Caractérisation moléculaire et physiologique des facteurs de réponse à l’éthylène (ERF) chez la tomate (Solanum lycopersicon)

La phytohormone éthylène, contrôle de nombreux processus physiologique durant le développement des plantes, ainsi que la réponse aux stress biotiques et abiotiques. Les ERF (Facteurs de Réponse à l’Ethylène) sont les derniers facteurs de transcription de la voie de transduction de cette hormone. Ils sont par leur nombre, de bons candidats pour expliquer la diversité de réponse à l’éthylène. Dans cette étude 28 ERF de tomates ont été isolés, caractérisés et renommés. Des études d’interaction ADN/protéine montrent que l’environnement du cis-élément est déterminant pour l’interaction GCC/ERF. Des expériences d’expression transitoire des ERF ont permis de démontrer que leur activité transcriptionnelle est indépendante de leur classe d’appartenance. Leur profil d’expression suggère une spécificité de réponse au cours du développement végétatif ou de la maturation, ainsi qu’un rôle prépondérant dans l’initiation du fruit. L’analyse fonctionnelle est illustrée par deux exemples. D’une part, la surexpression de SlERF2 dans la tomate induit une germination précoce des graines où il a été montré que la MANNANASE2, un marqueur de la germination, est fortement induit dans les graines transgéniques. D’autre part, la surexpression d’ATERF13 fusionné à un domaine répresseur dominant induit une insensibilité partielle à l’éthylène et une hypersensibilité au stress salin. ABSTRACT: The phytohormone ethylene controls many physiological aspects of the plant development and stress response. ERFs (Ethylene Response Factors) are the last transcription factors of the ethylene transduction pathway. By their number, they are good candidates to explain the diversity of ethylene response. In this work 28 tomato ERFs have been isolated, characterized and renamed. DNA/protein interaction studies indicate that flanking regions of the ciselement are decisive for the GCC/ERF binding. Transient expression studies of ERFs demonstrated that the transcriptional activity is independent of the class they belong to. The study of their expression pattern revealed a specific response for some ERFs during the vegetative growth whereas others are preferentially expressed in fruit, from fruit set to ripening. The physiological significance of the ERFs is addressed through two examples. First, over-expression of the SlERF2 gene in the tomato resulted in premature seed germination in which MANNANASE2, a germination marker, is dramatically enhanced in the transgenic seeds. Second, over-expression of AtERF13 fused to a dominant repressor domain induces a partial insensitivity to ethylene and hypersensitivity to salt stress

Regulation of tomato fruit ripening

Fruit ripening is a sophisticatedly orchestrated developmental process, unique to plants, that results in major physiological and metabolic changes, ultimately leading to fruit decay and seed dispersal. Because of their strong impact on fruit nutritional and sensory qualities, the ripeningassociated changes have been a matter of sustained investigation aiming at unravelling the molecular and genetic basis of fruit ripening. Tomato rapidly emerged as the model of choice for fleshy fruit research and a wealth of genetic resources and genomics tools have been developed, providing new entries into the regulatory mechanisms involved in the triggering and coordination of the ripening process. Some of the key components participating in the control of tomato fruit ripening have been uncovered, but our knowledge of the network of signalling pathways engaged in this complex developmental process remains fragmentary. This review highlights the main advances and emphasizes issues still to be addressed using the rapidly developing ‘omics’ approaches

Silencing Sl-EBF1 and Sl-EBF2 expression causes constitutive ethylene response phenotype, accelerated plant senescence, and fruit ripening in tomato

The hormone ethylene regulates a wide range of plant developmental processes and EBF (EIN3-binding F-box) proteins were shown to negatively regulate the ethylene signalling pathway via mediating the degradation of EIN3/EIL proteins. The present study reports on the identification of two tomato F-box genes, Sl-EBF1 and Sl-EBF2 from the EBF subfamily. The two genes display contrasting expression patterns in reproductive and vegetative tissues and in response to ethylene and auxin treatment. Sl-EBF1 and Sl-EBF2 genes are actively regulated at crucial stages in the development of the reproductive organs. Their dynamic expression in flowers during bud-to-anthesis and anthesis-to-post-anthesis transitions, and at the onset of fruit ripening, suggests their role in situations where ethylene is required for stimulating flower opening and triggering fruit ripening. VIGS-mediated silencing of a single tomato EBF gene uncovered a compensation mechanism that tends to maintain a threshold level of Sl-EBF expression via enhancing the expression of the second Sl-EBF gene. In line with this compensation, tomato plants silenced for either of the Sl-EBF genes were indistinguishable from control plants, indicating functional redundancy among Sl-EBF genes. By contrast, co-silencing of both Sl-EBFs resulted in ethylene-associated phenotypes. While reports on EBF genes to date have focused on their role in modulating ethylene responses in Arabidopsis, the present study uncovered their role in regulating crucial stages of flower and fruit development in tomato. The data support the hypothesis that protein degradation via the ubiquitin/26S proteasome pathway is a control point of fruit ripening and open new leads for engineering fruit quality

