Dr Alain Pech

Prof Jean-Paul Latché

Roustan

Corinne, Christian, Simo, Pierre Isabelle Benoit

Les derniers acteurs de la voie de signalisation à l'éthylène sont des facteurs de transcription appelés ERF (Ethylene Response Factors). La connaissance de leur rôle spécifique dans la régulation des processus développementaux dépendant de l'éthylène reste limitée. Les travaux présentés dans la thèse concernent la caractérisation fonctionnelle du gène Sl-ERF.B3, un membre de cette grande famille de régulateurs transcriptonnels dans la tomate (Solanum lycopersicum). Utilisant une stratégie répresseur dominant ; il est montré en particulier que ce gène intervient dans la mise en place de la réponse à l'éthylène et dans le contrôle de la maturation du fruit. L'expression d'une construction ERF.B3-SRDX, une version chimérique de Sl-ERF.B3 fusionné à un domaine répresseur de type EAR, entraine des phénotypes pléotropiques aussi bien dans la signalisation de l'éthylène que dans le développement des parties végétatives et des organes reproducteurs. Ainsi, une altération de la triple réponse à l'éthylène est constatée chez les lignées transgéniques et au stade adulte, les plantes présentent des phénotypes d'épinastie des feuilles, de sénescence prématurée des fleurs et d'abscission accélérée des fruits. L'ensemble de ces observations est corrélée avec une modification de l'expression de gènes impliqués dans la biosynthèse et la réponse à l'éthylène. Ces données suggèrent que ERF.B3 intervient dans un mécanisme de rétro-control de la réponse à l'éthylène en agissant à la fois sur les gènes de biosynthèse et de signalisation de l'hormone. Au niveau du fruit, la sur-expression d'ERF.B3-SRDX entraine une modification du processus de maturation avec un retard notable de l'avènement de l'acquisition de la compétence à murir. Cependant, une fois la maturation initiée, elle s'accompagne d'une forte production d'éthylène et d'une accélération du ramollissement du fruit. A l'inverse, l'accumulation de pigment est inhibée par altération de la voie de biosynthèse des caroténoïdes. Ces données phénotypiques sont corrélées avec le niveau d'expression des gènes clés impliqués dans ces processus. Les résultats indiquent que dans les lignées transgéniques, il y a découplage de certaines caractéristiques de la maturation du fruit et permettent de mettre en lumière le rôle d'ERF.B3 dans la régulation des processus de développement dépendant de l'éthylène chez la tomate.

Main components of the thesis

The plant hormone ethylene is involved in many developmental processes and plays a critical role in a wide range of physiological responses, including seed germination, cell elongation, flowering, fruit ripening, organ senescence, abscission, root nodulation, programmed cell death, and response to abiotic stresses and pathogen attacks. After its biosynthesis, ethylene is perceived by a receptor which generates a signal leading to the activation of a transduction machinery that triggers specific biological responses. To improve our understanding of how ethylene is able to mediate plant growth and fruit The body of this thesis consists of four main chapters. The first chapter is a bibliographic review on ethylene. In this chapter, up-to-date knowledge regarding ethylene biosynthesis, perception and signal transduction is presented. The main roles of ethylene in regulating different plant growth and development processes and the interaction of this hormone with other phytohormones were described. The advantage of using tomato as plant model in my study dealing with ethylene-mediated fruit development processes is emphasized.

Chapter II focuses on addressing the physiological significance of Sl-ERF.B3 and its potential role in mediating ethylene responses. This chapter is presented on the form of an article which has been recently accepted for publication in The Plant Journal. Using a dominant repressor strategy, it is demonstrated in this work that Sl-ERF.B3 gene controls ethylene sensitivity via feedback regulation of ethylene signaling and response components. It is shown that the expression of a dominant repressor version of Sl-ERF.B3 (ERF.B3-SRDX) in the tomato results in pleiotropic ethylene responses and vegetative and reproductive growth phenotypes. The multiple symptoms related to enhanced ethylene sensitivity correlate with the altered expression of ethylene biosynthesis and signaling genes, suggesting the involvement of Sl-ERF.B3 in a feedback mechanism regulating components of ethylene production and response. Moreover, Sl-ERF.B3 is shown to modulate the transcription of a set of ERFs revealing the existence of a complex network interconnecting different ERF genes.

Chapter III mainly describes the putative role of ERF family genes in controlling fruit maturation and ripening in tomato and more particularly the role of Sl-ERF.B3 in fruit development and ripening. In this chapter, it is shown that most of ERF family genes display a ripening-associated expression pattern suggesting their involvement in the ripening process. Specific roles for ERF gene family members in fruit development and ripening is further revealed by the functional characterization of Sl-ERF.B3, a member tomato ERF genes. Sl-ERF.B3 displays a pattern of expression that is quite distinctive from other ERF genes, its transcript accumulation being induced at the breaker stage and maintained at a high level in all stages of fruit ripening, suggesting that its expression is continuously required for the modulation of the ripening-regulated genes all along the ripening process. The study indicate that over-expression of a chimeric repressor construct of Sl-ERF.B3 (ERF.B3-SRDX) in tomato results in alterations in fruit shape and size, abnormal seed morphology, orange ripe fruits, and accelerated fruit senescence.

Moreover, genes involved in different metabolic pathways, such as carotenoid biosynthesis, ethylene synthesis, and cell wall metabolism exhibit altered mRNA accumulation patterns in transgenic lines during fruit ripening process. Further characterization at the molecular level of the dominant repressor lines, indicated that Sl-ERF.B3 impacts the ripening process through mediating ripening-associated genes.

The last chapter presents general conclusions and perspectives of the work performed in this thesis.

Objectives of the study

Ethylene mediates diverse developmental and physiological processes throughout the entire life cycle of plants. After its synthesis, ethylene is perceived and its signal transduced through a transduction machinery that triggers specific biological responses.

Significant progress has been made in our understanding of how plants perceive and transduce the ethylene signal [START_REF] Benavente | Molecular mechanisms of ethylene signaling in Arabidopsis[END_REF]Ju et al., 2012). Studies on components of ethylene signaling have revealed a linear transduction pathway that leads to the activation of transcriptional regulators belonging to the Ethylene Response Factor (ERF) type. ERF proteins which are responsible for regulating the transcription of primary ethylene-responsive genes are the last known downstream components of ethylene transduction pathway. Since the upstream components of the ethylene transduction pathway are common to all ethylene responses, the apparent simplicity of the ethylene signaling pathway cannot account for the wide diversity of ethylene responses. A tempting hypothesis is that differential responses to ethylene are directed at the transcriptional levels. Being encoded by one of the largest family of plant transcription factors, ERF proteins are the most suited step of the ethylene signaling pathway where the diversity and specificity of ethylene responses may originate.

In an attempt to understand the molecular basis of ethylene-regulated plant growth and fruit development, 28 tomato ERF genes have been isolated in the laboratory of Genomics and Biotechnology of the Fruit (GBF) and these genes have been shown to fall into 9 subclasses defined by distinct structural features. Previous studies on these genes have provided some molecular clues on how ERFs can contribute to the specificity and selectivity of ethylene responses through (i) the differential expression of gene family members, (ii) the ability to negatively or positively impact transcriptional activity and, (iii) the capacity to select with specificity target genes based on the nucleotide environment of the GCC-box [START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF]. The diversity of their transcriptional activity and expression patterns suggest that ERFs possess the necessary features for channeling ethylene signaling to a selected set of genes required for the appropriate developmental responses or the desired responses to environmental cues. To date, however, most of the members of the ERF family have yet to be studied, the specific role of individual ERF in controlling ethylene responses and plant developmental processes remains poorly understood.

The aim of this study was to unravel the molecular mechanism underlying the specificity of ethylene responses during plant development and fruit ripening. The function significance of ERF genes is addressed in the tomato using advanced reverse genetics and genomics methodologies, with a special focus given to the role of ERFs in fruit development and ripening. My own project was dedicated to the functional characterization of Sl-ERF.B3, a tomato ethylene-inducible ERF gene previously shown to display a strong binding affinity to GCC-box-containing promoters. Specifically, the study addresses two main questions: (i) Is Sl-ERF.B3 involved in mediating ethylene responses and if so by which molecular mechanism it performs this function? (ii) Does Sl-ERF.B3 impact ethylene-dependent developmental processes such as fruit ripening?

Chapter I Bibliographic review 6 Ethylene, the simplest olefin, has been recognized as a plant hormone for over a century.

The discovery of the biological activity of ethylene came about in the 19th century as leaks in illuminating gas caused premature senescence and defoliation of plants in the greenhouse and of trees near gas lines [START_REF] Abeles | Ethylene in plant biology 2nd edn[END_REF]. In 1901, the Russian physiologist Dimitri Neljubow demonstrated that ethylene causes the "triple response" in dark-grown pea seedlings with inhibited epicotyl elongation, radial swelling, and a horizontal growth habit. The first evidence that ethylene was produced by plants was the observation that the ripening of bananas was promoted by gases produced from oranges.

Definitive chemical proof that ethylene is produced by plants was reported by the English scientist Gane in 1934. It is now known that ethylene is produced by all cells during plant development with the highest rates being associated with meristematic, stressed, or ripening tissues [START_REF] Abeles | Ethylene in plant biology 2nd edn[END_REF].

In the plant life cycle, ethylene is involved in a wide range of plant growth and developmental processes, including seed germination, cell elongation, flowering, fruit ripening, organ senescence, abscission, root nodulation, programmed cell death, and response to abiotic stresses and pathogen attacks [START_REF] Johnson | THE ETHYLENE GAS SIGNAL TRANSDUCTION PATHWAY: A Molecular Perspective[END_REF][START_REF] Bleecker | Ethylene: a gaseous signal molecule in plants[END_REF][START_REF] Lin | Recent advances in ethylene research[END_REF]. To better understand the roles of ethylene in plant functions, it is important to know how this gaseous hormone is synthesized, transduced and able to regulate so many plant development processes.

Ethylene biosynthesis

Ethylene biosynthesis has been intensively studied in plants and the establishment of Sadenosyl-L-methionine (S-Ado Met) and ACC as the biological precursors of ethylene is thought to be the main breakthroughs in the ethylene biosynthesis pathway. As it is shown in Figure 1, ethylene is synthesized from the amino acid methionine. In the ethylene biosynthesis pathway, methionine is converted to S-AdoMet by S-AdoMet synthetase and S-AdoMet is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) and 5'-deoxy-5 methylthioadenosine (MTA) by the enzyme 1aminocyclopropane-1-carboxylase synthase (ACS) [START_REF] Adams | Ethylene biosynthesis: Identification of 1aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene[END_REF]. Through the Yang cycle, MTA can be recycled to methionine, which allows high rates of ethylene production without depletion of the endogenous methionine pool [START_REF] Miyazaki | Metabolism of 5-methylthioribose to methionine[END_REF]. At the last step, ACC is further converted to ethylene by ACC oxidase (ACO), with CO2 and cyanide as by-products. In ethylene biosynthesis pathway, the rate-limiting step of ethylene synthesis is the conversion of S-AdoMet to ACC by ACC synthase, but there are situations where ACO is absent and ACS and ACO are induced, for example by wounding and the ripening stimulus (Alexander and Grierson, 2002a;[START_REF] Lin | Recent advances in ethylene research[END_REF]. In most of the species investigated, including Arabidopsis, ACS and ACO are members of large and small multigene families, respectively (De Paepe and Van der Straeten, 2005).

