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Plant Physiol. (1998) 118: 1295-1305
Differential Expression and Internal Feedback Regulation
of
1-Aminocyclopropane-1-Carboxylate Synthase,
1-Aminocyclopropane-1-Carboxylate Oxidase, and
Ethylene Receptor
Genes in Tomato Fruit during Development and Ripening1
Akira Nakatsuka2,
Shiho Murachi,
Hironori Okunishi,
Shinjiro Shiomi,
Ryohei Nakano,
Yasutaka Kubo, and
Akitsugu Inaba*
Laboratory of Postharvest Agriculture, Faculty of
Agriculture, Okayama University, Tsushima, Okayama, 700-8530 Japan
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ABSTRACT |
We
investigated the feedback regulation of ethylene biosynthesis in tomato
(Lycopersicon esculentum) fruit with respect to the
transition from system 1 to system 2 ethylene production. The abundance
of LE-ACS2, LE-ACS4, and
NR mRNAs increased in the ripening fruit concomitant
with a burst in ethylene production. These increases in mRNAs with
ripening were prevented to a large extent by treatment with
1-methylcyclopropene (MCP), an ethylene action inhibitor. Transcripts
for the LE-ACS6 gene, which accumulated in
preclimacteric fruit but not in untreated ripening fruit, did accumulate in ripening fruit treated with MCP. Treatment of young fruit
with propylene prevented the accumulation of transcripts for this gene.
LE-ACS1A, LE-ACS3, and
TAE1 genes were expressed constitutively in the fruit
throughout development and ripening irrespective of whether the fruit
was treated with MCP or propylene. The transcripts for
LE-ACO1 and LE-ACO4 genes already existed in preclimacteric fruit and increased greatly when ripening commenced. These increases in LE-ACO mRNA with ripening were also
prevented by treatment with MCP. The results suggest that in tomato
fruit the preclimacteric system 1 ethylene is possibly mediated via constitutively expressed LE-ACS1A and
LE-ACS3 and negatively feedback-regulated LE-ACS6 genes with preexisting LE-ACO1
and LE-ACO4 mRNAs. At the onset of the climacteric
stage, it shifts to system 2 ethylene, with a large accumulation of
LE-ACS2, LE-ACS4, LE-ACO1,
and LE-ACO4 mRNAs as a result of a positive feedback
regulation. This transition from system 1 to system 2 ethylene
production might be related to the accumulated level of
NR mRNA.
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INTRODUCTION |
Fruits can be classified as climacteric or nonclimacteric
depending on the presence or absence of massive ethylene production during ripening and on their response to exogenous ethylene (Biale and
Young, 1981 ). Even in climacteric fruit, ethylene production is
generally very low until the commencement of ripening. At the onset of
ripening, fruit exhibit a climacteric increase in respiration, with a
concomitant burst of ethylene production. Based on the level of
ethylene production during fruit development, McMurchie et al. (1972)
introduced the concept of system 1 and system 2 ethylene. System 1 is
the basal low rate of ethylene production present in preclimacteric
fruits. The basal level of ethylene produced by vegetative tissues and
nonclimacteric fruits can be classified as system 1 (Oetiker and Yang,
1995). System 2 is the high rate of ethylene production observed during
ripening in climacteric fruits and in certain senescent flowers
(Oetiker and Yang, 1995).
In the ethylene-biosynthetic pathway, ACC synthase
and ACC oxidase catalyze the reaction from
Sadenosylmethionine to ACC and from ACC to ethylene,
respectively (Yang, 1987). In this pathway it is well known that
biosynthesis is subject to both positive and negative feedback
regulation (Kende, 1993). Positive feedback regulation of ethylene
biosynthesis is a characteristic feature of ripening fruits and
senescing flowers. In tomato (Lycopersicon esculentum) and
cantaloupe fruits (Liu et al., 1985), banana fruit (Inaba and Nakamura,
1986), and carnation flowers (Wang and Woodson, 1989), a large increase
in ethylene production is triggered by exposure to exogenous ethylene,
with activation of ACC synthase and/or ACC oxidase. From these
observations system 2 ethylene was thought to be regulated by a
positive feedback mechanism. A significant amount of ethylene is also
induced by auxin or stress in a number of plant tissues, and in many
cases it has been shown to be under negative feedback regulation (Yang
and Hoffman, 1984). Therefore, since there are two types of large
ethylene production regulated in opposite feedback directions,
the term system 2 ethylene should be limited to the ethylene produced
from ripening fruits.
In recent molecular studies it has been demonstrated that both ACC
synthase and ACC oxidase are encoded by multigene families in various
plants (Kende, 1993; Zarembinski and Theologis, 1994; Fluhr and Mattoo,
1996). These genes have been isolated and structurally characterized
and are differentially expressed in various tissues at different stages
of development and in response to internal or external stimuli such as
ripening, senescence, wounding, and auxin (Fluhr and Mattoo, 1996). In
tomato fruit a large body of evidence demonstrates that massive
ethylene production is responsible for increases in LE-ACS2,
LE-ACS4, and LE-ACO1 transcripts (Van Der
Straeten et al., 1990; Olson et al., 1991; Rottmann et al., 1991; Yip
et al., 1992; Lincoln et al., 1993; Barry et al., 1996). Expression of
these genes in preclimacteric tomato fruit is rapidly induced and/or
enhanced by treatment with ethylene (Maunders et al., 1987; Rottmann et
al., 1991; Lincoln et al., 1993). Therefore, the expression of the
genes related to system 2 ethylene may be under a positive feedback
regulation mechanism in tomato fruit, at least at the initiation of
ripening.
We previously demonstrated the involvement of a strong positive
feedback regulation mechanism in tomato fruit even at the stage with a
burst of ethylene production (Nakatsuka et al., 1997). The increases in
the abundance of LE-ACS2, LE-ACS4, and
LE-ACO1 mRNAs in ripening fruit were prevented to a large
extent by treatment with MCP, an inhibitor of ethylene action. However,
ethylene production, ACC content, and the activities of ACC synthase
and ACC oxidase in the fruit were not inhibited to the expected level
with respect to suppression of the expression of the ACC synthase and
ACC oxidase genes, suggesting an involvement of a negatively regulated
gene(s) in ethylene biosynthesis in tomato fruit.