Ethylene Control of Fruit Ripening: Revisiting the Complex Network of Transcriptional Regulation

The plant hormone ethylene plays a key role in climacteric fruit ripening. Studies on components of ethylene signaling have revealed a linear transduction pathway leading to the activation of ethylene response factors. However, the means by which ethylene selects the ripening-related genes and interacts with other signaling pathways to regulate the ripening process are still to be elucidated. Using tomato (Solanum lycopersicum) as a reference species, the present review aims to revisit the mechanisms by which ethylene regulates fruit ripening by taking advantage of new tools available to perform in silico studies at the genomewide scale, leading to a global view on the expression pattern of ethylene biosynthesis and response genes throughout ripening. Overall, it provides new insights on the transcriptional network by which this hormone coordinates the ripening process and emphasizes the interplay between ethylene and ripening-associated developmental factors and the link between epigenetic regulation and ethylene during fruit ripening

TdERF1, an ethylene response factor associated with dehydration responses in durum wheat (Triticum turgidum L. subsp. durum)

Water deficit and increasing salinization reduce productivity of wheat, the leading crop for human diet. While the complete genome sequence of this crop has not been deciphered, a BAC library screening allowed the isolation of TdERF1, the first ethylene response factor gene from durum wheat. This gene is putatively involved in mediating salt stress tolerance and its characterization provides clues towards understanding the mechanisms underlyning the adaptation/tolerance of durum wheat to suboptimal growth conditions. TdERF1 expression is differentially induced by high salt treatment in two durum wheat varieties, the salt-tolerant Grecale (GR) and the salt-sensitive Om Rabiaa (OR). To further extend these findings, we show here that the expression of this ERF is correlated with physiological parameters, such as the accumulation of osmo-regulators and membrane integrity, that discriminate between the two contrasted wheat genotypes. The data confirm that GR and OR are two contrasted wheat genotypes with regard to salt-stress and show that TdERF1 is also induced by water stress with an expression pattern clearly discriminating between the twogenotypes. These findings suggest that TdERF1 might be involved in responses to salt and water stress providing a potential genetic marker discriminating between tolerant and sensitive wheat varieties

Ethylene Response Factor Sl-ERF.B.3 Is Responsive to Abiotic Stresses and Mediates Salt and Cold Stress Response Regulation in Tomato

Sl-ERF.B.3 (Solanum lycopersicum ethylene response factor B.3) gene encodes for a tomato transcription factor of the ERF (ethylene responsive factor) family. Our results of real-time RT-PCR showed that Sl-ERF.B.3 is an abiotic stress responsive gene, which is induced by cold, heat, and flooding, but downregulated by salinity and drought. To get more insight into the role of Sl-ERF.B.3 in plant response to separate salinity and cold, a comparative study between wild type and two Sl-ERF.B.3 antisense transgenic tomato lines was achieved. Compared with wild type, Sl-ERF.B.3 antisense transgenic plants exhibited a salt stress dependent growth inhibition. This inhibition was significantly enhanced in shoots but reduced in roots, leading to an increased root to shoot ratio. Furthermore, the cold stress essay clearly revealed that introducing antisense Sl-ERF.B.3 in transgenic tomato plants reduces their cell injury and enhances their tolerance against 14 d of cold stress. All these results suggest that Sl-ERF.B.3 gene is involved in plant response to abiotic stresses and may play a role in the layout of stress symptoms under cold stress and in growth regulation under salinity

Sl-ERF2, a Tomato Ethylene Response Factor Involved in Ethylene Response and Seed Germination

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 MCGGAAII/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 darkgrown 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

Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene

Background : The phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Ethylene Response Factor genes (ERF)which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins. Results : A set of 28 cDNA clones encoding ERFs in the tomato (Solanum lycopersicon) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis-acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters. Based on their expression pattern, ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones. Conclusions : The data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner