Four new members of the ERF (ethylene-response factor) family of plant-speci¢c DNA-binding (GCC box) factors were isolated from tomato fruit (LeERF1^4). Phylogenetic analysis indicated that LeERF2 belongs to a new ERF class, characterized by a conserved N-terminal signature sequence. Expression patterns and cis/trans binding a⁄nities di¡ered between the LeERFs. Combining experimental data and modeled three-dimensional analysis, it was shown that binding a⁄nity of the LeERFs was a¡ected by both the variation of nucleotides surrounding the DNA cis-element sequence and the nature of critical amino acid residues within the ERF domain. ß

Ethylene response factors (ERFs) are plant transcriptional regulators mediating ethylene-dependent gene expression via binding to the GCC motif found in the promoter region of ethylene-regulated genes. We report here on the structural and functional characterization of the tomato Sl-ERF2 gene that belongs to a distinct class of the large ERF gene family. Both spliced and unspliced versions of Sl-ERF2 transcripts were amplified from RNA samples and the search in the public tomato expressed sequence tag (EST) database confirmed the existence of the two transcript species in a number of cDNA libraries. The unspliced transcript contains two open reading frames yielding two hypothetical proteins, a small highly truncated version lacking the APETALA2 domain and a bigger protein lacking the N-terminal MCGGAAI(I)/(L) consensus peptide specific to ERF members from subfamily IV. Nevertheless, functional Sl-ERF2 protein may only derive from spliced transcripts since, depending on the tissue, the level of the spliced transcript is much higher than that of the unspliced transcript. Sl-ERF2 is expressed in all plant tissues tested, though its transcript accumulates preferentially in germinating seeds and ripening fruit. Overexpression of the Sl-ERF2 gene in transgenic tomato lines results in premature seed germination and enhanced hook formation of dark-grown seedlings, which is indicative of increased ethylene sensitivity. The expression of the mannanase2 gene is upregulated in Sl-ERF2-overexpressing seeds, suggesting that Sl-ERF2 stimulates seed germination through the induction of the mannanase2 gene. It is noteworthy that the exaggerated hook phenotype is abolished when ethylene perception is blocked, strongly suggesting that Sl-ERF2 requires other ethylene-dependent components to impact the hook formation process.

SummaryThe term 'RNA silencing' describes a process that results in the specific degradation of an RNA target. In plants, silenced tissues can initiate the spreading of the process into non-silenced regions by a mobile signal that can be transmitted over long distances. In the present work, we made use of a modified grafting approach to elucidate the driving force behind long-distance transport of the silencing signal. We made reciprocal grafts of two GFP-transgenic Nicotiana benthamiana lines, the non-silenced line 16c (sensor) and the silenced line 6.4 (inducer). We show that the direction of systemic spread of silencing from inducer to sensor can be manipulated by altering sink/source relations in the plant. Using radioactive phosphate as a phloem tracer, we demonstrated that plants that transmitted silencing from silenced scion to non-silenced rootstock had developed a persisting phloem flow from scion to rootstock. These data provide experimental proof of what has been hypothesized so far, that the silencing signal travels via phloem from source to sink. We present here evidence that the appearance of systemic silencing is not an accidental stochastic process, but can be predicted on the basis of the direction of phloem flow.