d
after flowering. In ripening fruit, the levels of LeETR4
were the highest among all of the tomato ETR1 homologs,
indicating that this gene, if encoding a functional ethylene receptor,
should play an important role in ethylene perception in tomato.
LeETR5 mRNA levels in ripening fruit were somewhat lower
than NR mRNA levels. LeETR4 was calculated to
constitute approximately 0.06% of total RNA in 58-d-old fruit, whereas
LeETR1, LeETR2, and NR mRNAs
constituted approximately 0.01%, 0.002%, and 0.03%, respectively, of
total RNA in ripening fruit (Lashbrook et al., 1998). In wild-type
fruit, NR mRNA levels increased rapidly at the onset of
ripening and continued to increase until fruit reached the pink stage.
In comparison, the levels of NR mRNA expression in
ethylene-insensitive Nr fruit were greatly reduced. These
results suggest that the levels of ethylene sensitivity and ethylene
evolution are both regulated during fruit ripening (Wilkinson et al.,
1995). We examined LeETR4 and LeETR5 mRNA levels in tissues of Nr plants to determine the effects of
diminished ethylene sensitivity on RNA expression. Overall,
LeETR4 and LeETR5 mRNA expression patterns were
similar in Nr and wild-type fruit, flowers, and vegetative
tissues (data not shown), suggesting that the regulation of these genes
is not affected by ethylene insensitivity of the Nr plants.
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| Figure 5.
LeETR4 and LeETR5
mRNA expression in fruit, flowers, and vegetative tissues of cv Pearson
tomato plants. Fruit at 30, 40, 50, and 58 d after anthesis are
approximately immature, mature green, turning, and red ripe,
respectively. RNase protection assays were performed using 5 and 10 µg of total RNA for LeETR4 and LeETR5,
respectively.
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DISCUSSION |
Tomato is an excellent system in which to study the biochemistry
and physiology of ethylene perception because hormone sensitivity is
developmentally regulated in many processes, such as fruit ripening,
petal senescence, and leaf and flower abscission (Liu et al., 1985).
The roles of the ethylene biosynthetic enzymes ACC synthase and ACC
oxidase in fruit ripening are well established. Elimination of either
ACC synthase or ACC oxidase gene expression results in fruit that ripen
only after exogenous ethylene application. However, ethylene perception
is less well understood. Because ethylene is readily diffusible within
the plant, the regulation of differential tissue and developmental
responses to ethylene is essential to the plant's survival. An
understanding of the tomato putative ethylene-receptor family's
functions, expression patterns, and interactions is essential to
understanding responses to ethylene. Previously, we examined the
expression patterns of LeETR1, LeETR2, and
NR mRNAs in tomato plant development (Lashbrook et al.,
1998). In the experiments reported here, we cloned two novel tomato
ethylene-receptor candidates using the Arabidopsis ETR2 cDNA
as a hybridization probe. These genes bring the total number of cloned
tomato ethylene-receptor homologs to five.
Structurally, the known tomato and Arabidopsis ethylene-receptor
homologs can be divided into three classes (Figs. 1-3). The first
class, incorporating two subclasses, includes the tomato proteins most
closely related to ETR1. The first subclass consists of AtETR1, LeETR1,
and LeETR2. The second subclass consists of proteins that, although
similar to ETR1, do not have a response-regulator domain (LeNR and
AtERS1). The proteins of the first class are highly homologous in the
five conserved regions of residues characteristic of bacterial His
kinases, each of which includes the autophosphorylated His (Fig. 2)
(Parkinson and Kofoid, 1992). The Asp suggested to act as a phosphate
receiver is also conserved in the proteins that have the
response-regulator domain (Fig. 2). The second class consists of
AtETR2, AtEIN4, LeETR4, and LeETR5, proteins that are structurally
divergent from those in the first class. These proteins are also less
similar in the conserved regions of His kinases, especially the third
and fourth conserved regions. The autophosphorylated His is absent in
LeETR5, indicating that it may not be an active His kinase. This His is
also absent from AtETR2 and AtERS2 (Hua et al., 1998; Sakai et al.,
1998). Although a high level of sequence similarity is evident in
the membrane-spanning region, an additional predicted membrane-spanning
domain is present in LeETR5, AtETR2, AtERS2, AtEIN4, and possibly
LeETR4. The sole member of the third class of receptors, AtERS2, is
highly divergent from all other tomato and Arabidopsis
ethylene-receptor homologs, with only 59% similarity and 38% identity
to AtETR1. The role of this protein in ethylene signal perception has
not been determined.
Of the known tomato proteins, only NR lacks the response-regulator
domain. The significance of this domain for signal transduction has yet
to be determined. In bacteria, many two-component sensors lack a
contiguous response-regulator domain. Rather, the phosphate is
transferred to an Asp on a separate protein responsible for signal
transduction (Parkinson and Kofoid, 1992). Several response-regulator proteins capable of accepting a phosphate have been identified in
Arabidopsis (Imamura et al., 1998). The yeast osmosensing pathway contains a multistep phosphorelay system consisting of a two-component His kinase/receiver protein (SLN1), a protein (YPD1) that accepts the
phosphate from the receiver of SLN1, and a second receiver protein
(SSK1) that accepts the phosphate from YPD1 (Posas et al., 1996). A
family of proteins with significant homology to YPD1 has been
identified in the Arabidopsis expressed sequence tag database (H. Klee,
unpublished data). The absence of a response-regulator domain in a
subset of putative ethylene receptors, the possibility of heterodimer
formation, and the presence of multiple separate response regulators
and YPD1-like proteins suggest that the phosphorelay mechanism may be
quite complex and vary with different ethylene receptors.
