Journal of Experimental Botany, Vol. 54, No. 393, pp. 2775-2776,
December 1, 2003
© 2003 Oxford University Press
Characterization of a novel tomato EIN3-like gene (LeEIL4)
Received 1 May 2003; Accepted 19 September 2003
1 Graduate School of Natural Science and Technology, Okayama University, Tsushima, Okayama 700-8530, Japan
2 Laboratory of Postharvest Agriculture, Faculty of Agriculture, Okayama University, Tsushima, Okayama 700-8530, Japan
* To whom correspondence should be addressed. Fax: +81 86 251 8338. E-mail: ykubo{at}cc.okayama-u.ac.jp
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A novel cDNA clone encoding a putative EIN3-like protein (LeEIL4) was identified from ripening tomato (Lycopersicon esculentum) fruit. The predicted amino acid sequence contained conserved domains of EIN3-like proteins in the N-terminal half. In phylogenetic analysis, LeEIL4 was classified into the cluster consisting of EIN3 and EIN3-like proteins known to be involved in ethylene signal transduction. LeEIL4 transcripts accumulated in all tissues examined, with higher levels in ripening fruit.
Key words: EIL, EIN3, ethylene, signal transduction, tomato.
The plant hormone ethylene is involved in a variety of developmental and physiological processes in plants, including senescence, fruit ripening and abscission (Abeles et al., 1992). In climacteric fruit, ethylene plays a crucial role in the regulation of fruit ripening (Alexander and Grierson, 2002). Furthermore, it has been reported that ethylene activates many ripening-related proteins both at the transcriptional and at the translational levels (Giovannoni, 2001). The unravelling of the molecular basis of the ethylene perception and signal transduction pathway(s) has been enhanced by the use of Arabidopsis mutants altered in the seedling triple response, and several genes encoding ethylene signal components have been identified (Stepanova and Ecker, 2000). EIN3 (ETHYLENE INSENSITIVE 3) has been identified from the the ethylene-insensitive Arabidopsis mutant, ein3 (Chao et al., 1997; Solano et al., 1998). It encodes nuclear protein that acts as a trans-activation factor during a downstream stage of the ethylene signal transduction pathway. Recently, three cDNAs encoding EIN3-like protein were cloned from tomato and designed as LeEIL1, 2 and 3 (Tieman et al., 2001). Each of these genes complemented the ein3 mutation in transgenic Arabidopsis, indicating that they are involved in ethylene signalling. While antisense suppression of a single LeEIL in the tomato plant did not reduce ethylene sensitivity, reduction of all three LeEILs resulted in the loss of ethylene responses, including leaf epinasty, flower abscission, flower senescence, and fruit ripening. Thus, it is suggested that the LeEILs are functionally redundant and that they regulate a wide range of developmental processes. In this note, the isolation and expression of LeEIL4, a novel tomato EIN3 homologue gene, are described.
cDNA synthesized from ripening tomato (Lycopersicon esculentum cv. Momotaro) fruit was used as a template for RT-PCR with degenerated primers, 5'-MRNGGNTTYGTNTAYGGNAT-3' and 5'-YTTYTTNARRTCRTGNTTYTT-3'. These primers were designed from conserved N-terminal amino acid sequences among Arabidopsis EIN3 and Arabidopsis EIN3-like proteins, AtEILs (Chao et al., 1997). Two cDNA fragments corresponding to LeEIL2 and LeEIL3 were cloned. By screening of a cDNA library using these fragments as probes, an additional EIN3 homologue clone LeEIL4 was isolated. Since the screened LeEIL4 clone lacked the 5'-end sequences, full-length cDNA was obtained by RACE-PCR.
LeEIL4 was 2402 bp long and contained a 1815 bp open reading frame encoding predicted 605 amino acids. LeEIL4 shared 79.7%, 73.8%, and 54.2% identity at deduced amino acid levels with LeEIL1, 2 and 3, respectively. The predicted protein contained the conserved acidic domain, a proline-rich domain and five basic domains in the N-terminal half (Fig. 1A). Phylogenetic analysis was performed using the CLUSTAL W (Thompson et al., 1994) and TreeView software (Fig. 1B). Except AtEIL3, all the EILs used for the analysis have been shown to be involved in ethylene signalling (Chao et al., 1997; Kosugi and Ohashi, 2000; Tieman et al., 2001; Alonso et al., 2003). The tree showed that LeEIL4 was grouped into the cluster consisting of EIN3 and functional EILs, but not AtEIL3.
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RNA gel-blot analysis using gene-specific probes of LeEILs showed that LeEIL4 was expressed in all the tomato tissues tested, similar to the other LeEILs (Fig. 2). Interestingly, accumulation of LeEIL4 mRNA increased during fruit ripening. This increase might be involved in the stimulation of ripening processes in response to ethylene, therefore further research evaluating the physiological role of the gene would contribute to an understanding of fruit ripening. Tieman et al. (2001) pointed out that ethylene sensitivity in each tomato tissue may be determined by the total amount of LeEIL mRNAs. It is proposed that LeEIL4 be taken into account, together with other LeEILs, for studying the role of LeEILs in ethylene sensitivity and/or responses.
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This work was supported in part by Grant-in-Aid for Scientific Research (B) (no. 14360023 to AI) from the Japan Society for the Promotion of Science. The sequence of LeEIL4 has been submitted to DDBJ under the accession number AB108840 [GenBank] .
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