Both positive and negative feedback regulation of ethylene biosynthesis have been reported in different plant species [START_REF] Wang | Ethylene biosynthesis and signaling networks[END_REF].

Ethylene signaling pathway

After its synthesis, ethylene is perceived and its signal transduced through transduction machinery to trigger specific biological responses. Insight into the ethylene signaling pathway has been mainly provided by molecular genetic studies in Arabidopsis thaliana.

Dissection of the ethylene signaling pathway began with the isolation of ethylene- S-adenosylmethionine (S-AdoMet) is synthesized from the methionine by the S-adenosylmethionine synthetase (SAM synthetase) with one ATP molecule expensed per S-AdoMet synthesized. S-AdoMet is then converted to 1-aminocyclopropane-1carboxylicacid (ACC) by ACC synthase (ACS), 5'-methylthioadenosine (MTA) being a by-product. MTA is recycled to methionine by successive enzymatic reactions involving various intermediates (MTR, 5-methylthioribose; KMB, 2-keto-4-methylthiobutyrate), which constitute the methionine (Yang) cycle. Ethylene production is catalyzed by the ACC oxidase using ACC as substrate, and generates carbon dioxide and hydrogen cyanide [START_REF] Arc | ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination[END_REF]. response mutants, using genetic screens that are based on the "triple response" of ethylene-treated seedlings [START_REF] Bleecker | Insensitivity to Ethylene Conferred by a Dominant Mutation in Arabidopsis thaliana[END_REF][START_REF] Guzmán | Exploiting the triple response of Arabidopsis to identify ethylene-related mutants[END_REF]. The triple response is a striking morphology adopted by germinating seedlings exposed to ethylene in the dark. In Arabidopsis, the triple response consists of shortening and thickening of the hypocotyl and root, exaggeration of the apical hook curvature and proliferation of root hairs (Figure 2). Decades of scientific research devoted to deciphering how plants are able to sense and respond to ethylene have culminated in the establishment of one of the best characterized signal transduction pathways in Arabidopsis [START_REF] Bleecker | Insensitivity to Ethylene Conferred by a Dominant Mutation in Arabidopsis thaliana[END_REF][START_REF] Guzmán | Exploiting the triple response of Arabidopsis to identify ethylene-related mutants[END_REF][START_REF] Chang | Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators[END_REF][START_REF] Roman | Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway[END_REF][START_REF] Chao | Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins[END_REF][START_REF] Sakai | ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis[END_REF][START_REF] Alonso | EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis[END_REF]. In the currently accepted model, ethylene signaling pathway starts with the ethylene perception by their specific receptors, which have been shown to activate the hormone transduction pathway through releasing the block exerted by CTR1 on EIN2 (Solano and Ecker, 1998;Ju et al., 2012).

The release of EIN2 then activates EIN3/EIL1 primary transcription factors, resulting in the expression of secondary transcription factors, namely ERFs, which regulate the expression of downstream ethylene-responsive genes (Solano et al., 1998;[START_REF] Alonso | Genome-wide insertional mutagenesis of Arabidopsis thaliana[END_REF]. Through using a combination of genetic, biochemical and molecular approaches, a [START_REF] Benavente | Molecular mechanisms of ethylene signaling in Arabidopsis[END_REF].

pathway that transduces the ethylene signal from the endoplasmic reticulum membrane to the nucleus has been uncovered (Figure 3). C terminus being cleaved and localizing to the nucleus where it can activate the downstream transcriptional cascade (Ju et al., 2012).

Ethylene perception is mediated by a small family of receptors

Perception of ethylene in plants is achieved by several related membrane-bound histidine kinases. In Arabidopsis, ethylene is perceived by a family of five receptors (ETR1, ETR2, ERS1, ERS2 and EIN4) that share similarity with bacterial twocomponent regulators [START_REF] Chang | Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators[END_REF][START_REF] Hua | Ethylene insensitivity conferred by Arabidopsis ERS gene[END_REF][START_REF] Sakai | ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis[END_REF]Hua and Meyerowitz, 1998;[START_REF] Chang | Ethylene hormone receptor action in Arabidopsis[END_REF]. All of the initial receptor mutants identified where gain-of-function mutants exhibiting dominant ethylene insensitivity.

Single loss-of-function receptor mutants in Arabidopsis show no visible phenotypes likely due to functional redundancy. The presence of triple, quadruple or etr1/ers1 double mutants results in a constitutive ethylene response in the absence of increased ethylene production [START_REF] Wang | Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission[END_REF]. This evidence is consistent with a model where the receptors are negative regulators of ethylene response and that ethylene receptors actively suppress the response in the absence of ethylene (Hua and Meyerowitz, 1998). Ethylene was found to bind receptor through a transition metal copper co-factor. Ethylene binding results to a modification of the coordination chemistry of the copper in the N-terminal region. This modification is transmitted to the C-terminal region (Rodriguez et al., 1999) and initiates the ethylene response. On the basis of structural similarities, the receptor family can be divided into two subfamilies (Figure 4). Subfamily 1, consisting of ETR1 and ERS1, features three hydrophobic transmembrane domains in the N-terminal region, where ethylene binding occurs [START_REF] Schaller | Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene[END_REF][START_REF] Hall | Ethylene perception by the ERS1 protein in Arabidopsis[END_REF], and a well conserved histidine kinase domain in C-terminal region. Subfamily 2, which includes ETR2, ERS2, and EIN4, has four hydrophobic domains in the N-terminal region and a non-conserved His-kinase domain, in which some consensus amino acid residues essential for His-kinase activity are lacking [START_REF] Moussatche | Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family[END_REF][START_REF] Xie | Receptor signal output mediated by the ETR1 N terminus is primarily subfamily I receptor dependent[END_REF]. The fact that the subfamily 2 receptors ETR2, ERS2 and EIN4 have Ser/Thr kinase activity in vitro [START_REF] Gamble | Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis[END_REF][START_REF] Moussatche | Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family[END_REF] supports the notion that the subfamily 2 of receptors may function not as histidine kinases but possibly as serine/threonine kinases. In addition, ETR1, ETR2 and EIN4 possess a C-terminal receiver (Figure 4). [START_REF] Shakeel | Mechanisms of signal transduction by ethylene: overlapping and non-overlapping signalling roles in a receptor family[END_REF].

The C-terminal domains of the ethylene receptors show sequence homology to bacterial two-component system histidine kinases. These systems are generally constituted of a sensor molecule containing an histidine kinase domain which autophosphorylates itself in reaction to a stimuli, and a response regulator containing a receiver domain which accept, the residue phosphate from the histidine sensor [START_REF] Pirrung | Histidine kinases and two-component signal transduction systems[END_REF]. The histidine kinase domain of the ethylene receptors has been shown to be important for the association of the receptors with CTR1 [START_REF] Clark | Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors[END_REF][START_REF] Gao | Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes[END_REF][START_REF] Zhong | Tomato ethylene receptor-CTR interactions: visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum[END_REF]. It was demonstrated that a truncated ETR1 lacking the histidine kinase and receiver domain failed to rescue a etr1-6;etr2-3;ein4-4 triple loss-of-function mutant, while the truncated ETR1 lacking only the receiver domain was able to restore normal growth of the triple mutant in air, and the transgenic plants show ethylene hypersensitivity [START_REF] Qu | Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1[END_REF]. These results demonstrate that the kinase domain is necessary for signal transmission by the receptor and that the receiver domain was not essential for restoring ethylene responsiveness. However, while ethylene may inhibit His kinase activity in ETR1 [START_REF] Voet-Van-Vormizeele | Ethylene controls autophosphorylation of the histidine kinase domain in ethylene receptor ETR1[END_REF], it seems that His kinase activity is not needed for signaling [START_REF] Wang | Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission[END_REF][START_REF] Qu | Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1[END_REF] but does have a role in growth recovery after ethylene removal [START_REF] Binder | Arabidopsis seedling growth response and recovery to ethylene. A kinetic analysis[END_REF] and in regulation of growth [START_REF] Qu | Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1[END_REF][START_REF] Cho | ETHYLENE RESPONSE 1 histidine kinase activity of Arabidopsis promotes plant growth[END_REF]. Kim et al., (2011) also showed that ETR1 histidine kinase activity and phosphotransfer through the receiver domain are not required to rescue ethylene-mediated nutations. [START_REF] Hall | Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis[END_REF] demonstrated that the histidine kinase activity of ETR1 is not required for but plays a modulating role in the regulation of ethylene responses. By comparing the dominant-negative effect of ETR1-1 in a two receptor double LOF mutant background: etr1-7;ers1-2 and etr1-7;ers1-3, [START_REF] Xie | Receptor signal output mediated by the ETR1 N terminus is primarily subfamily I receptor dependent[END_REF] showed that the truncated ETR1 without the histidine kinase domain must rely on the remaining ERS1, which has the histidine kinase activity, to repress ethylene signaling. Indeed, up till now, the exact function of the receptor histidine kinase domains and the role of the receptor heterodimer interaction in ethylene signaling are still open questions. In tomato, there are six ethylene receptors (LeETR1-2, NR, and LeETR4-6) which can be broken down into two subfamilies. The predicted structures of these tomato receptors are very similar to those in Arabidopsis (Klee and Tieman, 2002).

LeETR1, 2 and NR are members of Subfamily I which contain three transmembrane domains and all of the conserved residues of known histidine kinases. Subfamily II, consisting of LeETR4-6, contains four transmembrane domains and degenerate histidine kinase domains with LeETR5 containing none of the conserved residues. Moreover, NR is the only member of the family that does not contain the carboxy-terminal receiver domain whose function in ethylene signaling is still unknown. In contrast to the Arabidopsis ethylene receptors that have been considered to be functionally redundant, the tomato ethylene receptors NR, LeETR4, and LeETR6 are preferentially expressed in fruit and have been suggested to have unique roles during ripening (Tieman et al., 2000;Kevany et al., 2007).

