New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element

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

Climacteric fruit ripening: Ethylene-dependent and independent regulation of ripening pathways in melon fruit

Cantaloupe melons have a typical climacteric behaviour with ethylene playing a major role in the regulation of the ripening process and affecting the ripening rate. Crossing of Cantaloupe Charentais melon with a non-climacteric melon indicated that the climacteric character is genetically dominant and conferred by two duplicated loci only. However, other experiments made by crossing two non-climacteric melons have generated climacteric fruit, indicating that different and complex genetic regulation exists for the climacteric character. Suppression of ethylene production by antisense ACC oxidase RNA in Charentais melon has shown that, while many ripening pathways were regulated by ethylene (synthesis of aroma volatiles, respiratory climacteric and degreening of the rind), some were ethylene-independent (initiation of climacteric, sugar accumulation, loss of acidity and coloration of the pulp). Softening of the flesh comprised both ethylene-dependent and independent components that were correlated with differential regulation of cell wall degrading genes. These results indicate that climacteric (ethylene-dependent) and non-climacteric (ethylene-independent) regulation coexist during climacteric fruit ripening. In addition, ethylenesuppressed melons allowed demonstrating that the various ethylene-dependent events exhibited differential sensitivity to ethylene and that ethylene was promoting sensitivity to chilling injury. Throughout this review, the data generated with melon are compared with those obtained with tomato and other fruit

Two negative cis-regulatory regions involved in fruit-specific promoter activity from watermelon (Citrullus vulgaris S.)

A 1.8 kb 5′-flanking region of the large subunit of ADP-glucose pyrophosphorylase, isolated from watermelon (Citrullus vulgaris S.), has fruit-specific promoter activity in transgenic tomato plants. Two negative regulatory regions, from –986 to –959 and from –472 to –424, were identified in this promoter region by fine deletion analyses. Removal of both regions led to constitutive expression in epidermal cells. Gain-of-function experiments showed that these two regions were sufficient to inhibit RFP (red fluorescent protein) expression in transformed epidermal cells when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter. Gel mobility shift experiments demonstrated the presence of leaf nuclear factors that interact with these two elements. A TCCAAAA motif was identified in these two regions, as well as one in the reverse orientation, which was confirmed to be a novel specific cis-element. A quantitative β-glucuronidase (GUS) activity assay of stable transgenic tomato plants showed that the activities of chimeric promoters harbouring only one of the two cis-elements, or both, were ∼10-fold higher in fruits than in leaves. These data confirm that the TCCAAAA motif functions as a fruit-specific element by inhibiting gene expression in leaves

Ethylene-regulated gene expression in tomato fruit: characterization of novel ethylene-responsive and ripeningrelated genes isolated by differential display.

Differential display was used to isolate early ethyleneregulated genes from late immature green tomato fruit in order to obtain a broader understanding of the molecular basis by which ethylene coordinates the ripening process. Nineteen novel ethylene-responsive (ER) cDNA clones were isolated that fell into three classes: (i) ethylene up-regulated (ii) ethylene downregulated, and (iii) transiently induced. Expression analysis revealed that ethylene-dependent changes in mRNA accumulation occurred rapidly (15 min) for most of the ER clones. The predicted proteins encoded by the ER genes are putatively involved in processes as diverse as primary metabolism, hormone signalling and stress responses. Although a number of the isolated ER clones correspond to genes already documented in other species, their responsiveness to ethylene is described here for the ®rst time. Among the ER clones sharing high homology with regulatory genes, ER43, a putative GTP-binding protein, and ER50, a CTR1-like clone, are potentially involved in signal transduction. ER24 is homologous to the multiprotein bridging factor MBF1 involved in transcriptional activation, and ®nally, two clones are homologous to genes involved in post-transcriptional regulation: ER49, a putative translational elongation factor, and ER68, a mRNA helicase-like gene. Six ER clones correspond to as yet unidenti®ed genes. The expression studies indicated that all the ER genes are ripening-regulated, and, depending on the clone, show changes in transcript accumulation either at the breaker, turning, or red stage. Analysis of transcript accumulation in different organs indicated a strong bias towards expression in the fruit for many of the clones. The potential roles for some of the ER clone