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.

Fine-tuning of ethylene production plays an important role in developmental processes and in plant responses to stress, but very little is known about the regulation of ethylene response factor (ERF) proteins in ethylene biosynthesis genes and ethylene production. Identifying cis-acting elements and transcription factors that play a role in this process, therefore, is important. Previously, a tomato (Solanum lycopersicum [f. sp. Lycopersicon esculentum]) ERF protein, LeERF2, an allele of TERF2, was reported to confer ethylene triple response on plants. This paper reports the transcriptional modulation of LeERF2/TERF2 in ethylene biosynthesis in tomato and tobacco (Nicotiana tabacum). Using overexpressing and antisense LeERF2/TERF2 transgenic tomato, we found that LeERF2/TERF2 is an important regulator in the expression of ethylene biosynthesis genes and the production of ethylene. Expression analysis revealed that LeERF2/TERF2 is ethylene inducible, and ethylene production stimulated by ethylene was suppressed in antisense LeERF2/TERF2 transgenic tomato, indicating LeERF2/TERF2 to be a positive regulator in the feedback loop of ethylene induction. Further research showed that LeERF2/TERF2 conservatively modulates ethylene biosynthesis in tobacco and that such regulation in tobacco is associated with the elongation of the hypocotyl and insensitivity to abscisic acid and glucose during germination and seedling development. The effects on ethylene synthesis were similar to those of another ERF protein, TERF1, because TERF1 and LeERF2/TERF2 have overlapping roles in the transcriptional regulation of ethylene biosynthesis in tobacco. Biochemical analysis showed that LeERF2/TERF2 interacted with GCC box in the promoter of NtACS3 and with dehydration-responsive element in the promoter of LeACO3, resulting in transcriptional activation of the genes for ethylene biosynthesis in tomato and tobacco, which is a novel regulatory function of ERF proteins in plant ethylene biosynthesis.