CTR1 acts as a negative regulator of ethylene signaling pathway

In the ethylene linear signaling pathway, acting directly downstream of the ethylene receptors is the mitogen-activated protein kinase kinase kinase (MAPKKK) constitutive triple-response1 (CTR1) protein. CTR1 was identified by mutants that displayed the triple response morphology in the absence of exogenous ethylene. Loss-offunction ctr1 mutations result in the constitutive activation of the ethylene response pathway in seedlings and adult plants, which indicates that the encoded protein acts as a negative regulator of ethylene signaling (Kieber et al., 1993). The binding of ethylene to the receptors results in the inactivation of CTR1 and in turn activation of the downstream components of the pathway, thereby leading to ethylene responses (Kieber et al., 1993;[START_REF] Huang | Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis[END_REF]; Figure 5). CTR1 consists of a unique N-terminal regulatory domain and a C-terminal serine/threonine kinase domain. Although CTR1 contains no predicted transmembrane domains (Kieber et al., 1993;[START_REF] Huang | Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis[END_REF], CTR1 is found at the ER membrane due to its association with the ethylene receptors [START_REF] Clark | Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors[END_REF][START_REF] Huang | Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis[END_REF][START_REF] Gao | Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes[END_REF][START_REF] Zhong | Tomato ethylene receptor-CTR interactions: visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum[END_REF]. It has been demonstrated that the N-terminal regulatory domain of CTR1 can interact directly with the subfamily I ethylene receptors (ETR1 and ERS1) in the yeast two-hybrid assay [START_REF] Clark | Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors[END_REF]. [START_REF] Zhong | Tomato ethylene receptor-CTR interactions: visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum[END_REF] also showed that ethylene receptors recruit tomato LeCTR proteins to the ER membrane through direct protein-protein interaction. Moreover, [START_REF] Mayerhofer | Protein kinase domain of CTR1 from Arabidopsis thaliana promotes ethylene receptor cross talk[END_REF] proposed that the interaction of CTR1 dimers with the ethylene receptor dimers reinforces the receptor complex by promoting associations between neighboring ethylene receptors (Figure 6). [START_REF] Mayerhofer | Protein kinase domain of CTR1 from Arabidopsis thaliana promotes ethylene receptor cross talk[END_REF].

Although the serine/threonine kinase activity of CTR1 was demonstrated in vitro and shown to be essential for proper functioning of the receptors/CTR1 signaling complex, as kinase-inactive alleles of CTR1 also resulted in a constitutive response phenotype [START_REF] Huang | Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis[END_REF], the molecular mechanism by which the receptors control CTR1 kinase activity remains unclear. The ethylene receptor-CTR1 association represents a novel combination of proteins that do not fit the existing paradigms for either the Raf-like CTR1 or the two-component receptors [START_REF] Wang | Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission[END_REF][START_REF] Wellbrock | The RAF proteins take centre stage[END_REF][START_REF] Schaller | Two-component systems and their co-option for eukaryotic signal transduction[END_REF]. A likely mechanism for CTR1 activation could be that the receptors interact with CTR1 in an active conformation in the absence of ethylene. When the receptors bind ethylene and presumably undergo a conformational change, there could be a concomitant alteration in the conformation of CTR1 that turns off the CTR1 kinase activity [START_REF] Gao | Localization of the Raf-like kinase CTR1 to the endoplasmic reticulum of Arabidopsis through participation in ethylene receptor signaling complexes[END_REF][START_REF] Ju | Advances in ethylene signalling: protein complexes at the endoplasmic reticulum membrane[END_REF]. It is conceivable that the histidine autophosphorylation induced by ethylene binding, as suggested by [START_REF] Hall | Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis[END_REF], plays a role in the conformational change that terminates CTR1 activation. Because structural studies have shown that the CTR1 kinase domain is a dimer when active [START_REF] Mayerhofer | Protein kinase domain of CTR1 from Arabidopsis thaliana promotes ethylene receptor cross talk[END_REF], a conformational change causing monomerization of CTR1 could be a possible mechanism for inactivation of CTR1.

While the main ethylene signaling pathway involves CTR1, it is worth pointing out that subtle effects of ethylene receptor signaling might occur via the two-component system's phosphotransfer proteins and response regulators in Arabidopsis (known as AHPs and ARRs, respectively). This is based on evidence that the ethylene receptors can interact with AHP proteins [START_REF] Urao | Possible His to Asp phosphorelay signaling in an Arabidopsis two-component system[END_REF][START_REF] Scharein | Ethylene signaling: identification of a putative ETR1-AHP1 phosphorelay complex by fluorescence spectroscopy[END_REF] and that a response regulator, ARR2, might have a role as a positive regulator in modulating ethylene responses downstream of ETR1 [START_REF] Hass | The response regulator 2 mediates ethylene signalling and hormone signal integration in Arabidopsis[END_REF][START_REF] Mason | Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis[END_REF]. Thus, ethylene receptor signaling through AHPs and ARRs might represent an ethylene response pathway that bypasses CTR1 [START_REF] Ju | Advances in ethylene signalling: protein complexes at the endoplasmic reticulum membrane[END_REF].

EIN2 is a central component of the signaling pathway and positively regulates ethylene responses

Based on genetic analyses, downstream of the receptors/CTR1 complexes there acts a positive regulator of ethylene responses, ETHYLENE INSENSITIVE2 (EIN2).

EIN2 is required for all ethylene responses studied and constitutes a critical step in the signal transduction [START_REF] Alonso | EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis[END_REF]. EIN2 consists of an N-terminal integral membrane domain of 12 predicted transmembrane helices (residues 1-461) with sequence similarity to Nramp metal ion transporters, followed by a hydrophilic Cterminal domain (residues 462-1294) believed to be cytosolic [START_REF] Alonso | EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis[END_REF]Ju et al., 2012; Figure 7). Qiao et al., (2009) reported that EIN2 can be stabilized by ethylene at the protein level, which protects it from proteasomal degradation mediated by two Fbox proteins ETP1/2. EIN2 was also shown to reside at the ER membrane in tobacco leaves and is capable of interacting with the kinase domain of ethylene receptors [START_REF] Bisson | EIN2, the central regulator of ethylene signalling, is localized at the ER membrane where it interacts with the ethylene receptor ETR1[END_REF][START_REF] Bisson | New insight in ethylene signaling: autokinase activity of ETR1 modulates the interaction of receptors and EIN2[END_REF]. Moreover, EIN2 was found to be required for the ethylene-induced EIN3/EIL1 protein stabilization [START_REF] Guo | Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor[END_REF], by promoting the proteasomal degradation of EBF1/EBF2 in the nucleus [START_REF] An | Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis[END_REF]. For more than a decade, although it has been well deciphered by genetic and doublemutant analyses that EIN2 is a central and most critical element of the ethylene signaling pathway and acts between the soluble serine/threonine kinase CTR1 and the EIN3/EILs transcription factors, two key mysteries (i) how is the ethylene signal from CTR1 transmitted to EIN2, (ii) how is the signal transmitted from the ER-localized EIN2 to the nuclear-localized transcription factors remain unknown. Recent research progress by three groups (Ju et al., 2012;[START_REF] Qiao | Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas[END_REF][START_REF] Wen | Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus[END_REF] has shown that CTR1, instead of operating through an intermediary MAPK cascade, directly phosphorylates EIN2 to inhibit its activity. The most significant sites of EIN2 phosphorylation are on Ser645 and Ser924 (Chen et al., 2011;[START_REF] Qiao | Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas[END_REF]Ju et al., 2012), with Ser924 playing a predominant role in EIN2 regulation (Ju et al., 2012). Following ethylene binding to the receptors, CTR1 becomes inactivated, resulting in the dephosphorylation and proteolytic cleavage of EIN2, the cleaved C-terminal portion (CEND) of EIN2 then translocating to the nucleus to regulate transcriptional events (Ju et al., 2012;[START_REF] Qiao | Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas[END_REF]. These findings uncover a mechanism of subcellular communication whereby ethylene stimulates phosphorylation-dependent cleavage and nuclear movement of the EIN2-C' peptide, linking hormone perception and signaling components in the ER with nuclear-localized transcriptional regulators (Figure 8). proteasomal degradation, and consequently leading to the activation of downstream ethylene response [START_REF] Wen | Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus[END_REF].

A transcriptional cascade modulates the expression of ethyleneresponsive genes

Genetically acting downstream of EIN2 are several nuclear-localized transcription factors (EIN3/EILs and ERFs) that mediate ethylene response at transcriptional levels [START_REF] Chao | Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins[END_REF]Solano et al., 1998;[START_REF] An | Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis[END_REF]. After translocating to the nucleus, EIN2 either directly or indirectly activates the EIN3 family of transcription factors to initiate the transcriptional response to ethylene (Solano et al., 1998;[START_REF] Alonso | Genome-wide insertional mutagenesis of Arabidopsis thaliana[END_REF][START_REF] An | Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis[END_REF]. EIN3 belongs to a small gene family that in Arabidopsis also includes five EIN3-LIKE (EIL) proteins [START_REF] Alonso | Genome-wide insertional mutagenesis of Arabidopsis thaliana[END_REF]. EIN3/EILs type of transcription factors are positive regulators of the ethylene signaling that function as trans-activating factors to trigger ethylene responses [START_REF] Chao | Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins[END_REF]Solano et al., 1998).

Overexpression of EIN3 or EIL1 results in a constitutive ethylene phenotype in Arabidopsis, while ein3 eil1 double LOF mutants show complete ethylene insensitivity in all known ethylene responses [START_REF] Chao | Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins[END_REF][START_REF] Alonso | Genome-wide insertional mutagenesis of Arabidopsis thaliana[END_REF]. None of EIN3/EIL genes identified to date is transcriptionally regulated in response to ethylene [START_REF] Chao | Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins[END_REF]Tieman et al., 2001;[START_REF] Rieu | Expression analysis of five tobacco EIN3 family members in relation to tissue-specific ethylene responses[END_REF], suggesting that the activities of these genes are regulated by ethylene through a posttranscriptional mechanism. Studies have demonstrated the involvement of the SCF/26S proteasome to regulate the level of EIN3/EILs [START_REF] Guo | Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor[END_REF][START_REF] Potuschak | EIN3-dependent regulation of plant ethylene hormone signaling by two arabidopsis F box proteins: EBF1 and EBF2[END_REF][START_REF] Gagne | Arabidopsis EIN3-binding F-box 1 and 2 form ubiquitin-protein ligases that repress ethylene action and promote growth by directing EIN3 degradation[END_REF][START_REF] An | Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis[END_REF]Figure 9). In the absence of ethylene, EIN3/EILs are targeted for ubiquitination by the SCF complex containing one of the two F-box proteins, EBF1 and EBF2. The ubiquitinated form of EIN3/EIL proteins is thus recruited by the 26S proteasome for degradation. whereas, in the presence of ethylene, EIN3/EIL proteins accumulate in the nucleus and bind to EIN3 binding site (EBS) located in target gene promoters leading to the activation of the expression of downstream genes [START_REF] Guo | Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor[END_REF][START_REF] Potuschak | EIN3-dependent regulation of plant ethylene hormone signaling by two arabidopsis F box proteins: EBF1 and EBF2[END_REF][START_REF] Binder | The Arabidopsis EIN3 binding F-Box proteins EBF1 and EBF2 have distinct but overlapping roles in ethylene signaling[END_REF]. Indeed, EIN3/EIL proteins were shown to bind in a sequence specific manner to the primary ethylene-response element (PERE) of the ERF genes which are the last known actors of ethylene transduction pathway (Solano et al., 1998;[START_REF] Chang | Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis[END_REF]. This binding triggers the primary ethylene response through a transcriptional cascade that first includes the activation of target ERF genes which in turn through binding to GCC box cis-element in the promoter of target genes modulate the expression of ethylene-responsive genes. In the absence of ethylene, the receptor family activates the negative regulator CTR1, which leads to inhibition of EIN2. EIN3 and EIL1 levels are kept low by selective ubiquitination of the proteins by SCF EBF1 and SCF EBF1 , which induces their subsequent breakdown by the 26S proteasome. In the presence of ethylene, the receptors are inhibited, thus reducing the output of CTR1 and its subsequent inhibition of EIN2. EIN2 acts in part to directly or indirectly block the interaction of EIN3 and EIL1 with the SCF E3s containing EBF1 and EBF2. The reduction in ubiquitination allows EIN3 and EIL1 levels to rise to mediate ethylene responses. [START_REF] Binder | The Arabidopsis EIN3 binding F-Box proteins EBF1 and EBF2 have distinct but overlapping roles in ethylene signaling[END_REF].

Ethylene Response Factors (ERFs)

In the linear ethylene signal transduction pathway, ERFs are shown to be the last known downstream components which are responsible for modulating the transcription of early ethylene-regulated genes in plants. Being encoded by one of the largest family of plant transcription factors, ERF proteins are the most suited step of ethylene signaling where the diversity and specificity of ethylene responses may originate.

ERFs are part of AP2 (APETALA2)/ERF super-family which also contains AP2 and RAV family genes [START_REF] Riechmann | Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[END_REF]. The AP2/ERF superfamily is characterized by the presence of the AP2/ERF domain [START_REF] Riechmann | The AP2/EREBP family of plant transcription factors[END_REF][START_REF] Sakuma | DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and coldinducible gene expression[END_REF], which consists of about 59-60 amino acids and is involved in DNA binding.

ERF family proteins contain only one AP2/ERF domain, whereas, AP2 family genes have two such domains. RAV family proteins contain an additional B3 DNA binding domain along with AP2/ERF domain. The AP2 domain was first identified as a repeated motif within the Arabidopsis AP2 protein, which is involved in flower development [START_REF] Jofuku | Control of Arabidopsis flower and seed development by the homeotic gene APETALA2[END_REF]. The ERF domain was first identified as a conserved motif in four DNA-binding proteins from tobacco (Nicotiana tabacum), namely, EREBP1, 2,3,and 4 (currently renamed ERF1,2,3,and 4), and was shown to specifically bind to a GCC box, which is a DNA sequence involved in the ethylene-responsive transcription of genes [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF]. In the case of the RAV family, RAV1 and RAV2 were first identified as full-length cDNAs encoding proteins that contain a B3-like domain and an AP2/ERF domain in Arabidopsis [START_REF] Kagaya | RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants[END_REF]. Using heteronuclear multidimensional Nuclear Magnetic Resonance, [START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF] described the three dimensional structure of AP2/ERF domain from AtERF1. It consists of three antiparallel β-sheets and an α-helix. In the DNA-TF complex, tryptophan and arginine residues present in the b-sheets have been found to make contact with the DNA in its major groove. Based upon the binding of ERF domain to DNA sequence element, ERF family has been further divided into two subfamilies, i.e., ERF and CBF/DREB (C-repeat binding factor/dehydration responsive element binding factor). ERF subfamily is characterized by the presence of an alanine and aspartic acid respectively at position 14 and 19 in the AP2 domain, whereas valine and glutamic acid are conserved in the corresponding positions for CBF/DREB [START_REF] Sakuma | DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and coldinducible gene expression[END_REF]. ERFs have been shown to bind the GCC-box sequence (AGCCGCC) found in ethylene-responsive genes and DREBs to the DRE/CRT cis-regulatory element (A/GCCGAC) [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF][START_REF] Stockinger | Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[END_REF][START_REF] Hao | Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor (ERF domain) in plant[END_REF][START_REF] Hao | Determinants in the sequence specific binding of two plant transcription factors, CBF1 and NtERF2, to the DRE and GCC motifs[END_REF][START_REF] Oñate-Sánchez | AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense[END_REF].

In Arabidopsis, it was shown that the ERF subfamily contains 65 members and is divided into 5 subclasses based on the conservation of the AP2/ERF domain [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF]. Recently, genome-wide study also showed that the tomato ERF gene family comprises 9 subclasses (Figure 10) defined by distinct structural features and this work proposed a new nomenclature for tomato ERFs [START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF] which complies with the most complete classification available in Arabidopsis and clarifies the correspondence between ERF subclasses in different species [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF]. Based on functional analysis of 28 tomato ERFs and through testing their ability to activate or repress transcriptional activity of target genes, suggested that functional activity is conserved among ERF proteins sharing the same structural features [START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF].

It was reported that ERF subgroups which are characterized by the presence of conserved domain enriched in acidic amino acids, such as, glutamine, and proline are putative transcriptional activators [START_REF] Liu | Transcription factors and their genes in higher plants functional domains, evolution and regulation[END_REF]. More recently, a novel short motif termed EDLL was described by (Tiwari et al., 2012). This motif is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. It has a unique arrangement of acidic amino acids and hydrophobic leucines, and functions as a strong activation domain, even to heterologous DNA binding proteins. These results suggest that most of ERF transcription factors identified so far are activators [START_REF] Zhou | The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes[END_REF][START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF][START_REF] Ohta | Three ethylene-responsive transcription factors in tobacco with distinct transactivation functions[END_REF][START_REF] Oñate-Sánchez | Identification of Arabidopsis ethyleneresponsive element binding factors with distinct induction kinetics after pathogen infection[END_REF][START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF][START_REF] Oñate-Sánchez | AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense[END_REF][START_REF] Wu | ERF protein JERF1 that transcriptionally modulates the expression of abscisic acid biosynthesis-related gene enhances the tolerance under salinity and cold in tobacco[END_REF][START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF]Tiwari et al., 2012). However, a few of them, such as some members in group VIII, act as repressors [START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF][START_REF] Ohta | Repression domains of class II ERF transcriptional repressors share an essential motif for active repression[END_REF]Kazan, 2006;[START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF][START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF]. In contrast to the ERF activators, the ERF repressors contain a conserved (L/F)DLN(L/F)xP sequence, also called the ERF-associated amphiphilic repression (EAR) motif, in their Cterminal regions [START_REF] Ohta | Repression domains of class II ERF transcriptional repressors share an essential motif for active repression[END_REF]. Indeed tobacco ERF3, Arabidopsis AtERF3, AtERF4 and AtERF7 were shown to repress the expression of a GCC-box-containing reporter gene [START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF][START_REF] Ohta | Three ethylene-responsive transcription factors in tobacco with distinct transactivation functions[END_REF][START_REF] Ohta | Repression domains of class II ERF transcriptional repressors share an essential motif for active repression[END_REF][START_REF] Song | Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses[END_REF][START_REF] Yang | Arabidopsis ERF4 is a transcriptional repressor capable of modulating ethylene and abscisic acid responses[END_REF]. letters (A to J). Tomato genes for which the corresponding cDNA has been successfully isolated and that were subjected to functional analysis in this paper are named using the Sl-ERF nomenclature while other tomato ERFs are named using International Tomato Annotation Genome (ITAG 2.3) nomenclature.

Phylogenetic trees were constructed with the whole protein sequences using neighbor joining method [START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF].

ERF proteins are ubiquitous in plant kingdom. It has been demonstrated that the expression of many ERF genes can be regulated by plant hormones such as ethylene (ET), salicylic acid (SA) and jasmonic acid (JA), as well as by biotic and abiotic stresses [START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF][START_REF] Park | Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco[END_REF][START_REF] Gu | Tomato transcription factors pti4, pti5, and pti6 activate defense responses when expressed in Arabidopsis[END_REF][START_REF] Chen | Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses[END_REF][START_REF] Oñate-Sánchez | Identification of Arabidopsis ethyleneresponsive element binding factors with distinct induction kinetics after pathogen infection[END_REF][START_REF] Brown | A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis[END_REF][START_REF] Cheong | BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor[END_REF][START_REF] Lorenzo | ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense[END_REF][START_REF] Mcgrath | Repressor-and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression[END_REF][START_REF] Yang | Arabidopsis ERF4 is a transcriptional repressor capable of modulating ethylene and abscisic acid responses[END_REF][START_REF] Jin | Molecular cloning, expression profile and promoter analysis of a novel ethylene responsive transcription factor gene GhERF4 from cotton (Gossypium hirstum)[END_REF]Trujillo et al., 2008;Liu et al., 2010;[START_REF] Pan | Ectopic expression of an EAR motif deletion mutant of SlERF3 enhances tolerance to salt stress and Ralstonia solanacearum in tomato[END_REF][START_REF] Pan | An ethylene response factor (ERF5) promoting adaptation to drought and salt tolerance in tomato[END_REF][START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF]Figure 11) suggesting their putative roles in these responses. Indeed, in different plant species, the ERF proteins have been shown to be involved in the transcriptional regulation of a wide range of processes including response to biotic and abiotic stresses, hormonal signal transduction, regulation of metabolism, and in developmental processes [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF][START_REF] Fits | ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[END_REF][START_REF] Graaff | Activation tagging of the LEAFY PETIOLE gene affects leaf petiole development in Arabidopsis thaliana[END_REF][START_REF] Banno | Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration[END_REF][START_REF] Chuck | The control of spikelet meristem identity by the branched silkless1 gene in maize[END_REF][START_REF] Broun | WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis[END_REF][START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF][START_REF] Li | LeERF1 positively modulated ethylene triple response on etiolated seedling, plant development and fruit ripening and softening in tomato[END_REF][START_REF] Hu | Overexpression of OsERF1, a novel rice ERF gene, up-regulates ethylene-responsive genes expression besides affects growth and development in Arabidopsis[END_REF]Zhang et al., 2009;[START_REF] Pan | Ectopic expression of an EAR motif deletion mutant of SlERF3 enhances tolerance to salt stress and Ralstonia solanacearum in tomato[END_REF][START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF]. Constitutive expression of ERF1 increases the resistance of Arabidopsis to B. cinerea and P. cucumerina and induces the expression of several defense-related genes, including PLANT DEFENSIN1.2 (PDF1.2) and BASIC CHITINASE [START_REF] Berrocal-Lobo | Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi[END_REF][START_REF] Lorenzo | ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense[END_REF]. [START_REF] Pré | The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense[END_REF] also demonstrated that ORA59, a prominent representative of the ERF group IX in Arabidopsis, acts as an essential integrator of the JA and ethylene signal transduction pathways and overexpression of ORA59 causes increased resistance against the fungus Botrytis cinerea, whereas ORA59-silenced plants were more susceptible.

