Journal of Experimental Botany, Vol. 53, No. 368, pp. 415-422,
March 1, 2002
© 2002 Oxford University Press
Original Papers |
Detection of ethylene receptor protein Cm-ERS1 during fruit development in melon (Cucumis melo L.)
1 Plant Biotechnology Institute, Ibaraki Agricultural Centre, Ago 3165–1, Iwama, Nishi-ibaraki, Ibaraki, 319-0292 Japan
2 Institute of Agriculture and Forestry, Gene Research Centre, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, 305-8572 Japan
3 Laboratory of Photobiology-2, Photodynamics Research Centre, The Institute of Physical and Chemical Research (Riken), Sendai, 980-0845 Japan
Received 29 May 2001; Accepted 27 September 2001
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Abstract |
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Antibodies against melon ethylene receptor, Cm- ERS1 was prepared. Cm-ERS1 protein formed a disulphide-linked homodimer and it was present in microsomal membranes but not in soluble fractions. Cm-ERS1 protein was present at high levels in melon fruit during early developmental stages. This transition pattern was also observed in another melon cultivar.
Key words: Antibody, ethylene receptor, fruit development, melon, regulation.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ethylene is a gaseous plant hormone that plays an important role in many aspects of plant growth and development, such as seed germination, root hair formation, and leaf and flower senescence and abscission. Ethylene is also involved in fruit ripening (Yang and Hoffman, 1984
; Abeles et al., 1992; Kende, 1993). To understand the regulation of plant cell processes by ethylene, both the production and perception of ethylene must be examined. However, little was known about the process of ethylene perception until the isolation of ethylene receptor genes because attempts to isolate ethylene receptors through biochemical approaches were unsuccessful (Sisler, 1991).
A candidate ethylene receptor gene, ETR1, was first isolated from Arabidopsis by molecular and genetic approaches (Chang et al., 1993), and ethylene binding to the ETR1 gene product has been confirmed (Schaller et al., 1995). Since then, four additional homologues, ETR2, ERS1, ERS2, and EIN4, have been isolated from Arabidopsis (Sakai et al., 1998; Hua et al., 1995, 1998). Each of the five ethylene receptors contains a putative transmembrane domain near its amino-terminus and a histidine kinase domain in the middle of the protein. In addition to these domains, ETR1, ETR2, and EIN4 each have a signal receiver domain near their carboxyl-termini, whereas ERS1 and ERS2 lack this domain. When expressed in yeast, ETR1 and ERS1 proteins form dimers through disulphide bonds linking the amino-termini of the two molecules (Schaller et al., 1995; Hall et al., 2000). Mutations in the transmembrane domains of each of these receptors commonly result in dominant ethylene insensitivity (Hua et al., 1998; Sakai et al., 1998). Analysis of loss-of-function mutants of ETR1, ETR2, EIN4, and ERS1 has shown that these receptors are functionally redundant and that they negatively regulate the ethylene response (Hua and Meyerowitz, 1998). Many homologues of ethylene receptor genes have been isolated from various plant species by library screens with heterologous probes and by RT-PCR with degenerate primers (Chang and Shockey, 1999; Sato-Nara et al., 1999a). The isolation of ethylene receptor genes has enabled studies of ethylene perception and sensitivity, thus enhancing an understanding of the mechanism by which ethylene regulates fruit development.
Melon is an excellent organism for such studies for several reasons. First, melon fruit development can be clearly divided into three stages, phases I, II, and III, as in tomato (Gillaspy et al., 1993). Second, the structure of the fruit is simple and obvious, and the embryo, flesh, placenta, and seeds are well ordered. Third, melon fruit development can be clearly divided into ethylene-insensitive and ethylene-sensitive stages: the developing fruit has a lower sensitivity to ethylene than does the ripening fruit. Finally, the genes encoding ACC synthase and ACC oxidase, which are key enzymes in ethylene production, have already been isolated from melon, and the expression of their mRNAs and proteins during fruit development has been studied (Balagué et al., 1993; Lasserre et al., 1996; Miki et al., 1995; Yamamoto et al., 1995; Kato et al., 1997).
To elucidate the role of ethylene receptor proteins in melon fruit development, the ethylene receptor homologue genes Cm-ERS1 and Cm-ETR1, which had previously been isolated from melon, and the expression of their mRNAs was analysed (Sato-Nara et al., 1999b). These mRNAs were differentially expressed during fruit development, suggesting that each of these proteins has a specific role in fruit development. However, it was unclear whether the amount of ethylene receptor mRNA accurately reflects the level of the corresponding proteins.