The involvement of positive feedback regulation in ethylene
biosynthesis has been elucidated at the molecular level for ACC synthase and/or ACC oxidase in plants such as carnation (Jones and
Woodson, 1997), orchid (O'Neill et al., 1993), and petunia (Tang and
Woodson, 1996) flowers and mung bean (Kim and Yang, 1994) and pea (Peck
and Kende, 1995) seedlings. The negative feedback regulation of
ethylene biosynthesis at the molecular level has been reported in
winter squash fruit (Nakajima et al., 1990), mung bean seedlings (Kim
et al., 1997; Yoon et al., 1997), transgenic petunia flowers
(Wilkinson et al., 1997), and leaves of the tomato cv Never
ripe (Lund et al., 1998). Although it has been suggested that
different ACC synthases may be involved in the two systems of ethylene
production (McGlasson, 1985), it has not been clarified which members
of the ACC synthase and/or ACC oxidase gene families are responsible
for system 1 ethylene synthesis.
We demonstrate the involvement of positive and negative feedback
regulated and constitutively expressed ACC synthase genes in tomato
fruit, in which system 1 and system 2 ethylene production are regulated
toward opposite directions of feedback, with differential expression of
some members of the ACC synthase gene family.
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MATERIALS AND METHODS |
Plant Material and Treatments
Greenhouse-grown tomato (Lycopersicon esculentum Mill.
cv Momotaro) fruit were harvested from a commercial farm at the
following stages: immature green (about 2 weeks after flowering),
mature green (pale-green color on fruit surface), turning (first
appearance of pink color at blossom end), pink (pink color in
approximately one-third of fruit surface), red (red color in
approximately two-thirds of fruit surface), and full ripe (red color on
entire fruit surface). Ethylene production by the fruit was measured at
22°C. Turning and pink fruits were treated with 10 to 20 nL
L 1 MCP for 6 h and then ripened at 22°C.
Ripening stages of MCP-treated fruit were monitored with reference to
the color development of control fruit. Immature green fruit were
treated with 5000 µL L1 propylene for 2 and
4 d at 22°C. Respiration and ethylene production rates, ACC
content, and in vivo ACC oxidase activity were measured in the fruit
treated with propylene. Mature green fruit were divided into three
stages based on the basal level of ethylene production: MG1, MG2, and
MG3. After the determination of ethylene production, pericarp tissues
from the fruit equatorial region were frozen in liquid nitrogen and
stored at  80°C until RNA extraction. All experiments except RNA
extraction were repeated at least three times. MCP synthesis and
treatment were carried out as described previously (Nakatsuka et al.,
1997).
Determination of Ethylene Biosynthesis and
CO2 Production
Ethylene and CO2 production from fruit were
measured by enclosing samples in an airtight chamber for 1 h at
22°C, withdrawing for each determination 1 mL of headspace gas from
the chamber, and injecting into a gas chromatograph (model GC-4CMPF,
Shimadzu, Kyoto, Japan) fitted with a flame-ionization detector and an
activated alumina column for ethylene and into another gas
chromatograph (model GC-3BT, Shimadzu) fitted with a thermal
conductivity detector and a Porapack Q column for
CO2. For immature and mature green fruits, the
basal level of ethylene production was measured using the mercuric
perchloride method described by Akamine and Goo (1978). ACC content was
measured by the method of Lizada and Yang (1979), with 80% ethanol
extracts from pericarp tissues. In vivo ACC oxidase activity was
assayed by the method of Moya-Leon and John (1994), with minor
modifications. Enzyme activity was expressed as the amount of ethylene
(in nanomoles) produced per gram per hour.
RNA Extraction and RT-PCR
RNA was extracted by the hot borate method (Wan and Wilkins,
1994). Poly(A)+ RNA was isolated using
Oligotex-dT30 (Takara, Kyoto, Japan) according to the manufacturer's
protocol. The first-strand cDNAs synthesized by RT from 2 µg of the
poly(A)+ RNA isolated from ripe tomato fruit with
or without MCP treatment were used as a template for RT-PCR with
degenerated primers A and B for ACC synthase (LE-ACS1A,
LE-ACS2, LE-ACS4, and
LE-ACS6), primers C and D for ACC oxidase
(LE-ACO1 and LE-ACO4), and primers E and F for
the ethylene receptor (Table I). These
primers were designed with reference to the conserved amino acid
sequences of ACC synthase and ACC oxidase (Kende, 1993) with
restriction site sequences of BamHI or PstI
(indicated in parentheses in Table I). Primers for the ethylene
receptor were designed with reference to the nucleotide sequences of
NR (accession no. U38666) and eTAE1 (accession
no. U41103) registered in the nucleotide sequence databases with
restriction site sequences of BamHI. Reactions for the
RT-PCR mentioned above were subjected to 30 cycles of 94°C for 1 min,
55°C for 2 min, and 72°C for 3 min. For amplification of the cDNA
fragment of LE-ACS3, we used specific primers K (bp 175-201) and L (bp 822-848) designed from the given nucleotide sequences registered on the database (accession no. U17972) with
restriction site sequences of BamHI and KpnI.
Reactions were subjected to 30 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min.
Amplification of Full-Length cDNA by RACE-PCR
To determine the full-length nucleotide sequences for
LE-ACS6 and LE-ACO4, RACE-PCR was performed using
a cDNA amplification kit (Marathon, Clontech, Palo Alto, CA) according
to the manufacturer's protocol. The 5 -end fragments were amplified
using specific primers N and P for LE-ACS6 and
LE-ACO4, respectively (Table I). To amplify 3-end
fragments, specific primers M and O were used for LE-ACS6 and LE-ACO4, respectively (Table I). Each primer was
designed based on the nucleotide sequences of the cDNA fragments for
LE-ACS6 and LE-ACO4 obtained from the RT-PCR
described above.
Cloning and DNA Sequencing
The PCR products were either ligated into vector pUC118 (Takara,
Kyoto, Japan) or TA-cloned in pCR (Invitrogen, Carlsbad, CA) and then
introduced into Escherichia coli JM109. After screening, target cDNAs were sequenced using a DNA sequencer (model DSQ-1000, Shimadzu) with either the 21M13 or the M13 sequencing primers according to the manufacturer's instructions (Amersham).
Confirmation of LE-ACS1A and
LE-ACS1B Expression
To determine whether LE-ACS1A and LE-ACS1B,
which have very high sequence similarity, were expressed in fruit
tissue, a cDNA fragment was amplified on RT-PCR with a template of the
combined single-strand cDNAs prepared from preclimacteric and ripening fruits in a ratio of 1:1 using specific primer pairs of G and H and I
and J for LE-ACS1A and LE-ACS1B, respectively.
These primers were synthesized with reference to the nucleotide
sequences registered in the database (primers G and H, bp 958-985 and
bp 1311-1334 for LE-ACS1A [accession no. U72389]; primers
I and J, bp 958-985 and bp 1311-1337 for LE-ACS1B
[accession no. U72390]). Competence of primers was confirmed by PCR
with a template of genomic DNA extracted from tomato leaves. The PCR
products were ligated into a plasmid, introduced into E. coli, and sequenced as described above. The resulting plasmids
inserted with the fragments of LE-ACS1A or
LE-ACS1B were used as a template to ascertain the
specificity of each primer pair in PCR. Reactions were subjected to 25 cycles of 94°C for 1 min, 63°C for 2 min, and 72°C for 3 min.
RNA Blotting and Hybridization
Three-microgram samples of mRNA isolated from pericarp tissues
were separated by electrophoresis on 1% agarose gels containing 0.66 M formaldehyde, blotted onto nylon membranes (Hybond N,
Amersham), and fixed with a UV cross-linker (Amersham). The membranes
were hybridized with 32P-labeled cDNA probes
obtained from the RT-PCR products mentioned above and hybridized as
described previously (Nakatsuka et al., 1997). Following hybridization,
membranes were washed once at 60°C in 2× SSPE (1× SSPE = 0.15 M NaCl, 10 mM
NaH2PO4, and 1 mM EDTA, pH 7.4) and 0.1% SDS for 30 min, in 0.5× SSPE
and 0.1% SDS for 30 min, and in 0.2× SSPE and 0.1% SDS for 30 min.
cDNA probes were labeled with a randomly primed DNA-labeling kit
(Boehringer Mannheim) with [32P]dCTP. The
membranes were then exposed to an imaging plate (Fuji Photo Film,
Tokyo, Japan) at room temperature. Equal reactivity and amount of RNA
in all samples were verified by hybridization with
32P-labeled actin (Nakatsuka et al., 1997).
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RESULTS |
Isolation and Identification of cDNA Clones
Using degenerate and specific oligonucleotide primers (Table I),
we cloned nine fragments from ripe tomato fruit without or treated with
MCP, including five different cDNAs for ACC synthase (LE-ACS1A, LE-ACS2, LE-ACS3,
LE-ACS4, and LE-ACS6), two for ACC oxidase (LE-ACO1 and LE-ACO4), and two for the
ethylene receptor (NR and TAE1). Nucleotide
sequences of each fragment except LE-ACO4 were more than
99.6% identical to those of corresponding cDNA previously registered
in the databases: LE-ACS1A; LE-ACS2 (accession no. X59145); LE-ACS3 (accession no. U17972);
LE-ACS4 (accession no. X59146); LE-ACS6
(accession no. U74461); LE-ACO1 (accession no. X58273);
NR; and TAE1. The mismatch of sequences between fragments and the registered cDNAs were probably due to PCR errors or
differences in tomato cultivars. One fragment for ACC oxidase cloned in
this study had low sequence similarity compared with other genes
encoding ACC oxidase already known in tomato (Barry et al., 1996), with
76% to 77% and 80% to 84% at the nucleotide and deduced amino acid
levels, respectively (Table II).
Therefore, we considered this fragment as a new member of the ACC
oxidase gene family in tomato and registered it in the database as
LE-ACO4 (accession no. AB013101).
The full-length cDNA of LE-ACO4, which was obtained by
RACE-PCR, contained an open reading frame of 960 bp encoding a sequence of 320 amino acids. The amino acid sequence comparison among the four
tomato ACC oxidase proteins is shown in Figure
1. The LE-ACS6 fragment cloned
in this study had a completely identical sequence to an already
registered ACC synthase gene (Oetiker et al., 1997; accession no.
U74461) except for the degenerate primer regions. The registered
sequence length is limited to 308 bp and we determined full-length
sequences of its cDNA using the RACE-PCR method. The full-length cDNA
of LE-ACS6 contained an open reading frame of 1431 bp
encoding a sequence of 477 amino acids.
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| Figure 1.
Comparison of the deduced amino acid sequences
among the four tomato ACC oxidase proteins (LE-ACO1,
LE-ACO2 [accession no. Y00478], LE-ACO3
[accession no. Z54199], and LE-ACO4). The asterisks
indicate sequence identity. Highly conserved regions for ACC oxidase
are boxed, and the nine shaded amino acid residues are conserved in all
members of the Fe(II) ascorbate family of dioxygenases (Lasserre et
al., 1996).
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Ethylene Production during Fruit Development and Ripening and
Effect of MCP
Figure 2 shows the rate of ethylene
production by the fruit immediately after harvest at the indicated
stages and by the fruit treated with MCP at the turning or pink stages.
In the control fruit ethylene production was very low at the basal
level at the preclimacteric stage and increased during ripening,
reaching a peak at the red stage and declining slightly thereafter.
This increase in ethylene production was inhibited by about 66% and 75% 2 d after MCP treatment at the turning and pink stages,
respectively. Thereafter, ethylene production recovered slowly without
any decline to the basal level, contrary to the expectation from the
action of MCP (Sisler and Serek, 1997).
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| Figure 2.
Changes in the rate of ethylene production in
tomato fruit during development and ripening and the effect of MCP.
Fruit were harvested at six stages: immature green (IM), mature green
(MG), turning (T), pink (P), red (R), and full ripe (FR), based on the
observations described in ``Materials and Methods''. Fruit harvested
at the turning and pink stages were treated with 10 to 20 nL
L1 MCP for 6 h and then ripened at 22°C.