Moreover, it was reported that a transcriptional activator, AtERF14, has a prominent role in the plant defense response. AtERF14 loss-of-function mutants showed impaired induction of defense genes and increased susceptibility to Fusarium oxysporum [START_REF] Oñate-Sánchez | AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense[END_REF]. In tomato, the first ERFs which have been isolated are Pti4, 5 and Pti6 and the expression of these ERFs are induced by pseudomonas syringae [START_REF] Zhou | The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes[END_REF]Thara et al., 1999;[START_REF] Gu | Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase[END_REF]. Overexpression of Pti5 or Pti5-VP16, a translational fusion with a constitutive transcriptional activation domain, in tomato accelerated pathogen-induced expression of GluB and Catalase and enhanced resistance to Pseudomonas syringae pv. tomato [START_REF] He | Overexpression of Pti5 in tomato potentiates pathogen-induced defense gene expression and enhances disease resistance to Pseudomonas syringae pv. tomato[END_REF]. The promoter elements that they bind to and the effects of their over expression in plants are shown.

Studies have also shown that ERF proteins play important roles in the response to environmental stresses such as high salinity, drought and low temperature conditions via regulation of stress responsive genes (van der [START_REF] Fits | ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[END_REF][START_REF] Park | Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco[END_REF][START_REF] Aharoni | The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis[END_REF][START_REF] Huang | Tomato TERF1 modulates ethylene response and enhances osmotic stress tolerance by activating expression of downstream genes[END_REF]Taketa et al., 2008;Zhang et al., 2009;[START_REF] Boer | APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis[END_REF][START_REF] Fukao | The submergence tolerance regulator SUB1A mediates crosstalk between submergence and drought tolerance in rice[END_REF][START_REF] Wan | Transcriptional activation of OsDERF1 in OsERF3 and OsAP2-39 negatively modulates ethylene synthesis and drought tolerance in rice[END_REF]. It was shown that the tomato ethylene responsive factor 1 (TERF1) was induced by both ethylene and NaCl treatment (Huang et al., 2004). Overexpression of TERF1 in tobaccos activates constitutive expression of PR genes like Prb-1b, GLA, osmotin and CHN50 [START_REF] Huang | Tomato TERF1 modulates ethylene response and enhances osmotic stress tolerance by activating expression of downstream genes[END_REF] and activates genes involved in ABA/osmotic stress known to be involved in response to ABA, cold-, drought-, salt-stress. Transgenic tobacco plants constitutive expressing TERF1 displayed typical ethylene triple response and enhanced both salt and drought tolerance (Huang et al., 2004;[START_REF] Zhang | Expressing TERF1 in tobacco enhances drought tolerance and abscisic acid sensitivity during seedling development[END_REF]. These results suggest that TERF1 may make the link between ethylene and salt response and it may also integrate different signaling pathway. [START_REF] Gao | Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity[END_REF] also reported that expression of TERF1 gene in rice induces expression of stress responsive genes and enhances tolerance to drought and high-salinity. In tomato and tobacco, it was demonstrated that overexpression of LeERF2/TERF2 regulates the expression of genes involved in ethylene synthesis and resulted in increased ethylene synthesis and increased tolerance to cold (Zhang et al., 2009;[START_REF] Zhang | Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis[END_REF].

The tomato JERF1 mRNA was rapidly accumulated within 10 min, and peaked after 1 h, 40 min, 8 h or 4 h under ethylene, MeJA, ABA or high salt treatment, respectively [START_REF] Zhang | The ethylene-, jasmonate-, abscisic acid-and NaCl-responsive tomato transcription factor JERF1 modulates expression of GCC box-containing genes and salt tolerance in tobacco[END_REF] suggesting that JERF1 as a transcriptional factor may play important roles in the regulation of plant stress and defense responses through different signaling pathways. Constitutive expression of JERF1 in tobacco caused an increase in the transcript levels of GCC box-containing PR genes such as osmotin, GLA, Prb-1b and CHN50, and subsequently resulted in enhanced tolerance to salt stress during germination. This suggests that JERF1 modulates osmotic tolerance by activation of downstream gene expression via interaction with the GCC-box cis-elements. Moreover, it was also shown that JERF1 also enhances tolerance to drought, salinity and cold in tobacco by modulating the expression of an abscisic acid (ABA) biosynthesis-related gene [START_REF] Wu | ERF protein JERF1 that transcriptionally modulates the expression of abscisic acid biosynthesis-related gene enhances the tolerance under salinity and cold in tobacco[END_REF]. Recently, in Arabidopsis, an ethylene response factor, RAP2.2, which functions in an ethylene-controlled signal transduction pathway, was reported to involve in plant survival under hypoxia (low-oxygen) stress [START_REF] Hinz | Arabidopsis RAP2.2: an ethylene response transcription factor that is important for hypoxia survival[END_REF]. It was also demonstrated that AtERF98 regulates the response to salt stress in Arabidopsis by increasing ascorbic acid (AsA) synthesis [START_REF] Zhang | The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in Arabidopsis[END_REF].

In addition to the functions in response to biotic and abiotic stresses, ERF proteins have also been shown to play an important role in plant development and fruit ripening. It was shown that expression of an ERF gene, TINY, impacted plant height, hypocotyl elongation, and fertility in Arabidopsis and resulted in a "tiny" phenotype [START_REF] Wilson | A Dissociation insertion causes a semidominant mutation that increases expression of TINY, an Arabidopsis gene related to APETALA2[END_REF]. Results of [START_REF] Banno | Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration[END_REF] indicated that the ERF gene, ESR1, specifically regulates the induction of shoot regeneration after the acquisition of competence for organogenesis in Arabidopsis. Moreover, transgenic plant overexpressing Sl-ERF2 shows an early germinating phenotype, probably due to an over expression of mannanase genes involved in the radicle protrusion [START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF]. Recent studies demonstrated that SlERF36, an EAR-motif-containing ERF gene from tomato, alters stomatal density and modulates plant growth, flowering time and senescence in tobacco (Upadhyay et al., 2013). In tomato, it was shown that LeERF1 is involved in leaf morphology, fruit ripening and softening [START_REF] Li | LeERF1 positively modulated ethylene triple response on etiolated seedling, plant development and fruit ripening and softening in tomato[END_REF]. Furthermore, SlERF6 was also reported to play an important role in fruit ripening by integrating the ethylene and carotenoid synthesis pathways in tomato [START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF].

Role of ethylene

The gaseous phytohormone ethylene plays multiple roles in regulating plant growth and development. Alone or in content with the other phytohormones, ethylene regulates a wide range of plant activities, including seed germination, root growth and development, flowering, fruit ripening, organ senescence and abscission, and response to abiotic stresses and pathogen attacks [START_REF] Johnson | THE ETHYLENE GAS SIGNAL TRANSDUCTION PATHWAY: A Molecular Perspective[END_REF][START_REF] Bleecker | Ethylene: a gaseous signal molecule in plants[END_REF][START_REF] Lin | Recent advances in ethylene research[END_REF].

Seed germination

Seed germination is a complex physiological process under the control of plant hormones that play important and manifold roles [START_REF] Bewley | Seed Germination and Dormancy[END_REF]. Among the phytohormones ethylene is regarded as one of the key regulators in the process of seed germination. The addition of ethylene in the germination medium to Arabidopsis seeds results in accelerated germination, while adding norbornadiene, an inhibitor of ethylene action, delays germination. Analysis of mutant lines altered in ethylene biosynthesis or signaling pathway also demonstrated the involvement of ethylene in regulating seed germination. In Arabidopsis, constitutive ethylene insensitive mutant, etr1-1, show a delayed germination phenotype [START_REF] Bleecker | Insensitivity to Ethylene Conferred by a Dominant Mutation in Arabidopsis thaliana[END_REF]. Mutation in ETHYLENE INSENSITIVE2 (EIN2) gene results in poor germination and deeper dormancy, in contrast constitutive triple response 1 (ctr1) seeds germinate slightly faster compared to wild type [START_REF] Leubner-Metzger | Ethylene-responsive element binding protein (EREBP) expression and the transcriptional regulation of class I beta-1,3-glucanase during tobacco seed germination[END_REF]Subbiah and Reddy, 2010). Ethylene response factor (ERF) genes which act as the last known components of ethylene signaling pathway play a key role in seed germination regulation [START_REF] Leubner-Metzger | Ethylene-responsive element binding protein (EREBP) expression and the transcriptional regulation of class I beta-1,3-glucanase during tobacco seed germination[END_REF][START_REF] Song | Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses[END_REF][START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF]. The ABA-insensitive Arabidopsis mutant abi4 affected in seed germination displays altered expression of seed-specific genes [START_REF] Finkelstein | The Arabidopsis abscisic acid response locus ABI4 encodes an APETALA 2 domain protein[END_REF] and the abi4 mutation is caused by a single pair deletion within an AP2 family gene. It was reported that AtERF7 acts as a transcriptional repressor of the ABA response and that transgenic Arabidopsis lines expressing an RNAi construct targeted to downregulate the AtERF7 gene are more sensitive to ABA and germinate later than the wildtype seeds [START_REF] Song | Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and drought stress responses[END_REF]. Beechnut FsERF1 is almost undetectable in dormant seeds incubated under high temperature conditions that maintain dormancy, or in the presence of germination inhibitors, either ABA or AOA, an inhibitor of ethylene biosynthesis, but increases during moist chilling that progressively breaks dormancy [START_REF] Arc | ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination[END_REF]. [START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF] also demonstrated that SlERF2 transcript accumulation is higher in germinating seeds than in non-germinating ones and overexpression of this transcription factor in transgenic tomato lines results in enhanced ethylene sensitivity and premature seed germination. In seeds of Arabidopsis and other species, ethylene has a demonstrated antagonism to ABA, a hormone that inhibits germination [START_REF] Arc | ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination[END_REF].

In Arabidopsis, ethylene counteracts the inhibitory effects of ABA on endosperm cap weakening and endosperm rupture [START_REF] Linkies | Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana[END_REF]. ABA also increases the ethylene requirement to release primary and secondary dormancies. Mutants with enhanced response to ABA were found to be ethylene insensitive alleles in known genes of the ethylene pathway [START_REF] Beaudoin | Interactions between abscisic acid and ethylene signaling cascades[END_REF][START_REF] Ghassemian | Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis[END_REF]. Therefore, ethylene's effects in regulating germination may be explained, at least partially, by the ABA antagonism (Figure 12). Ethylene does not affect the seed ABA levels and therefore must counteract the ABA-induced inhibition of endosperm rupture by interfering with ABA signaling [START_REF] Linkies | Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana[END_REF].