In this study, antibodies against Cm-ERS1 protein were prepared in order to examine the temporal and spatial expression pattern of Cm-ERS1 protein during fruit development. In addition, the localization of the ethylene receptor in plant cells was investigated. The results revealed that a post-transcriptional regulation of Cm-ERS1 expression affects stage- and tissue-specific accumulation of this protein. Evidence for membraneous localization, dimerization of the protein and for possible involvement in cell division and elongation at the early stage of fruit development is discussed.
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Materials and methods |
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Plant material
Seeds of two cultivars of muskmelon (Cucumis melo L. reticulatus), cvs Fuyu A and Natsu 4, were sown and grown in a greenhouse as described previously (Sato-Nara et al., 1999
b). Fruits setting after self-pollination were harvested for experiment at the indicated days after pollination (DAPs).
Preparation of polyclonal antisera
Four oligonucleotide primers were designed based on the nucleotide sequences of the Cm-ERS1 gene (Sato-Nara et al., 1999b). To amplify the Cm-ERS1-KD-encoding DNA fragment, oligonucleotides F1 (5'-GGATCCAAAACTCGAGAATTGATTCTTAAAAATAAGGC-3') and R1 (5'-AAGCTTTCATCAGTCCACGAGCTGATCACGCGCCCGC-3') were used. To amplify the DNA fragments encoding Cm-ETR1-KE, a primer set of F2 (5'-GGATCCAAAACTAGAGAGCTCTTTTTGAAGAACAAG-3') and R2 (5'-AAGCTTTCATCACTCCATTAAGAGATCTCTAGCCCTC-3') was used. In these sequences, underlined nucleotides indicate BamHI and HindIII restriction sites. Nucleotides in italics indicate sequences complementary to the stop codon.
PCR amplification was carried out with a program of 94 °C for 9 min followed by 25 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min, and a final step of 72 °C for 2 min. The amplified fragments were TA-cloned into vector pCR2.1 (Invitrogen, Carlsbad, CA, USA), and the nucleotide sequences of both strands were confirmed. Each clone was then excised with BamHI and HindIII and ligated into E. coli expression vector pQE30 (Qiagen, Chatsworth, CA, USA), which placed a 6x-histidine tag at the amino termini. The fusion proteins were overexpressed in E. coli by induction with isopropyl-ß-D-thiogalactopyranoside (IPTG) and were affinity-purified using a His–Trap column (Amersham Pharmacia Biotech, Buckinghamshire, England) according to the manufacturer's instructions. The sample of Cm-ERS1-KD was further fractionated on 12% (w/v) SDS-polyacrylamide gels (Laemmli, 1970) of 20x20 cm. The gels were stained with Coomassie Brilliant Blue (CBB), and gel bands containing the fusion proteins were excised and used to raise antisera in rabbits with the assistance of Shibayagi Co. Ltd. (Gunma, Japan). The IgG fraction was recovered from each antiserum using a MAb Trap GII Kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The IgG fraction was subsequently cleared by adsorption of non-specific IgG to powdered E. coli proteins, as described earlier (Sambrook et al., 1989).
Preparation of samples for SDS-PAGE
E. coli cells were pelleted by centrifugation at 3000 rpm for 5 min, dissolved in 1x SDS-PAGE sample buffer (62.5 mM TRIS-HCl, pH 6.8, 10% (w/v) glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) containing 5% (v/v) 2-mercaptoethanol and boiled for 5 min. Since the affinity-purified proteins were already dissolved in affinity column elution buffer, a 1/3 volume of 4x sample buffer with 2-mercaptoethanol was added to each of these samples before boiling.