The lack of the autophosphorylated His and the presence of a fourth
membrane-spanning region in LeETR5 may indicate a divergent function
for this protein. How the presence of a fourth membrane-spanning domain
might affect dimerization is not known; however, the Cys residues
involved in dimerization are present in a region predicted to be
outside of the membrane, between the first and second membrane-spanning domains of LeETR5 (Fig. 2). The missing autophosphorylated His in
LeETR5 also presents the interesting possibility of an ethylene receptor that cannot interact with the next step in the signal transduction pathway. This protein does have the Asp that is essential for response-regulator activity; therefore, conceivably it could accept
a phosphate from an active His kinase. This possibility would depend on
formation of heterodimers, something that has not yet been formally
demonstrated. Mutations in the membrane-spanning regions of LeETR4 and
LeETR5 result in ethylene insensitivity in transgenic Arabidopsis,
suggesting involvement in ethylene signal perception (Table I; Fig. 4).
Similar results have been seen with mutated versions of the Arabidopsis
ETR2 and ERS2 genes that are missing the
autophosphorylated His (Hua et al., 1998; Sakai et al., 1998). These
results suggest that His kinase activity is not essential to the
function of this protein. Alternatively, the missing His in LeETR5 is
not the His that is autophosphorylated; however, mutation of the
analogous His in the Arabidopsis ETR1 protein resulted in the loss of
autophosphorylation in vitro (Gamble et al., 1998). There is a His
conserved in all of the tomato and Arabidopsis proteins seven amino
acids from the autophosphorylated His.
Although the expression of the mutant LeETR4 and
LeETR5 genes in Arabidopsis resulted in a phenotype
consistent with ethylene insensitivity, the introduction of the
wild-type versions of LeETR4 and LeETR5 into
Arabidopsis also resulted in a change in phenotype in some transgenic
lines (data not shown). The shorter phenotype of Arabidopsis plants
expressing LeETR4 and the longer phenotype in some lines
expressing LeETR5 may be the result of an
ethylene-independent role of the putative receptors, as suggested by
the etr1 loss-of-function mutants of Arabidopsis that have a
shorter phenotype in the presence of ethylene (Hua and Meyerowitz,
1998). The altered phenotype may also be the result of cosuppression of
gene expression by the introduction of the transgene under the control
of a strong promoter. A constitutive ethylene phenotype has been seen
in Arabidopsis loss-of-function mutants of multiple ethylene-receptor
genes (Hua and Meyerowitz, 1998).
The presence of structurally divergent ethylene receptors in plants may
be one mechanism whereby plants coordinate and distinguish their many
responses to a single phytohormone. The differential expression
patterns of members of the tomato putative ethylene-receptor gene
family suggest another mechanism for regulating ethylene insensitivity.
For example, tomato fruit undergo a fundamental change in ethylene
responsiveness just before the onset of ripening (Liu et al., 1985).
NR expression is known to increase concomitantly with the
developmental shift in responsiveness (Wilkinson et al., 1995) and may
contribute to the shift in ethylene sensitivity. In contrast, published
expression data indicate that LeETR1 and LeETR2 are expressed at
similar levels in most tissues, including fruit (Zhou et al., 1996a;
Lashbrook et al., 1998). LeETR4 and LeETR5 are also present at
relatively constant levels during fruit development. However, LeETR4 is
expressed at a very high level, accounting for more than 90% of the
putative receptor expression in green fruit and approximately 50% of
the putative receptor expression in ripening fruit. LeETR4 and LeETR5
are also the predominantly expressed putative receptors in flowers, but
they are expressed at relatively low levels in vegetative tissues.
In conclusion, we have shown that the tomato ethylene-receptor homolog
gene family consists of at least five members that exhibit overlapping
expression in virtually all tissues. However, relative levels of
expression of the individual genes vary significantly during
development. The individual putative receptor proteins exhibit
significant structural divergence. This divergence within the
functional domains of the proteins could greatly affect the mechanisms
and efficiencies with which they transduce the ethylene signal. The
differential responses to ethylene in response to developmental and
environmental stimuli may be a result of several factors working singly
or in combination, including the differential regulation of gene
expression, the differential binding affinities of the receptors for
ethylene, the formation of receptor heterodimers or homodimers from the
complement of receptors found in a tissue, the absence of the
response-regulator domain in NR, and the differential interactions with
downstream components of the ethylene signal transduction pathway. If
the members of the ethylene-receptor gene family act as negative
regulators of the ethylene signal transduction pathway (Hua and
Meyerowitz, 1998), high levels of receptors in fruit tissues may act to
modulate the fruit response to the high levels of ethylene produced
during fruit ripening. Although the actual mechanisms for modulating
ethylene sensitivity have yet to be elucidated, it is clear that there
is ample opportunity for plants to adjust ethylene responses at the
receptor level.
| |
FOOTNOTES |
*
Corresponding author; e-mail hjklee{at}gnv.ifas.ufl.edu; fax
1-352-846-2063.
Received November 9, 1998;
accepted February 7, 1999.
1
This work was supported in part by the U.S.
Department of Agriculture (grant no. 95-37304-2326 to H.J.K.). This is
Florida Agricultural Experiment Station journal series no. R-06316.
| |
ACKNOWLEDGMENTS |
We thank Dr. K.G. Raghothama for the phosphate-stressed root
cDNA library and Dr. A. Bleecker for the ETR2 gene. We also
thank Dr. Elliot Meyerowitz for sharing the unpublished sequences of EIN4 and ETR2, Dr. Mark Taylor for excellent care of the plants, and
Dr. David Clark for assistance with the figures.
| |
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Abstract
Introduction