Root development

In recent years, studies of the effect of ethylene on root growth and development has achieved substantial progress, with identification of both inhibitory effects of ethylene on root elongation and lateral root development and stimulatory effects of ethylene on root hair initiation. The inhibition of root elongation in the presence of ethylene or its precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), has been well known in different species [START_REF] Abeles | Ethylene in plant biology 2nd edn[END_REF][START_REF] Negi | Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato[END_REF]Figure 13). Ethyleneinsensitive mutants, including etr1, ein2, ein3, and eil1 in Arabidopsis and never ripe (nr) and green ripe (gr) in tomato, have elevated primary root growth rates compared to the wild type [START_REF] Růzicka | Ethylene regulates root growth through effects on auxin biosynthesis and transport-dependent auxin distribution[END_REF][START_REF] Stepanova | Multilevel interactions between ethylene and auxin in Arabidopsis roots[END_REF][START_REF] Negi | Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato[END_REF]. By contrast, roots of seedlings with elevated ethylene signaling or synthesis, ctr1 and eto1, respectively, display reductions in the rate of root elongation (Kieber et al., 1993). These results clearly indicate the inhibitory effect of ethylene on root growth. Five-day-old Arabidopsis seedlings were transferred to medium containing 1 μM ACC and the tip of the roots at the time of transfer was marked by a black dot. When their roots were imaged five days later, ethylene had decreased the rate of root elongation relative to an untreated control, as judged by the length of root that had formed below the black dots. By contrast, ethylene treatment prevented lateral root formation in the region formed after transfer [START_REF] Muday | Auxin and ethylene: collaborators or competitors?[END_REF] Ethylene has been shown to play a negative role in lateral root formation through the studies in Arabidopsis and tomato using the diversity of mutants with altered ethylene signaling or synthesis [START_REF] Ivanchenko | Ethylene-auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana[END_REF][START_REF] Negi | Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana[END_REF][START_REF] Negi | Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato[END_REF].

Treatments or mutations to elevate ethylene levels inhibit lateral root formation.

Arabidopsis wild type Columbia seedlings show a dose dependent decrease in lateral root numbers when grown on the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) and this is reversed by the treatment with ethylene antagonist, silver nitrate [START_REF] Negi | Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana[END_REF]. The ctr1 mutant, with enhanced ethylene signaling (Kieber et al., 1993;[START_REF] Huang | Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis[END_REF], and the eto1 mutant, with enhanced ethylene synthesis (Kieber et al., 1993), exhibited significant reductions in lateral root numbers compared with wild-type seedlings. In contrast the Arabidopsis and tomato ethylene-insensitive mutants exhibit elevated numbers of lateral roots [START_REF] Negi | Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana[END_REF][START_REF] Negi | Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato[END_REF][START_REF] Strader | Ethylene directs auxin to control root cell expansion[END_REF]. The ethylene-insensitive mutants etr1, which has a dominant negative receptor mutation (Hua et al., 1998;[START_REF] Sakai | ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis[END_REF], and ein2, which has a defect in an ethylene signaling protein (Kendrick and [START_REF] Gendron | Chemical genetic dissection of brassinosteroid-ethylene interaction[END_REF], showed enhanced lateral root formation and was insensitive to the inhibitory effect of ACC on lateral root numbers [START_REF] Negi | Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana[END_REF]. Interestingly, although most of these studies examined root growth on agar medium, the increase in lateral root formation seen in nr is even more striking in seedlings grown in soil for several weeks [START_REF] Negi | Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato[END_REF], suggesting that ethylene may have even more profound effects on roots during standard cultivation. These effects of ethylene are on the earliest stages of lateral root initiation [START_REF] Ivanchenko | Ethylene-auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana[END_REF] and alter auxin transport, suggesting that crosstalk with auxin should be a critical component of the activity of ethylene in lateral root development [START_REF] Negi | Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana[END_REF].

The role of ethylene in the formation of adventitious roots has been examined in a variety of plant species, but the results have been contradictory. In tomato, elevated exogenous or endogenous ethylene levels increase adventitious root formation, while ethyleneinsensitive nr produces fewer adventitious roots [START_REF] Clark | Root formation in ethylene-insensitive plants[END_REF]Kim et al., 2008;[START_REF] Negi | Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato[END_REF]. In contrast in Arabidopsis, ACC treatment, as well as the eto1 and ctr1 mutations, results in reduced adventitious root formation (Sukumar, 2010). These contradictory findings may be due to variation in the different tissues, growth conditions, and methods of quantifying adventitious root formation.

In contrast to the negative effects of ethylene on root elongation and lateral root formation, root hair development is positive regulated by ethylene (Tanimoto et al., 1995;[START_REF] Rahman | Auxin and ethylene response interactions during Arabidopsis root hair development dissected by auxin influx modulators[END_REF]. In Arabidopsis, the ethylene precursor ACC induces ectopic root hair formation (Tanimoto et al., 1995;[START_REF] Pitts | Auxin and ethylene promote root hair elongation in Arabidopsis[END_REF], whereas application of an ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG) and Ag + (an ethylene action inhibitor) reduce root hairs [START_REF] Masucci | The rhd6 Mutation of Arabidopsis thaliana Alters Root-Hair Initiation through an Auxin-and Ethylene-Associated Process[END_REF]Tanimoto et al., 1995). The initiation and elongation of root hairs may synergistically induced by ethylene and auxin. Since auxin is able to rescue root hair elongation defects in ethyleneinsensitive mutants, and inhibition of auxin influx exacerbates the ein2 root hair phenotype [START_REF] Rahman | Auxin and ethylene response interactions during Arabidopsis root hair development dissected by auxin influx modulators[END_REF]. Application of ACC or IAA to the root hair-deficient mutant root hair defective 6 (rhd6) can restore root hair initiation [START_REF] Masucci | The rhd6 Mutation of Arabidopsis thaliana Alters Root-Hair Initiation through an Auxin-and Ethylene-Associated Process[END_REF]. Moreover, auxin-insensitive mutants that also show ethylene insensitivity, such as axr2, axr3 and aux1, exhibit reduced root hair initiation (Pickett et al., 1990;[START_REF] Wilson | A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid[END_REF][START_REF] Leyser | Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter[END_REF]. Interestingly, ethylene also affects the positioning of root hairs and ethylene-insensitive mutants having apically shifted root hairs [START_REF] Fischer | Planar polarity of root hair positioning in Arabidopsis[END_REF].

Flowering

The transition from vegetative growth to flowering is the most drastic change in plant development. Ethylene has been known to be involved in flowering process for a long time [START_REF] Abeles | Ethylene in plant biology 2nd edn[END_REF]. Experiments applying ethylene and chemical inhibitors of its biosynthesis and function have demonstrated that ethylene differentially regulates flowering in different plant species. Ethylene plays a role in floral promotion in pineapple, mango, lychee and Plumbago indica, and in floral inhibition in short-day plants such as cocklebur, Japanese morning glory, chrysanthemum, tobacco and Chenopodium [START_REF] Abeles | Ethylene in plant biology 2nd edn[END_REF]. It has been reported that ethylene induced flowering in pineapple (Ananas comosus) and that silencing of the AcACC synthase gene caused delayed flowering in pineapple (Trusov and Botella, 2006). In rice, overexpression of ETR2 leads to reduced ethylene sensitivity and late flowering, whereas T-DNA insertion mutant etr2 showed enhanced ethylene sensitivity and early flowering [START_REF] Wuriyanghan | The ethylene receptor ETR2 delays floral transition and affects starch accumulation in rice[END_REF]. It is showed that ethylene represses flowering in Arabidopsis and inactivation of specific ACS gene products enhances flowering time (Tsuchisaka et al., 2009). In Arabidopsis, it is found that wild-type plants grown in the presence of the ethylene precursor ACC, or in an ethylene-rich atmosphere, flowered late and the constitutive ethylene response Arabidopsis mutant ctr1-1 showed a late flowering phenotype [START_REF] Achard | The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristemidentity genes[END_REF].

Moreover, enhanced ethylene response mutant eer2 also displayed a delay in bolting and flowering time compared with wild type plants (De Paepe et al., 2005). Based on the data obtained in acs mutants, it was proposed that ethylene exerts its effect on flowering by regulating the expression of the FLC which acts as a rheostat to repress flowering through repression of the floral pathway integrators FT and SOC1. Interestingly, on the other hand, based on the observation that constitutively active ethylene signaling in the ctr1 mutant reduces GA levels, and the late flowering phenotype of the ctr1 mutant is partially rescued by loss-of-function mutations in DELLA genes, [START_REF] Achard | The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristemidentity genes[END_REF] showed that ethylene delays flowering via modulating DELLA activity. The induced DELLA accumulation by ethylene in turn delays flowering via repression of the floral meristemidentity genes LEAFY (LFY) and SOC1 [START_REF] Achard | The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristemidentity genes[END_REF]. A different flowering pathway operates in the acs mutants and in the ctr1 mutant could be a possible explanation for this difference findings. In tomato, overexpression of SlTPR1 gene, a tomato tetratricopeptide repeat protein, results in enhanced ethylene response and delayed flowering time [START_REF] Lin | SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development[END_REF]. Further studies should disclose the mechanism by which ethylene differentially regulates flowering in different plant species.

Fruit ripening

Fruit ripening is a developmentally regulated process unique to plants during which the majority of the sensory quality attributes are elaborated including aroma, flavor, texture and nutritional compounds [START_REF] Carrari | Metabolic regulation underlying tomato fruit development[END_REF]. Biochemical and physiological changes that occur during fruit ripening are driven by a cascade of molecular events leading to the stimulation of specific transcriptional regulators responsible for the coordinated expression of fruit ripening-related genes directly involved in the biochemical processes [START_REF] Giovannoni | Genetic regulation of fruit development and ripening[END_REF]. The requirement for ethylene in the ripening of climacteric fruit has long been recognized [START_REF] Abeles | Ethylene in plant biology 2nd edn[END_REF] and discrimination between climacteric and non-climacteric fruits has been made on the basis of the presence or absence of the climacteric rise in respiration and of autocatalytic ethylene production. In climacteric fruit, the plant hormone ethylene is considered to be the major signaling molecule that controls most aspects of fruit ripening. By contrast, in non-climacteric fruit, ethylene is not the trigger of the ripening process, which appears to depend on signals not yet elucidated. It should be noted that, however, these distinctions are not absolute, as closely related melon and capsicum species can be both climacteric and non-climacteric and some non-climacteric fruits show enhanced ripening phenotypes in response to exogenous ethylene [START_REF] Lelièvre | Ethylene and fruit ripening[END_REF][START_REF] Alexander | Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening[END_REF][START_REF] Mailhac | Ethylene and grape berry ripening[END_REF]. Nevertheless, enhanced ethylene synthesis at the onset of ripening is required for the normal ripening of many fruits.