For detection of native Cm-ERS1 protein, a Fuyu A fruit was harvested at 15 DAP, and proteins were extracted in the following manner. To prepare total proteins, 500 mg of pericarp were homogenized at 4 °C with 1 ml of extraction buffer [100 mM TRIS-HCl, pH 8.0, 300 mM NaCl, 20 mM EDTA, 20% (w/v) glycerol] containing 5 mM dithiothreitol (DTT) and the protease inhibitors [phenylmethylsulphonyl fluoride (1 mM), leupeptin (0.5 µg ml-1) and pepstatin A (1 µg ml-1)]. To prepare soluble and microsomal membrane proteins, the total protein homogenate was strained through two layers of gauze and centrifuged at 12000 g for 15 min at 4 °C. The supernatant was carefully recovered and centrifuged again at 100000 g for 1 h at 4 °C. The supernatant thus obtained was used as the soluble fraction. The remaining pellet was resuspended in 100 µl of extraction buffer containing 1% (v/v) Triton X-100, 0.1% (w/v) SDS, and protease inhibitors at the concentrations listed above, and incubated for 30 min at 4 °C. This solubilized fraction was used as the microsomal membrane fraction. The protein concentrations in the total, soluble, and microsomal membrane protein samples were determined using a Bio-Rad Protein Assay Kit (Bio-Rad) and bovine serum albumin as a standard. Protein samples of 5 µg were mixed with 1/3 volume of 4x SDS-PAGE sample buffer. DTT was then added to a final concentration of 100 mM and the samples were boiled for 5 min.
To examine dimerization of Cm-ERS1 protein, 5 µg of the microsomal membrane samples from Fuyu A fruit at 15 DAP was mixed with 1/3 volume of 4x SDS-PAGE sample buffer with or without 100 mM DTT, and boiled for 5 min.
To examine the accumulation of ethylene receptor (Cm-ERS1) proteins during melon fruit development, Fuyu A fruit were harvested at 0, 4, 8, 15, 22, 29, 40, 50, 53, and 56 DAP, and Natsu 4 fruit were harvested at 0, 4, 8, 15, 22, and 29 DAP. Microsomal membrane proteins were prepared from each sample in the presence of 100 mM DTT as described above.
Western blot analysis
E. coli proteins and melon fruit proteins were separated by SDS-PAGE on polyacrylamide gels of 12% and 8% (w/v) (Laemmli, 1970), respectively. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes, which were then stained with Ponceau S to confirm efficient transfer. The membranes were destained in PBS buffer [8.1 mM Na2HPO4, 1.47 mM KH2PO4, (pH 7.5), 137 mM NaCl, 2.68 mM KCl] containing 0.2% Tween20 and then probed with primary antibody preparations. These preparations consisted of 1:1000 dilutions of polyclonal antibodies to Cm-ERS1-KD antigen. In control experiments, preimmune serum obtained from identical rabbits was used at a dilution of 1:1000. An anti-rabbit IgG-alkaline phosphatase conjugate (Bio-Rad) was used at a 1:3000 dilution for the secondary antibody. Immunoreactive species were detected using a 5-bromo-4-chloro-3-indolylphosphate-p-toluidine/nitroblue tetrazolium colour reaction in an Immun-Blot Assay Kit (Bio-Rad), according to the manufacturer's instructions.
Western tissue print analysis
The portions of the Fuyu A and Natsu 4 fruits remaining after Western blot analysis were cut into slices and placed on PVDF membranes for 5 min. The membranes were then air-dried and subjected to Western tissue print analysis. Immunoreaction and detection were performed in the same manner as described above.
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Results |
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Preparation of antisera and characterization of antibody specificity
To produce antibodies against Cm-ERS1 protein, the polypeptide region K117-D327 of Cm-ERS1 (Cm-ERS1-KD) was chosen as an antigen. This region lies between the transmembrane and histidine kinase domains for the polypeptide chain of Cm-ERS1 protein (Fig. 1A
). When the antigen was expressed as fusion proteins in E. coli, polypeptides of the predicted molecular masses were specifically amplified (lanes 1 and 2 in Fig. 1B). The fusion protein was affinity-purified (lane 3 in Fig. 1B) and used to raise antiserum in rabbits.
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To determine whether the crude antiserum was specific for Cm-ERS1 protein, Western blot analysis was performed on total protein preparations from melon. Some non-specific bands were observed in addition to the band corresponding to ethylene receptor protein (data not shown). Therefore IgG fractions were purified from the crude antiserum and the fraction used in the studies described below. When the purified antibodies were used against total proteins from E. coli cells overexpressing Cm-ERS1-KD, single bands representing the antigens were observed (lane 2 in Fig. 1C
). By contrast, no bands were observed for total proteins from uninduced E. coli cells (lane 1 in Fig. 1C). When preimmune serum was used, no bands were observed for either the induced or uninduced E. coli cells (data not shown). These results indicate that the purified antibodies specifically recognize the Cm-ERS1 antigen.
Experiments were then performed to rule out the possibility that anti-Cm-ERS1-KD antibodies would cross-react with another ethylene receptor, Cm-ETR1. When anti-Cm-ERS1-KD antibodies were used, no cross-reaction was observed with the Cm-ETR1-KE (K116-E325 of Cm-ETR1; lane 4 in Fig. 1C) which corresponds to Cm-ERS1-KD in the polypeptide chain of Cm-ETR1 (Fig. 1A). Therefore, anti-Cm-ERS1-KD antibodies were used in the following studies as the Cm-ERS1-specific antibodies.
Membrane localization and dimerization of Cm-ERS1 protein
Since Cm-ERS1 has three putative transmembrane domains at its amino-terminus (Fig. 1A), it has been suggested that the protein is integral membrane protein. To examine this possibility, Western blot analysis was performed against protein fractions from melon fruits. A single, 75 kDa band appeared in the microsomal membrane fraction but not in the soluble fraction (lanes M and S in Fig. 2B). The apparent mass of this species is in close agreement with the predicted mass of Cm-ERS1 (71.1 kDa), supporting the idea that Cm-ERS1 protein is present in membranes. No bands were observed in the sample of total proteins (lane T in Fig. 2B), possibly because Cm-ERS1 may be at too low a concentration in the total protein preparation to be detected.
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Structural features of Cm-ERS1 suggest that the proteins may form disulphide-linked dimer in melon cells, as does Arabidopsis ETR1 protein (Schaller et al., 1995
). To examine the native structures of Cm-ERS1, membrane fractions were extracted with or without the reducing agent dithiothreitol (DTT). In the absence of DTT, two species of 75 and 150 kDa, were detected in the membrane fraction (Fig. 3). By contrast, when DTT was present, the 150 kDa species could no longer be detected, and only the 75 kDa species appeared. These results imply that Cm-ERS1 forms a high molecular mass species that is converted to a lower molecular mass species by DTT. Since a reducing agent is required to induce this conversion, a disulphide bond is indicated, suggesting that Cm-ERS1 forms disulphide-linked homodimers.
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Temporal expression of Cm-ERS1 protein during fruit development in Fuyu A
In a previous study (Sato-Nara et al., 1999b), the accumulation pattern of Cm-ERS1 mRNA was investigated during melon fruit development. This study sought to clarify whether the amount of translational product correlates with the amount of the corresponding mRNA by performing Western blot analysis of proteins from Fuyu A fruit at various developmental stages. Accumulation of Cm-ERS1 protein was already observed at 0 DAP (Fig. 4A). The accumulation level gradually decreased until 8 DAP, when it dramatically increased and reached its highest level at 15 and 22 DAP. The protein then decreased again and it was no longer detectable at 40 DAP.
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Accumulation of a Cm-ERS1 homologue during early-stage fruit development in a different cultivar, Natsu 4
Western blot analysis revealed that Cm-ERS1 accumulated at an early developmental stage of Fuyu A such as fruit enlargement. To examine whether such early accumulation of Cm-ERS1 is a general phenomenon, another melon cultivar, Natsu 4, was examined. Although Natsu 4 is closely related to Fuyu A, it exhibits distinct differences in fruit development (Higashi et al., 1999). The fruit of Natsu 4 are smaller than those of Fuyu A because the former cultivar has a shorter period of cell proliferation at the beginning of fruit development. The enlargement of Natsu 4 fruit stops between 21 and 35 DAP, while that of Fuyu A stops between 35 and 49 DAP (Nara-Sato et al., 1999b).
Western blot analysis was performed against proteins from Natsu 4 fruit at various developmental stages (Fig. 4B), after it was first confirmed that antibodies to Cm-ERS1-KD immunoreact with the Natsu 4 Cm-ERS1 protein. At 0–8 DAP, a 75 kDa Cm-ERS1 protein was observed, which remained at almost constant levels during that period. After 8 DAP, the amount of the protein dramatically decreased until it became undetectable at 15 DAP, and it was not observed again during later stages of fruit development. The high accumulation immediately after pollination and the gradual decrease during subsequent fruit development are consistent with the accumulation pattern of Cm-ERS1 in Fuyu A (Fig. 4A). The faster disappearance of the Cm-ERS1 homologue in Natsu 4 than in Fuyu A may reflect the earlier completion of fruit enlargement in Natsu 4.
Spatial expression of Cm-ERS1 protein during fruit development in Fuyu A and Natsu 4
To analyse the localization patterns of Cm-ERS1 protein in these cultivars, a Western tissue print analysis was performed with emphasis on the early stages of fruit development when accumulation of the protein was observed by Western blot analysis (Fig. 4C, D). This analysis gave results similar to that found by Western blot analysis (Fig. 4A, B). In the case of Fuyu A (Fig. 4C), a weak accumulation of Cm-ERS1 was observed immediately after pollination throughout the fruit. Cm-ERS1 began to increase in pericarps as the fruit expanded and showed a marked accumulation at 22 DAP. Cm-ERS1 then decreased at 29 DAP.
In Natsu 4 fruit (Fig. 4D), Cm-ERS1 homologue protein was observed immediately after pollination throughout the fruit. The protein accumulated in the pericarps of developing Natsu 4 fruit from 4–15 DAP. Both the temporal and spatial expression patterns of the Cm-ERS1 homologue were similar to those observed in Fuyu A, suggesting that the role of Cm-ERS1 is highly conserved and the protein plays an important role during the early stage of fruit development.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Since the isolation of the ETR1 gene from Arabidopsis (Chang et al., 1993
), many homologues of ethylene receptor genes have been isolated from various plant species (Chang and Shockey, 1999; Sato-Nara et al., 1999a). All of their deduced amino acid sequences, including Cm-ERS1 have hydrophobic regions at their amino-termini. It has therefore been proposed that these receptor proteins are located in the membrane. Arabidopsis ETR1 and ERS1 proteins were revealed to be located in membrane fractions when expressed in a yeast system (Schaller et al., 1995; Hall et al., 2000). ETR1 protein was further demonstrated to be located in the membrane fraction in Arabidopsis (Schaller et al., 1995). However, it has been unclear where the ERS1-type proteins are located in plant cells. Using Western blot analysis, it was revealed that Cm-ERS1 is located in microsomal membrane fractions, but not in the soluble fractions as Arabidopsis ETR1 protein.
In addition to the question of intracellular location, how the ethylene receptor exists in cells has also been a matter of contention. Arabidopsis ETR1 and ERS1 proteins are known to form disulphide-linked homodimers when expressed in yeast (Schaller et al., 1995; Hall et al., 2000). Western blot analysis of total proteins from Arabidopsis has detected a dimer protein of almost twice the molecular mass of ETR1 (Schaller et al., 1995). In this study, it was revealed that Cm-ERS1 also forms disulphide-linked dimers in melon. This dimerization of Arabidopsis ETR1 and ERS1 are believed to be mediated by Cys-4 and Cys-6 of the proteins (Schaller et al., 1995; Hall et al., 2000). These two Cys residues are also conserved in Cm-ERS1, supporting an idea that these two Cys residues are important for dimerization. However, the potential heterodimerization of Cm-ERS1 and Cm-ETR1 cannot be completely ruled out. To examine this, further studies are needed. For example, multiple members of the ethylene receptor family could be expressed simultaneously in yeast and then immunoprecipitated to search for dimerized ethylene receptor proteins. Antibodies specific for Cm-ERS1 and Cm-ETR1, such as those used in this study, would greatly facilitate such studies.
In a previous study, the accumulation pattern of Cm-ERS1 mRNA during melon fruit development was investigated (Sato-Nara et al., 1999b). Here, the accumulation of Cm-ERS1 protein has been examined and some differences have been found between the accumulation pattern of Cm-ERS1 protein and that of its mRNA. For example, Cm-ERS1 mRNA was still present at the ripening stage, but the protein itself was not detected at that stage. The time points of maximum accumulation also differed for the protein and the mRNAs. Cm-ERS1 protein accumulated to its highest level at earlier stages than Cm-ERS1 mRNA. The difference in the accumulation patterns of mRNA and protein for Cm-ERS1 suggests that its expression is post-transcriptionally regulated.
The higher accumulation of Cm-ERS1 protein at the early stages of fruit development implies that Cm-ERS1 protein has a specialized function in melon fruit development. At the early stages, melon 3-hydroxy-3-methylglutaryl CoA reductase (Cm-HMGR) mRNA is present at a high level, and the Cm-HMGR protein displays high enzyme activity (Kato-Emori et al., 2001). HMGR is thought to be involved in satisfying the demand for mevalonate that is associated with cell division and growth (Jelesko et al., 1999), suggesting that cell division and elongation occur at high frequencies during this period in melon fruits. In this experiment, it was revealed that the Cm-ERS1 protein is highly accumulated during the same periods when Cm-HMGR was highly expressed. Comparison of Cm-ERS1 expression in Fuyu A and Natsu 4 demonstrated that a proportional relationship between fruit size and the Cm-ERS1 protein expression period exists. Furthermore, Western tissue print analysis showed that Cm-ERS1 protein was present at high levels in the pericarps of both cultivars at the early stages of fruit development, where frequent cell division occurs. These results suggest a role for Cm-ERS1 protein in cell division and expansion.