The ripening stages of MCP-treated fruit corresponding to the control
fruit were determined as described in ``Materials and Methods''.
Vertical bars are the SE of three replications; missing
error bars are smaller than the symbols.
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Confirmation of LE-ACS1A Expression in
Fruit Tissue
Since the twin LE-ACS1 cDNAs LE-ACS1A
and LE-ACS1B, which share very high sequence similarity,
have been cloned from a tomato genomic library (Oetiker et al., 1997),
we determined whether both were expressed in the fruit. As shown in
Figure 3, only the LE-ACS1A
cDNA fragment with the expected length of 377 bp was amplified by
RT-PCR when the specific primers designed to have a 2-base mismatch at
3 ends in both upstream and downstream primers (compare lanes 2 and 6)
were used. The LE-ACS1A and LE-ACS1B genomic DNA
fragments were amplified by PCR using each primer pair (Fig. 3, lanes 3 and 7), ligated into a plasmid, and then introduced into E. coli. The nucleotide sequences of each fragment were completely
identical to those of the corresponding regions for each cDNA (data not
shown). When these plasmids inserted with the LE-ACS1A or
LE-ACS1B fragments were used as templates for PCR, the
LE-ACS1A primer amplified the LE-ACS1A fragment
but not the LE-ACS1B fragment (Fig. 3, compare lanes 4 and
9) and vice versa (Fig. 3, compare lanes 5 and 8). These experiments
confirmed that, among the twin LE-ACS1 genes, only
LE-ACS1A mRNA was expressed in the fruit tissue.
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| Figure 3.
Agarose/ethidium bromide gel image of RT-PCR
products amplified using specific primers for LE-ACS1A
and LE-ACS1B. Each primer was designed to amplify the
corresponding region in LE-ACS1A and
LE-ACS1B but with two different nucleotides at the 3
ends either upstream or downstream set to avoid cross-amplification.
The LE-ACS1A primers were used for the reaction of lanes
2, 3, 4, and 9, and the LE-ACS1B primers were used for
lanes 5 to 8. Templates used for RT-PCR were the combined single-strand
cDNAs prepared from preclimacteric and ripening fruits in a ratio of
1:1 (lanes 2 and 6), the genomic DNA extracted from tomato leaves
(lanes 3 and 7), and the plasmid inserted with the
LE-ACS1A (lanes 4 and 5) or LE-ACS1B
(lanes 8 and 9) fragment. Lane 1 shows a 100-bp DNA ladder as a size
marker.
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Gene Expression during Fruit Development and Ripening and Effect of
MCP
Figure 4 shows the expression of
members of the gene families for ACC synthase, ACC oxidase, and the
ethylene receptor in tomato fruit during development and ripening and
in the fruit treated with MCP. Among the five members of the
LE-ACS gene family, the abundance of LE-ACS2 and
LE-ACS4 mRNAs in the fruit was undetectable in fruit at the
preclimacteric stage, increased from the turning to pink stages, and
thereafter slightly declined (Fig. 4, lanes 1-6). These increases in
the mRNA abundance associated with ripening were prevented to a large
extent by treatment of fruit with MCP at both the turning (Fig. 4,
lanes 7-9) and pink (Fig. 4, lanes 10 and 11) stages. In particular,
2 d after MCP treatment, the abundance of mRNA that hybridized
with the LE-ACS2 and LE-ACS4 probes was almost
completely eliminated (Fig. 4, compare lanes 4 and 5 with 7 and 10, respectively). This elimination recovered gradually in 2 and 4 d
(lanes 8, 9, and 11).
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| Figure 4.
Expression of LE-ACS,
LE-ACO, and ethylene receptor genes in tomato fruit
during development and ripening and effect of MCP. mRNAs were prepared
from the fruit immediately after the determination of ethylene levels
as shown in Figure 2. Lane 1, Control fruit at the immature stage; lane
2, control fruit at the mature green stage; lane 3, control fruit at
the turning stage; lane 4, control fruit at the pink stage; lane 5, control fruit at the red stage; lane 6, control fruit at the full-ripe
stage; lane 7, turning-stage fruit 2 d after MCP treatment; lane
8, turning-stage fruit 4 d after MCP treatment; lane 9, turning-stage fruit 6 d after MCP treatment; lane 10, pink-stage
fruit 2 d after MCP treatment; and lane 11, pink-stage fruit
4 d after MCP treatment. Each lane contained 3 µg of mRNA. Actin
was used as an internal control to normalize the amount of mRNA
loaded.
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In contrast, the LE-ACS6 gene was expressed in the fruit at
the immature green and mature green stages (Fig. 4, lanes 1 and 2),
whereas no signals for this gene were detected in the ripening fruit
(Fig. 4, lanes 3-6). However, accumulation of LE-ACS6 mRNA was detected in the fruit treated with MCP at both the turning and pink
stages (Fig. 4, lanes 7-11). LE-ACS1A and
LE-ACS3 genes were expressed weakly in the fruit throughout
development and ripening, and the abundance of their mRNAs was less
influenced by treatment with MCP. Although two LE-ACO genes
were expressed in immature green and mature green fruit (Fig. 4, lanes
1 and 2), the abundance increased further upon commencement of ripening (Fig. 4, lanes 3-6), particularly in LE-ACO1. The increases
in accumulation of the LE-ACO mRNAs with ripening were
prevented considerably by treatment of fruit with MCP at both the
turning and pink stages (Fig. 4, lanes 7-11). Of the two members of
the ethylene receptor gene family, the abundance of NR mRNA
in the fruit was at a very low level at the preclimacteric stage (Fig. 4, lanes 1 and 2), increased suddenly at the turning stage, and maintained its strong signals during ripening (Fig. 4, lanes 3-6). This increase of NR mRNA associated with ripening was also
lowered by MCP treatment in a manner similar to that observed for
LE-ACS2 (Fig. 4, lanes 7-11). Signals for the
TAE1 gene in the fruit were detected at the preclimacteric
stage (Fig. 4, lanes 1 and 2) and increased slightly during ripening
(Fig. 4, lanes 3-6). MCP decreased the abundance of TAE1
mRNA in ripening fruit (Fig. 4, lanes 7-11).