Two systems of ethylene biosynthesis have been proposed in climacteric plants [START_REF] Mcmurchie | Treatment of fruit with propylene gives information about the biogenesis of ethylene[END_REF]. System 1 functions during normal vegetative growth, is ethylene autoinhibitory and is responsible for producing basal ethylene levels that are detected in all tissues including those of non-climacteric fruit. System 2 operates during the ripening of climacteric fruit when ethylene production is autocatalytic. Through a combination of ethylene and inhibitor studies together with expression analysis of ethylene biosynthesis genes in ripening tomato fruits and various ripening mutants, the molecular mechanisms of autocatalytic ethylene production were investigated in tomato [START_REF] Barry | The regulation of 1aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[END_REF]. Expression analysis has revealed that at least four ACS (LEACS1A, LEACS2, LEACS4, and LEACS6) and three ACO (LEACO1, LEACO3, and LEACO4) genes are differentially expressed in tomato fruit [START_REF] Rottmann | 1-aminocyclopropane-1-carboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence[END_REF][START_REF] Barry | Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato[END_REF][START_REF] Nakatsuka | Differential expression and internal feedback regulation of 1aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening[END_REF][START_REF] Barry | The regulation of 1aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[END_REF]. It was shown that system 1 ethylene is regulated by the expression of LeACS1A and LeACS6 with the two genes being negatively regulated by ethylene. Subsequently, the up-regulation of LeACS2 and LeACS4 through positive feedback by ethylene is responsible for the activation of System 2 [START_REF] Nakatsuka | Differential expression and internal feedback regulation of 1aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening[END_REF][START_REF] Barry | The regulation of 1aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[END_REF]. LeACO1, LeACO3, and LeACO4 are all expressed at low levels in green fruit that are in a system 1 mode of ethylene synthesis, but the transcripts of each increase at the climacteric peak as the fruit ethylene production transition to system 2. Moreover, LEACO1 and LEACO4 are sustained in expression during fruit ripening, whereas the increase in LEACO3 expression is transient [START_REF] Barry | Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato[END_REF][START_REF] Nakatsuka | Differential expression and internal feedback regulation of 1aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening[END_REF]. In the case of LEACO1 and LEACO4, ripening-related increases in transcript abundance can be largely blocked by 1-MCP treatment, indicating that these genes are positively regulated by ethylene. The main changes in ACS and ACO gene expression associated with the ethylene synthesis from system 1 to system 2 during tomato fruit development and ripening are shown in Figure 14. It should be point out that because the expression of ethylene biosynthesis genes were shown to be also regulated by some regulator factors, such as, through binding to their promoters, RIN and LeHB-1 regulate the expression of LeACS2 and LeACO1, respectively [START_REF] Ito | DNA-binding specificity, transcriptional activation potential, and the rin mutation effect for the tomato fruit-ripening regulator RIN[END_REF][START_REF] Lin | Recent advances in ethylene research[END_REF], it is possible that the autocatalytic regulation is not the only mechanism of the system 2 ethylene synthesis. Autocatalytic ethylene synthesis at the onset of fruit ripening is mediated through ethylene-stimulated expression of LeACS2 and 4 and LeACO1 and 4 [START_REF] Barry | Ethylene and fruit ripening[END_REF].

Inhibition or delay in fruit ripening in tomato by antisense expression of tomato ACS2 and ACO1 genes was the first direct evidence that ethylene biosynthesis is essential for climacteric fruit ripening [START_REF] Oeller | Reversible inhibition of tomato fruit senescence by antisense RNA[END_REF]. Furthermore, effects of ethylene perception and signal transduction on fruit ripening were also well known. It was shown that the Never-ripe (Nr) mutant of tomato corresponding to a mutation in the ethylene receptor conferred ethylene insensitivity and thus produced non-ripening fruit [START_REF] Wilkinson | An ethylene-inducible component of signal transduction encoded by never-ripe[END_REF]. Moreover, antisense inhibition of production of the mutant mRNA in the Nr mutant resulted in failure to synthesize the mutant receptor protein, and partially or completely restored fruit ripening [START_REF] Hackett | Antisense inhibition of the Nr gene restores normal ripening to the tomato Neverripe mutant, consistent with the ethylene receptor-inhibition model[END_REF] supporting the evidence that the ethylene receptors act as negative regulators of ethylene action. Another mutant, Greenripe (Gr), a dominant ripening mutation that occurs in a gene encoding another component of ethylene signaling failed to fully ripen as a consequence of inhibition of ethylene responsiveness [START_REF] Barry | Ethylene insensitivity conferred by the Green-ripe and Never-ripe 2 ripening mutants of tomato[END_REF]. The GR protein which corresponds to the REVERSION TO ETHYLENE SENSITIVITY1 (RTE1) in Arabidopsis was proposed to mediate ethylene response via interacting with and regulating the ethylene receptor(s) [START_REF] Barry | Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling[END_REF][START_REF] Resnick | REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates ethylene receptor function in Arabidopsis[END_REF][START_REF] Zhou | RTE1 is a Golgi-associated and ETR1dependent negative regulator of ethylene responses[END_REF]. In addition, it was demonstrated that receptor level, during fruit development, determines the timing of ripening (Kevany et al., 2007) and fruit-specific suppression of LeETR4 resulted in earlyripening fruit in tomato (Kevany et al., 2008).

The ethylene transduction pathway leads to the regulation of fruit ripening through a transcriptional cascade including primary (ETHYLENE-INSENSITIVE3 (EIN3) and

EIN3-like (EIL)) and secondary response factors (ETHYLENE RESPONSE FACTORS

(ERF)). It was shown that transgenic tomato plants with reduction in expression of multiple tomato LeEIL genes significantly reduced ethylene sensitivity and thus affected fruit ripening (Tieman et al., 2001). [START_REF] Chen | Constitutive expression of EIL-like transcription factor partially restores ripening in the ethylene-insensitive Nr tomato mutant[END_REF] also demonstrated that overexpression of LeEIL1 in the Nr mutant partially restored normal fruit ripening and stimulated the expression of some ethylene-responsive genes. In Kiwifruit, AdEIL genes were constitutively expressed during fruit ripening and the transcription factors AdEIL2 and AdEIL3 can activate transcription of the ripening-related genes AdACO1 suggesting a role of EIL genes in fruit ripening via regulating ethylene biosynthesis genes. In addition, silencing of SlEBF1 and SlEBF2 which negatively regulate ethylene signaling by mediating the degradation of EIN3/EIL proteins resulted in a constitutive ethylene response phenotype and accelerated fruit ripening in tomato [START_REF] Yang | Silencing Sl-EBF1 and Sl-EBF2 expression causes constitutive ethylene response phenotype, accelerated plant senescence, and fruit ripening in tomato[END_REF]. These results indicated that ethylene-mediated climacteric fruit ripening is also controlled at the transcriptional levels.

The identification of several key ripening regulatory genes from tomato, such as MADSbox Ripening-Inhibitor (RIN) [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF], SBP-box Colourless Non-Ripening (CNR) [START_REF] Manning | A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening[END_REF], MADS-box AGAMOUS-LIKE1 (TAGL1) [START_REF] Itkin | TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network[END_REF][START_REF] Vrebalov | Fleshy fruit expansion and ripening are regulated by the Tomato SHATTERPROOF gene TAGL1[END_REF], and leucine zipper homeobox LeHB-1 [START_REF] Lin | SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development[END_REF], has led to new insights into understanding of ethylene and ripening control mechanisms. The RIN gene encodes a putative MADS box transcription factor that controls tomato fruit ripening, with its mutated version (rin) conferring a non-ripening character [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF]. Molecular and biochemical studies have shown that RIN participates in ethylene production by inducing many of the ethylene synthesis/signaling genes (e.g., ACS2 and ACS4), by upregulating NOR and CNR and by downregulating HB-1 [START_REF] Ito | DNA-binding specificity, transcriptional activation potential, and the rin mutation effect for the tomato fruit-ripening regulator RIN[END_REF][START_REF] Fujisawa | Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation[END_REF][START_REF] Fujisawa | Direct targets of the tomato-ripening regulator RIN identified by transcriptome and chromatin immunoprecipitation analyses[END_REF][START_REF] Martel | The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENINGdependent manner[END_REF][START_REF] Qin | Unraveling the regulatory network of the MADS box transcription factor RIN in fruit ripening[END_REF][START_REF] Zhou | Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening[END_REF]. Cnr is an epigenetic change that alters the promoter methylation of a SQUAMOSA promoter-binding (SPB) protein, resulting in a pleiotropic ripening inhibition phenotype and inhibited expression of ethylene-associated genes ACO1, E8, and NR, and several other ripening-related genes (Thompson et al., 1999). Tomato AGAMOUS-LIKE1 (TAGL1) gene whose down-regulation results in yellow fruit with reduced carotenoids and thin pericarp, has been shown to control fruit expansion and ripening [START_REF] Itkin | TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network[END_REF][START_REF] Vrebalov | Fleshy fruit expansion and ripening are regulated by the Tomato SHATTERPROOF gene TAGL1[END_REF]. Furthermore, TAGL1-suppressed fruit produce lower amounts of ethylene with a reduced expression of LeACS2 suggesting that TAGL1 may be another important regulator of ripening-related ethylene production. The transcription factor encoded by the LeHB-1 gene belonging to class-I HD-Zip proteins can bind the promoter of LeACO1 [START_REF] Lin | SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development[END_REF] and its silencing via virus-induced gene silencing (VIGS) strategy results in down-regulation of LeACO1 expression associated with delayed fruit ripening. The putative transcription factor, Sl-AP2a, a member of the AP2/ERF superfamily gene was also recently described as a negative regulator of fruit ripening and of ethylene production [START_REF] Chung | A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening[END_REF][START_REF] Karlova | Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening[END_REF]. The characterization of these transcriptional regulators indicates that transcription factors play key roles in relaying ripening-inducing signals and controlling ethylene biosynthesis. Thus, the regulation of transcriptional regulators by acting upstream of ethylene synthesis should be an important mechanism for controlling fruit ripening (Figure 15). [START_REF] Fujisawa | A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening[END_REF].

Ethylene Response Factors (ERFs) are the last identified downstream components of the ethylene signal transduction pathway known to regulate early ethylene-responsive genes.

Recently, accumulating studies have shown that ERF proteins play an important role in fruit ripening (Klee and [START_REF] Martel | The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENINGdependent manner[END_REF]. Most of the ERF genes identified in tomato were ethylene inducible and showed ripening-related expression pattern [START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF]. Tomato LeERF1 was reported to mediate ethylene response and thus control fruit ripening [START_REF] Li | LeERF1 positively modulated ethylene triple response on etiolated seedling, plant development and fruit ripening and softening in tomato[END_REF]. Overexpression of LeERF1 in tomato resulted in constitutive ethylene response and accelerated fruit ripening and softening [START_REF] Li | LeERF1 positively modulated ethylene triple response on etiolated seedling, plant development and fruit ripening and softening in tomato[END_REF]. A ripening-related pattern of expression has also been shown for LeERF2 and LeERF3b in tomato fruit (Tournier et al., 2003;[START_REF] Chen | Differential regulation of tomato ethylene responsive factor LeERF3b, a putative repressor, and the activator Pti4 in ripening mutants and in response to environmental stresses[END_REF]. LeERF2 is induced by ethylene and suppressed in ripening-inhibited mutants (Wu et al., 2002;Tournier et al., 2003).