In Arabidopsis, ERS1 mRNA accumulates predominantly in younger and smaller cells, rather than in older and larger cells (Hua et al., 1998). The mRNA of RP-ERS1, a Rumex ERS1 homologue, is known to accumulate to high levels during the leaf elongation response to flooding (Vriezen et al., 1997) as well as the petiole extension response to low CO2 concentrations (Voesenek et al., 1997). Furthermore, the Arabidopsis ers1-1 mutant strain, which is insensitive to ethylene, sets 50% larger leaves than wild-type strain (Hua et al., 1995). The primary cause of the larger leaves is cell enlargement to twice the normal size, indicating that ERS1 protein is involved in determining cell size. Through the mutant analyses of Arabidopsis and tomato, it is now widely accepted that ethylene receptors act as negative regulators in ethylene signal transduction (Hua and Meyerowitz, 1998; Tieman et al., 2000; Hackett et al., 2000). At the earlier stage of melon fruit development, ethylene production was not observed. Melon fruits harvested at these periods did not have an ability to ripen even if they were incubated in ethylene (H Ezura et al., unpublished data). These results suggest that Cm-ERS1 protein is a negative regulator in melon. The absence of ethylene and the accumulation of Cm-ERS1 protein at the earlier developmental stages will increase receptor signalling and promote cell expansion in melon fruits, as seen for the rosette leaves of Arabidopsis ers1-1 mutant. ERS1 and its homologues may be therefore generally involved in cell division and cell expansion independent of the particular plant species.
Although the roles of ethylene in the regulation of cell expansion in fruit remain unknown, ethylene has been shown to inhibit cell elongation in other systems perhaps by causing reorientation of the cytoskeleton and/or cell wall (Steen and Chadwick, 1981; Roberts et al., 1985; Dolan, 1997). As far as is known, there were few reports on the relationship between ethylene and the expression of HMGR genes although many HMGR genes have been isolated from various plant species. Chye et al. studied the expression of HMGR genes in Hevea brasiliensis and reported that hmg1 was induced by ethylene while hmg3 expression remained constitutive (Chye et al., 1992). Whether ethylene affects the expression of the HMGR gene in melon, and whether/how the signalling from Cm-ERS1 is involved in the regulation must be determined in future studies to clarify the roles of ethylene in the regulation of cell expansion in fruit.
Ethylene biosynthesis is regulated by 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase gene expression (Yang and Hoffman, 1984; Zarembinski and Theologis, 1994; Yamamoto et al., 1995; Lasserre et al., 1996). Once synthesized, however, ethylene gas is removed solely by dissipation (Abeles et al., 1992; Peiser and Yang, 1998). Therefore, a system for regulating the perception of ethylene is also required. It has been shown here that expression of the melon ethylene receptor Cm-ERS1 is temporally and spatially regulated not only at the transcriptional level (Sato-Nara et al., 1999b) but also at the translational level. This differential regulation allows the plant to muster a tissue-specific response to ethylene. On the other hand, it should be noted that functional compensation has been reported between ethylene receptors in tomato (Tieman et al., 2000). Whether all the members of an ethylene receptor family can compensate for each other in melon needs to be clarified. Future studies will also address whether each member has a different affinity for ethylene, and whether each member outputs signals of differing strengths and type.
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Acknowledgments |
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The authors thank Professor H Kamada (University of Tsukuba, Japan) for his critical comments on these experiments. This work was supported by a research fellowship to HT and TK from the Ibaraki prefectural government.
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Notes |
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4 Present address: Department of Applied Biological Sciences, Faculty of Science and Technology, Science University of Tokyo, Yamazaki 2641, Noda, Chiba, 278-8510 Japan.
5 Present address: National Institute for Agro-Environmental Sciences, Kannondai, Tsukuba, Ibaraki, 305-8604 Japan.
6 These two authors equally contributed to this work.
7 To whom correspondence should be addressed. Fax: +81298537263. E-mail: ezura{at}gene.tsukuba.ac.jp
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