| The plant hormone ethylene has a profound role in plant growth and development. A number of ethylene signaling elements have been isolated and identified. Although nuclear cascades in ethylene signal transduction pathway from EIN3 to ERF1 to PR proteins has been established in Arabidopsis thaliana, a similar route from EIN3 to EREBPs to fruit ripening and senescence has not been elucidated in Lycopersicon esculentum (tomato). EREBPs (ethylene-responsive element binding proteins) belong to a family of transcription factors, which regulate ethylene responses through binding to the promoter region of ethylene-regulated target genes. Studies on EREBPs in tomato, which is a model plant in fruit and vegetable ripening and senescence researches, will make us better understand the mechanism of ripening-related genes expression regulated by ethylene and further elucidate ethylene transduction pathway.A pair of degenerate primers were designed in the conserved domain (ERF domain) which based on the alignment of EREBPs gene family in tomato and other plant species. A 114 bp fragment was amplified by RT-PCR (Reverse Transcription Polymerase Chain Reaction), which was used as a probe for screening tomato fruit (pink stage) cDNA library. Two positive clones containing entire coding region were isolated, which were identified as new members of EREBPs family by BLAST Server, and named as LeERF1 and LeERF2. The LeERF1 cDNA is 1041 bp and the predicted translation product is 204 amino acids. The LeERF2 cDNA is 2898 bp and the predicted translation product is 210 amino acids. LeERFl was homologous with EREBP4 and DDTFR10A, with 33% similarity at the amino acid level. LeERF2 was homologous with EREBP3 and pti5, with 46% similarity at the amino acid level. The cDNA sequences of LeERFl and LeERF2 were deposited in GenBank (GenBank Accession Number AY077626, AY275554 respectively).Northern blot hybridization showed that LeERFl, LeERF2 and pti4 (a member of EREBPs in tomato) expressed differentially in different parts of wild tomato fruit during ripening, which was related with fruit ripening. In both Nr fruit and antisense ACS transgenic tomato fruit, expression levels of LeERFl, LeERF2 and pti4 were generally lower than those in wild fruit but still related with fruit ripening.The expression levels of of LeERF1, LeERF2 increased in the wild mature green tomato fruits after 0.5 h of treatment with ethylene (100 ul/L), and pti4 mRNA abundance increased after 12 h of exposure to ethylene (100 ul/L). After 10 h of treatment of breaker tomato fruits with 1-MCP (1 ul/L), a potent inhibitor of ethylene action, decreased the mRNA abundance of LeERF1, LeERF2 and pti4 . These results indicated that gene expression of LeERF1, LeERF2 and pti4 were positively regulated by ethylene.Treatment of mature green fruits with 0.1 mM ABA enhanced the ethylene production and gene expressions of LeERFl, LeERF2. Treatment of mature green fruits with 1 mM SA had slight inhibitionon ethylene production. SA treatment inhibited the expression of LeERF1 and had no effect on the expression of LeERF2. However, treatments with ABA and SA had no effects on the expression ofpti4. These results indicated that accumulation ofpti4 mRNA was not induced by ABA and SA.The expression levels of LeERF1, LeERF2 andpti4 in seedlings gradually increased after treatment with 0.1 mM ABA. The mRNAs of LeERFl, LeERF2 and pti4 accumulated in response to 1 mM SA within 30 min in seedlings. These results indicated that there existed tissue specificity with regard to accumulation of LeERFl, LeERF2 and pti4 mRNA induced by ABA and SA.The mRNA abundance of LeERF1, LeERF2 and pti4 decreased gradually in the mature green tomato fruits as the treatment concentration of CaCl2 increased, which indicated that Ca2+ involved in the ethylene signal transduction.Sense plant expression vectors of pGA643-LeERF1 and pGA643-LeERF2 and antisense plant expression vectors of pBI121-LeERFl and pBI121-LeERF2 were constructed and transformed into Agrobacterium tumefaciens strain EH A10...
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