Effect of Propylene on Gene Expression in Immature Green Fruit
The results presented above suggest that the expression of the
LE-ACS6 gene may be under negative feedback regulation in
tomato fruit. To test this hypothesis, immature green fruit were
treated with 5000 µL L1 propylene for 2 and
4 d. Neither autocatalytic ethylene production nor increases in
respiration rate and ACC content was induced by propylene in these
young fruit, whereas ACC oxidase was activated more than 2- to 3-fold
(Table III). The results of northern
analysis for mRNAs from these fruit are shown in Figure
5. The accumulation of LE-ACS6
transcript in the control fruit (Fig. 5, lanes 1-3) was strongly
prevented by treatment with propylene for 2 and 4 d (Fig. 5, lanes
4 and 5, respectively). Since there were no increases in ethylene
production or ACC content in the fruit, propylene did not induce the
accumulation of transcripts for LE-ACS2 and LE-ACS4. LE-ACS1A and LE-ACS3 were
expressed constitutively in the fruit irrespective of propylene
treatment. Although in vivo activity of ACC oxidase in the fruit was
increased by propylene treatment, we did not observe an
enhancement of the accumulation of LE-ACO1 mRNA. Signals for
the NR and TAE1 genes were weak in the control
fruit and were less influenced by treatment with propylene.
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Table III.
Effect of propylene on the rates of respiration
and ethylene production, ACC content, and in vivo ACC oxidase activity
in immature green fruit
Fruit were harvested about 2 weeks after flowering and then treated
with 5000 µL L1 propylene for 2 and 4 d at 22°C.
The values are the means ± SE of three replications.
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| Figure 5.
Effect of propylene on the accumulation of mRNAs
corresponding to LE-ACS and ethylene receptor gene
families and the LE-ACO1 gene in immature green fruit.
mRNAs were isolated from the same fruit sample shown in Table III. Lane
1, Control fruit at harvest; lane 2, control fruit 2 d after
harvest; lane 3, control fruit 4 d after harvest; lane 4, propylene-treated fruit for 2 d; lane 5, propylene-treated fruit for 4 d. Each lane contained 3 µg of mRNA. Actin was used as an internal
control to normalize the amounts of mRNAs loaded.
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Transition of Expression of Genes at Ripening Onset
It is possible that the elimination of LE-ACS6 and the
appearance of LE-ACS2 transcripts may have been responsible
for the transition from system 1 to system 2 ethylene production. To
examine this concept, northern analysis was performed in fruit at
stages from mature green to turning, all of which had different levels of basal ethylene production (Fig. 6).
The rates of ethylene production in the fruit were 0.18, 0.36, 0.96, and 1.46 nL g1 h1 at
the MG1, MG2, MG3, and turning stages, respectively. The abundance of
LE-ACS6 mRNA in the fruit decreased gradually with ripening, reaching undetectable levels at the turning stage. In contrast, the
LE-ACS2 transcript, which was undetectable at the MG1 stage, increased gradually when the rate of ethylene production was increased. Signals for the NR gene at the MG1 stage were very weak,
increasing from the MG2 stage to the turning stage. Signals for the
LE-ACS1A and LE-ACS3 genes changed little from
the MG1 stage to the turning stage. The abundance of LE-ACO1
and TAE1 mRNAs was also unchanged from the MG1 stage to the
turning stage. No signal for the LE-ACS4 gene was detected
in the turning fruit, which had a lower ethylene level (1.46 nL
g1 h1) than that used
in the fruit shown in Figures 2 and 4 (2.35 nL g1 h1).
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| Figure 6.
Changes in the accumulation of mRNAs corresponding
to LE-ACS and ethylene receptor gene families and the
LE-ACO1 gene in fruit with different rates of ethylene
production from the mature green stage to the turning stage. Lane 1, MG1 fruit (0.18 nL g1 h1 ethylene
production); lane 2, MG2 fruit (0.36 nL g1
h1 ethylene production); lane 3, MG3 fruit (0.96 nL
g1 h1 ethylene production); and lane 4, turning fruit (1.46 nL g1 h1 ethylene
production). Each lane contained 3 µg of mRNA. Actin was used as an
internal control to normalize the amounts of mRNAs loaded.
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| |
DISCUSSION |
The climacteric life of fruits is divided into preclimacteric and
climacteric stages depending on whether a massive production of
ethylene has commenced. In tomato fruit ethylene production during the
climacteric stage has been demonstrated to be due to the accumulation
of transcripts of two ACC synthase genes, LE-ACS2 and
LE-ACS4 (Rottmann et al., 1991; Lincoln et al., 1993), and one ACC oxidase gene, LE-ACO1 (Barry et al., 1996). Using
MCP, an ethylene action inhibitor, we previously demonstrated that the
expression of all three of these genes is highly regulated through a
positive feedback mechanism in ripening tomato fruit (Nakatsuka et al.,
1997). In that study we suggested the possible existence of a gene(s)
under negative feedback regulation, because the inhibitory effects of
MCP on the expression of the genes were not correlated with those on
ethylene biosynthesis. To provide experimental evidence to support our
hypothesis, we cloned nine cDNA fragments, including five members of
the ACC synthase gene family, two of the ACC oxidase family, and two of
the ethylene receptor family. Among the seven previously cloned genes
for ACC synthase (Rottmann et al., 1991; Yip et al., 1992; Lincoln et al., 1993; Spanu et al., 1993; Olson et al., 1995; Oetiker et al.,
1997), fragments of LE-ACS1B and LE-ACS5 could
not be amplified by RT-PCR used in this study, even by the use of
specific primers. Although the transcription of these two genes has
been demonstrated in tomato roots and suspension cultures (Yip et al.,
1992; Spanu et al., 1993; Oetiker et al., 1997), there is no evidence
demonstrating their expression in the fruit. Therefore, we concluded
that their transcripts were absent in the fruit tissue.
In the present study we observed large ethylene production in the fruit
from the turning stage with further increases toward the red stage
(Fig. 2). This increase in ethylene production was prevented to a large
extent by treatment with MCP at both the turning and pink stages. Using
mRNAs extracted from these fruit, we performed northern analysis with
the probes prepared from cDNA fragments cloned in this study (Fig. 4).
Among five members of the LE-ACS gene family, the abundance
of LE-ACS2 and LE-ACS4 mRNAs in the fruit
increased beginning at the turning stage, and MCP greatly suppressed
this increase in a manner similar to that observed in our previous
study (Nakatsuka et al., 1997). In mature green fruit the transcripts
of these genes were absent but were inducible by treatment with
ethylene through a positive feedback mechanism, resulting in the
induction of ripening (Lincoln et al., 1993).
Expression of LE-ACS2 during the natural progress of
ripening first appeared in MG2 fruit, the stage showing the first
elevation of ethylene production from the basal level (Fig. 6).
However, propylene did not induce the accumulation of
LE-ACS2 and LE-ACS4 transcripts in immature green
fruit within 4 d (Fig. 5) but did by 8 d of treatment (data
not shown), indicating a possible lack of a rapid, autocatalytic system
for ethylene biosynthesis in young fruit. This lack of a rapid response
to applied ethylene has been reported in young tomato fruit, in which
fruits harvested as early as 17 d after pollination required 12 to
15 d of continuous treatment with 1000 µL
L1 ethylene to develop red color (Lyons and
Pratt, 1964). Although expression of the LE-ACS2 and
LE-ACS4 genes is also inducible by wounding (Lincoln et al.,
1993), these are probably the major genes responsible for the system 2 ethylene production during ripening in tomato fruit. More direct
evidence for this is shown in transgenic tomatoes in which the
LE-ACS2 antisense fruits produce less ethylene and fail to
ripen, with complete inhibition of the LE-ACS2 and
LE-ACS4 genes during ripening (Oeller et al., 1991).
In contrast to LE-ACS2 and LE-ACS4, the
LE-ACS6 gene was expressed in fruit from the immature green
to the mature green stages, whereas no signals for this gene were
detected in the ripening fruit. Signals for this gene were detected in
the ripening fruit treated with MCP (Fig. 4), strongly suggesting that
the expression of the LE-ACS6 gene is regulated by a
negative feedback mechanism. This concept was clearly demonstrated in
immature green fruit, in which the previously detected signals for the
LE-ACS6 gene were eliminated by treatment with propylene, an
ethylene analog (Fig. 5). Furthermore, the abundance of this mRNA in
the fruit during the natural onset of ripening decreased gradually to
an undetectable level at the turning stage (Fig. 6).
Oetiker et al. (1997) isolated LE-ACS6 cDNA from tomato
roots, but theirs is the only available information concerning its expression, suggesting that it exhibits an elicitor-inducible feature.
Lincoln et al. (1993) also previously described the cloning of
LE-ACS6 cDNA and suggested the possible expression of this gene in ripe tomato fruit. However, their suggestion differs from our
present observation with respect to the characteristic features of the
LE-ACS6 gene. Therefore, LE-ACS6 reported by
Lincoln et al. (1993) may have been a different cDNA from that cloned
by Oetiker et al. (1997) and that obtained in the present study. Mori
(1995) described an expression pattern of LE-ACS6 in tomato fruit that is similar to ours, with an elimination of its transcripts in ripe fruit, but to our knowledge, no further information is available for this observation (in particular the gene sequences). The
present results clearly demonstrate the existence of an
ethylene-biosynthetic gene, the expression of which is regulated under
a negative feedback mechanism in fruit. The possible involvement of a
negative feedback regulation at the ethylene-production level has been
suggested in fruits such as banana (Vendrell and MacGlasson, 1971),
citrus (Riov and Yang, 1982), and winter squash (Hyodo et al., 1985).
LE-ACS1A and LE-ACS3 genes were expressed in the
fruit throughout development and ripening (Figs. 4 and 6). Furthermore,
the abundance of their mRNAs was not influenced by treatment with either MCP (Fig. 4) or propylene (Fig. 5), indicating that the expression of these genes is independent of ethylene action. Although these two genes resembled each other closely in expression pattern, LE-ACS3 had low sequence similarities (less than 62%) among
the LE-ACS gene family (data not shown). This may exclude a
possibility that the probe for LE-ACS3 could hybridize to
other transcripts encoding tomato ACC synthase. The full-length
sequence of LE-ACS1A mRNA together with its twin of
LE-ACS1B was previously registered on the database
(accession nos. U72389 and U72390), and their expression was first
examined in cultured cells using the RNase-protection assay, in which
LE-ACS1B was strongly and constitutively expressed but no
signals for LE-ACS1A were detectable (Oetiker et al., 1997).
However, only the LE-ACS1A cDNA fragment was amplified on
RT-PCR. LE-ACS5 was not amplified in the present study,
suggesting a tissue-specific expression of each ACC synthase gene
family. The transcript of LE-ACS3 has been detected in
fruits (Yip et al., 1992) and suspension cultures (Oetiker et al.,
1997). Among the members of the LE-ACS gene family studied,
LE-ACS1A, LE-ACS3, and LE-ACS6 genes
were expressed in the preclimacteric fruit, suggesting that system 1 ethylene in tomato fruit may be mediated via these three genes.
In tomato at least three genes encode ACC oxidase (Barry et al., 1996):
LE-ACO1 is the main gene expressed in ripening tomato fruit,
LE-ACO2 expression is mainly restricted to the tissues associated with the anther cone, and LE-ACO3 transcripts
accumulate in floral organs and transiently appear with a weak signal
in fruit at the breaker stage (Barry et al., 1996). In the present study we cloned a novel ACC oxidase gene and named it
LE-ACO4. Both LE-ACO1 and LE-ACO4
transcripts accumulated in preclimacteric fruit, and this accumulation
increased in ripening fruit. This increase was prevented to a large
extent by MCP treatment in a manner similar to that of the
LE-ACS2 and LE-ACS4 genes (Fig. 4).
Although feedback regulation of the ACC oxidase genes has not yet been
clarified, there is evidence that accumulation of the transcripts is
enhanced with increases in ethylene production and by exogenously
applied ethylene in fruits such as tomato (Barry et al., 1996), apple
(Ross et al., 1992), melon (Lasserre et al., 1996), banana (Huang et
al., 1997), kiwifruit (Whittaker et al., 1997), and pear
(Lelievre et al., 1997). In vegetative tissues ACC oxidase mRNA
has also been shown to be regulated by ethylene; the transcript for an
ACC oxidase gene in excised mung bean hypocotyls was enhanced by
exogenous ethylene and suppressed by aminooxyacetic acid, an ACC
synthase inhibitor, with a reduction of endogenous ethylene to the
basal level (Kim and Yang, 1994). From these observations, it may be
reasonable to assume that a positive feedback regulation is involved in
the expression of ACC oxidase gene in a manner similar to that in ACC
synthase. However, since propylene did not enhance the
already-accumulated LE-ACO1 transcript in immature green
fruit (Fig. 5), the responsiveness of LE-ACO1 to ethylene may be less than that of LE-ACS6.
Since the ETR1 gene in Arabidopsis was cloned and sequenced
as the gene related to ethylene receptors (Chang et al., 1993), five
homologs have been isolated from tomato (Lashbrook et al., 1998). We
cloned cDNA fragments corresponding to the NR (Wilkinson et
al., 1995) and TAE1 (Zhou et al., 1996) genes based on their reported sequences. Expression of the NR gene was extremely
low in immature and mature green fruit but suddenly increased greatly at the turning stage (Fig. 4). Investigations at the onset of ripening
revealed that this increase commenced in MG2 fruit, the stage of the
first increase in ethylene production from the basal level (Fig. 6).
Wilkinson et al. (1995) indicated that NR mRNA in tomato
fruit is positively regulated by ethylene in a development-specific manner from observations that the amount of NR mRNA
increases in ripening fruit and ethylene-treated mature green fruit but not in Nr mutant tomato.
A strong induction of NR mRNA at the onset of ripening has
also been demonstrated in tomato fruit (Lashbrook et al., 1998). In the
present study this accumulation of NR mRNA associated with ripening was prevented in the fruit treated with MCP (Fig. 4). It has
been proved that MCP is an ethylene-action inhibitor that binds to the
receptor site competitively, thereby preventing tissue response to
ethylene (Sisler and Serek, 1997). The present results demonstrate that
MCP prevents the accumulation of LE-ACS2,
LE-ACS4, LE-ACO1, and LE-ACO4 mRNAs in
the ripening fruit with an almost complete elimination of NR
transcripts (Fig. 4). Furthermore, inhibition of the accumulation of
LE-ACS and LE-ACO transcripts recovered after 2 to 4 d concomitantly with the recovery of NR transcripts. A similar observation has been reported for tomato fruit
using diazocyclopentadiene, another inhibitor of ethylene action (Tian
et al., 1997).
The above observations, together with the results presented here,
suggest that the NR protein may be synthesized successively in tomato
fruit during ripening, leading to the recovery of the gene transcripts
that are regulated under positive feedback. The present results also
suggest that this successive synthesis of NR protein might be under the
control of a positive feedback mechanism. However, the expression of
this gene was not inducible in immature green fruit by exposure to
ethylene for 1 d (Wilkinson et al., 1995) or propylene for 4 d (Fig. 5). These differences in NR gene expression in
response to ethylene treatment between immature and ripening fruits may
modulate the differential sensitivity to ethylene in maturing tomato
fruits (Wilkinson et al., 1995). McGlasson (1985) previously pointed
out that most fruit become increasingly sensitive to ethylene with time
after anthesis. The abundance of TAE1 mRNA accumulated
constitutively throughout development and ripening irrespective of
treatment with either MCP or propylene. Similar results have been
reported for tomato leaf, flower, and fruit tissues, in which
expression was unaffected by ethylene, silver ions, an ethylene-action
inhibitor, or auxin in leaf-abscission zones (Zhou et al., 1996). Using
the RNase-protection assay, Lashbrook et al. (1998) recently
demonstrated that the signals for three members of ETR1
homologs, including NR and TAE1, were detectable in tomato fruit throughout preclimacteric stages. Therefore, the presence of one or more ETR1 homologs prior to ripening may
contribute ripening-independent ethylene perception processes in
immature fruit, in which propylene eliminated the LE-ACS6
transcript but did not induce the LE-ACS2 transcript (Fig.
5).
In conclusion, the results presented here suggest that ethylene
biosynthesis in tomato fruit is regulated by the three different members of the ACC synthase gene family: (a) LE-ACS2 and
LE-ACS4 are the dominant genes responsible for system 2 ethylene production in ripening fruit and their expression is regulated
by a positive feedback mechanism, (b) the LE-ACS6 gene is
responsible for the low rates of system 1 ethylene production and is
negatively regulated in preclimacteric fruit, and (c) the
LE-ACS1A and LE-ACS3 genes are also responsible
for the preclimacteric system 1 ethylene production, and their
transcripts accumulate constitutively throughout fruit development
irrespective of the mode of feedback regulation.
In tomato fruit the preclimacteric system 1 ethylene production is
mediated by the LE-ACS1A, LE-ACS3, and
LE-ACS6 genes, together with LE-ACO1 and
LE-ACO4. Ethylene production shifts to system 2 at the
climacteric stage, with a burst in the accumulation of LE-ACS2, LE-ACS4, LE-ACO1, and
LE-ACO4 mRNAs as a result of positive feedback regulation.
This transition from system 1 to system 2 ethylene production may be
controlled by the accumulated level of NR protein from the mature green
stage to the turning stage. Considering the existence of multiple
ETR1 homologs in tomato (Yen et al., 1995), further work is
needed to clarify the induction mechanism of fruit ripening, especially
whether the expression of LE-ACS2 gene induces NR
transcript or vice versa.
| |
FOOTNOTES |
1
This work was supported in part by a
grant-in-aid (no. 08456020 to A.I.) from the Ministry of Education,
Science, Sports and Culture of Japan.
2
Present address: Laboratory of Horticultural
Breeding, Faculty of Life and Environmental Science, Shimane
University, Matsue, Shimane, 690-8504, Japan.
*
Corresponding author; e-mail enri{at}ccew2.cc.okayama-u.ac.jp; fax
81-86-251-8338.
Received May 26, 1998;
accepted August 28, 1998.
The accession numbers for the sequences reported in this article are
AB013100 (LE-ACO4) and AB013101 (LE-ACS6).
| |
ABBREVIATIONS |
Abbreviations:
MCP, 1-methylcyclopropene.
RACE, rapid
amplification of cDNA ends.
RT, reverse transcriptase.
| |
ACKNOWLEDGMENTS |
We thank Dr. Alan B. Bennett (University of California, Davis)
for providing the tomato actin cDNA. We also thank Dr. Francis M. Mathooko (Jomo Kenyatta University of Agriculture and Technology, Kenya) for his careful reading of the manuscript.
| |
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I. El-Sharkawy, S. Sherif, I. Mila, M. Bouzayen, and S. Jayasankar
Molecular characterization of seven genes encoding ethylene-responsive transcriptional factors during plum fruit development and ripening
J. Exp. Bot.,
March 1, 2009;
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[Abstract]
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I. El-Sharkawy, W. S. Kim, S. Jayasankar, A. M. Svircev, and D. C. W. Brown
Differential regulation of four members of the ACC synthase gene family in plum
J. Exp. Bot.,
May 1, 2008;
59(8):
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[Abstract]
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F.B. Flores, M.C. Martinez-Madrid, and F. Romojaro
Influence of Fruit Development Stage on the Physiological Response to Ethylene in Cantaloupe Charentais Melon
Food Science and Technology International,
February 1, 2008;
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[Abstract]
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A. Inaba, X. Liu, N. Yokotani, M. Yamane, W.-J. Lu, R. Nakano, and Y. Kubo
Differential feedback regulation of ethylene biosynthesis in pulp and peel tissues of banana fruit
J. Exp. Bot.,
March 1, 2007;
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[Abstract]
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A. Tassoni, C. B. Watkins, and P. J. Davies
Inhibition of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying ripening, but may moderate the rate of ripening or over-ripening
J. Exp. Bot.,
September 1, 2006;
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[Abstract]
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N. Ma, H. Tan, X. Liu, J. Xue, Y. Li, and J. Gao
Transcriptional regulation of ethylene receptor and CTR genes involved in ethylene-induced flower opening in cut rose (Rosa hybrida) cv. Samantha
J. Exp. Bot.,
August 1, 2006;
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[Abstract]
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S. Park, N. Sugimoto, M. D. Larson, R. Beaudry, and S. van Nocker
Identification of Genes with Potential Roles in Apple Fruit Development and Biochemistry through Large-Scale Statistical Analysis of Expressed Sequence Tags
Plant Physiology,
July 1, 2006;
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[Abstract]
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E. Katz, J. Riov, D. Weiss, and E. E. Goldschmidt
The climacteric-like behaviour of young, mature and wounded citrus leaves
J. Exp. Bot.,
May 1, 2005;
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[Abstract]
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H. J. Klee
Ethylene Signal Transduction. Moving beyond Arabidopsis
Plant Physiology,
June 1, 2004;
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[Full Text]
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A. Itai, K. Ishihara, and J. D. Bewley
Characterization of expression, and cloning, of {beta}-D-xylosidase and {alpha}-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit
J. Exp. Bot.,
December 1, 2003;
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[Abstract]
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H. P. J. de Wild, E. C. Otma, and H. W. Peppelenbos
Carbon dioxide action on ethylene biosynthesis of preclimacteric and climacteric pear fruit
J. Exp. Bot.,
June 1, 2003;
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[Abstract]
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C. Coenen, M. Christian, H. Luthen, and T. L. Lomax
Cytokinin Inhibits a Subset of Diageotropica-Dependent Primary Auxin Responses in Tomato
Plant Physiology,
April 1, 2003;
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[Abstract]
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K. Hiwasa, Y. Kinugasa, S. Amano, A. Hashimoto, R. Nakano, A. Inaba, and Y. Kubo
Ethylene is required for both the initiation and progression of softening in pear (Pyrus communis L.) fruit
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February 1, 2003;
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R. Nakano, E. Ogura, Y. Kubo, and A. Inaba
Ethylene Biosynthesis in Detached Young Persimmon Fruit Is Initiated in Calyx and Modulated by Water Loss from the Fruit
Plant Physiology,
January 1, 2003;
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[Abstract]
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V. Balbi and T. L. Lomax
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W. Moeder, C. S. Barry, A. A. Tauriainen, C. Betz, J. Tuomainen, M. Utriainen, D. Grierson, H. Sandermann, C. Langebartels, and J. Kangasjarvi
Ethylene Synthesis Regulated by Biphasic Induction of 1-Aminocyclopropane-1-Carboxylic Acid Synthase and 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Genes Is Required for Hydrogen Peroxide Accumulation and Cell Death in Ozone-Exposed Tomato
Plant Physiology,
December 1, 2002;
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[Abstract]
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L. Alexander and D. Grierson
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H. J. Klee
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J. Diaz, A. ten Have, and J. A.L. van Kan
The Role of Ethylene and Wound Signaling in Resistance of Tomato to Botrytis cinerea
Plant Physiology,
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[Abstract]
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K. L.-C. Wang, H. Li, and J. R. Ecker
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B. Ruperti, L. Cattivelli, S. Pagni, and A. Ramina
Ethylene-responsive genes are differentially regulated during abscission, organ senescence and wounding in peach (Prunus persica)
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March 1, 2002;
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[Abstract]
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J. A. Roberts, A. Hussain, I. B. Taylor, and C. R. Black
Use of mutants to study long-distance signalling in response to compacted soil
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January 1, 2002;
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[Abstract]
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I. Llop-Tous, C. S. Barry, and D. Grierson
Regulation of Ethylene Biosynthesis in Response to Pollination in Tomato Flowers
Plant Physiology,
July 1, 2000;
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[Abstract]
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C. S. Barry, M. I. Llop-Tous, and D. Grierson
The Regulation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase Gene Expression during the Transition from System-1 to System-2 Ethylene Synthesis in Tomato
Plant Physiology,
July 1, 2000;
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[Abstract]
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X. Liu, S. Shiomi, A. Nakatsuka, Y. Kubo, R. Nakamura, and A. Inaba
Characterization of Ethylene Biosynthesis Associated with Ripening in Banana Fruit
Plant Physiology,
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[Abstract]
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Top
Abstract
Introduction