Moreover, LeERF2 regulates ethylene response in tomato by modulating ethylene biosynthesis genes [START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF]Zhang et al., 2009), transcriptional regulation being achieved by interaction of LeERF2 with promoter of LeACO3 (Zhang et al., 2009).

Recently, SlERF6 was reported to play an important role in fruit ripening by integrating the ethylene and carotenoid synthesis pathways in tomato [START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF]. In banana, ERFs are also demonstrated to be involved in fruit ripening through their interactions with ethylene biosynthesis genes [START_REF] Xiao | Banana ethylene response factors are involved in fruit ripening through their interactions with ethylene biosynthesis genes[END_REF]. Nevertheless, so far only one ERF has been identified as direct regulator of ripening-associated genes via binding a ciselement present in the promoter of E4 [START_REF] Montgomery | Identification of an ethylene-responsive region in the promoter of a fruit ripening gene[END_REF], a ripening-regulated gene [START_REF] Lincoln | Diverse mechanisms for the regulation of ethylene-inducible gene expression[END_REF] encoding proteins of unknown function, and therefore the specific role of each ERF in ethylene response and the ripening process is still far from being well understood.

Although ripening control in non-climacteric fruit was thought to be independent of ethylene, some studies do show an increase in ethylene production in non-climacteric fruit which suggesting a role of ethylene in ripening. Indeed, the effect of ethylene in inducing color changes in the flavedo tissue of citrus fruit, a non-climacteric fruit, has long been known [START_REF] Goldschmidt | Probing the role of endogenous ethylene in the degreening of citrus fruit with ethylene antagonist[END_REF][START_REF] Goldschmidt | Ripening of citrus and other non-climacteric fruit: a role for ethylene[END_REF]. In strawberry, which is generally considered as non-climacteric fruit, an increase in ethylene production associated with a raise in respiration has been observed when the fruit reaches the redripe stage [START_REF] Iannetta | Ethylene and carbon dioxide production by developing strawberries show a correlative pattern that is indicative of ripening climacteric fruit[END_REF]. It was reported that, in grapes, a small increase in ethylene production occurs at the veraison stage when berries reach the onset of color changes and treatment of grape berries with 1-MCP, an inhibitor of ethylene perception, affected anthocyanin accumulation and berry swelling and caused a decrease in acidity [START_REF] Chervin | Stimulation of the grape berry expansion by ethylene and effects on related gene transcripts, over the ripening phase[END_REF] suggesting that ethylene might be required for the full accomplishment of the ripening process. Overall, these data suggest a putative involvement of ethylene in at least some aspects of the ripening process in nonclimacteric fruit.

Organ senescence and abscission

Senescence is a vitally important developmental step in the life cycle of a plant or a plant organ that determines yield and reproductive success. The most prominent symptom of leaf senescence is the visible yellowing, which correlates with physiological and biochemical changes such as dismantling of chloroplasts, drop of chlorophyll content and photosynthetic activities, and degradation of RNA and proteins [START_REF] Jing | Ageing in plants: Conserved strategies and novel pathways[END_REF].

Senescence is a complex developmental phase involving the actions of a complex network consisting of multiple pathways. In many species, and in different plant organs, ethylene has long been considered a key hormone in regulating the onset of leaf senescence [START_REF] Bleecker | Insensitivity to Ethylene Conferred by a Dominant Mutation in Arabidopsis thaliana[END_REF][START_REF] Zacarias | The role of growth regulators in the senescence of Arabidopsis thaliana leaves[END_REF]. In Arabidopsis, ethylene treatment advances the visible yellowing and senescence-associated genes (SAGs) induction in leaves that are primed to senesce [START_REF] Grbic | Ethylene regulates the timing of leaf senescence in Arabidopsis[END_REF]. Ethylene insensitive mutants, such as etr1 and ein2/ore3 display delayed leaf senescence [START_REF] Oh | Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana[END_REF]. It was reported that the lifespan of etr1-1 leaves is 30% longer than wild-type leaves and this delay is accompanied by a delayed induction of senescence associated genes (SAGs) which are used as molecular markers of leaf senescence [START_REF] Hensel | Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in arabidopsis[END_REF] and higher expression of photosynthesis-associated genes (PAGs) [START_REF] Grbic | Ethylene regulates the timing of leaf senescence in Arabidopsis[END_REF]. In tomato, reduced ethylene production due to antisense suppression of the genes involved in ethylene biosynthesis also resulted in a temporal delay in the onset of foliar senescence [START_REF] Jone | Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis[END_REF]. Recently, Chen et al., (2011) showed that a MADS box gene, FOREVER YOUNG FLOWER (FYF) acts as a repressor of organ senescence and abscission through suppressing ethylene response.

It was demonstrated that ethylene can induce senescence only when developmental changes controlled by leaf age are present and before senescence can be initiated, some age-related changes (ARCs) must have taken place in the leaf [START_REF] Hensel | Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in arabidopsis[END_REF][START_REF] Jing | Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence[END_REF][START_REF] Jing | Ethylene-induced leaf senescence depends on age-related changes and OLD genes in Arabidopsis[END_REF]. For example, the oldest leaves showed the greatest increase in SAG transcripts after ethylene treatment, and little or no effect of ethylene was observed in the youngest leaves [START_REF] Grbic | Ethylene regulates the timing of leaf senescence in Arabidopsis[END_REF]. These results strongly suggest that ethylene can induce leaf senescence only within specific age window. Indeed, at early leaf growth, ethylene does not induce leaf senescence, and this is the never senescence phase. This phase could be controlled by developmental signals or homeostatic genes such as so-called age-related factors. Only after a defined stage a leaf switches to the second phase, which allows the action of ethylene to promote leaf senescence. This promoting effect operates within a defined time span, marking the ethylene-dependent senescence phase [START_REF] Grbic | Ethylene regulates the timing of leaf senescence in Arabidopsis[END_REF][START_REF] Jing | Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence[END_REF][START_REF] Jing | Ethylene-induced leaf senescence depends on age-related changes and OLD genes in Arabidopsis[END_REF].

Abscission is a physiological process that involves the programmed separation of entire organs, such as leaves, petals, flowers, and fruit. It is the mechanism for the removal of senescing or damaged organs but also for the release of the fruit when it is ripe [START_REF] Bleecker | Last exit: senescence, abscission, and meristem arrest in Arabidopsis[END_REF]. The first demonstration that ethylene can promote abscission was documented by Wehmer in 1917 [START_REF] Abeles | Ethylene in plant biology 2nd edn[END_REF]. Application of exogenous ethylene hastens abscission by inducing expression of cellulase and polygalacturonase genes in different species and different plant organs [START_REF] Bonghi | Cellulase and polygalacturonase involvement in the abscission of leaf and fruit explants of peach[END_REF][START_REF] Kalaitzis | Cloning of a tomato polygalacturonase expressed in abscission[END_REF][START_REF] Campillo | Pedicel breakstrength and cellulase gene expression during tomato flower abscission[END_REF]. Inhibitors of ethylene action such as silver and 2, 5-norbornadiene have been found to block abscission and inhibitors of ethylene synthesis such as AVG have also been shown to retard abscission of leaves, flowers, and fruit (Kushad and Poovaiah, 1984;[START_REF] Abeles | Ethylene in plant biology 2nd edn[END_REF]. The first genetic evidence to substantiate an involvement of the ethylene signaling pathway in organ abscission was when the ETHYLENE RECEPTOR1 (ETR1) was identified [START_REF] Bleecker | Insensitivity to Ethylene Conferred by a Dominant Mutation in Arabidopsis thaliana[END_REF].

Ethylene insensitive etr1 mutant displayed a delayed capacity to undergo floral organ abscission [START_REF] Bleecker | Insensitivity to Ethylene Conferred by a Dominant Mutation in Arabidopsis thaliana[END_REF][START_REF] Bleecker | Last exit: senescence, abscission, and meristem arrest in Arabidopsis[END_REF]. Thanks to the identification of ethylene-signaling mutants in tomato, such as never ripe (nr), nr2, green ripe (gr), eil1, 2 and 3, our understanding of the effect of ethylene in mediating organ abscission has increased. The nr mutant which has a mutation in the ethylene receptor gene NR was shown to exhibit delayed pedicel abscission (Lanahan et al., 1994;Wilkinson et al., 1995b;Lashbrook et al., 1998;[START_REF] Hackett | Antisense inhibition of the Nr gene restores normal ripening to the tomato Neverripe mutant, consistent with the ethylene receptor-inhibition model[END_REF]. Moreover, both nr2 and gr dominant mutants which show reduced sensitivity to ethylene display delayed flower abscission even after exposure to exogenous ethylene [START_REF] Barry | Ethylene insensitivity conferred by the Green-ripe and Never-ripe 2 ripening mutants of tomato[END_REF]. By contrast, overexpressing the ethylene biosynthesis gene ACC synthase in tomato plants resulted in premature flower abscission [START_REF] Whitelaw | Delayed abscission and shorter Internodes correlate with a reduction in the ethylene receptor LeETR1 transcript in transgenic tomato[END_REF].

Up till now, several Arabidopsis mutants which show defects in organ abscission have been identified, including inflorescence deficient in abscission [START_REF] Butenko | Inflorescence deficient in abscission controls floral organ abscission in Arabidopsis and identifies a novel family of putative ligands in plants[END_REF], delayed floral organ abscission1, 2 and 3 [START_REF] Patterson | Ethylene-dependent and -independent processes associated with floral organ abscission in Arabidopsis[END_REF], hawaiian skirt [START_REF] González-Carranza | Hawaiian skirt: an F-box gene that regulates organ fusion and growth in Arabidopsis[END_REF], and the haesa (hae) haesa-like2 (hsl2) double mutant [START_REF] Cho | Regulation of floral organ abscission in Arabidopsis thaliana[END_REF][START_REF] Stenvik | The EPIP peptide of INFLORESCENCE DEFICIENT IN ABSCISSION is sufficient to induce abscission in arabidopsis through the receptor-like kinases HAESA and HAESA-LIKE2[END_REF]. All of these mutants display normal ethylene sensitivity. Moreover, exogenous ethylene, which promotes the cell separation phase of abscission in wild-type plants, does not alter the abscission defective phenotype in the MKK4-MKK5RAi and hae hsl2 plants [START_REF] Cho | Regulation of floral organ abscission in Arabidopsis thaliana[END_REF]. These results indicate that even if ethylene accelerates the abscission process, the perception of ethylene is not the unique process.

Pathogen resistance

Plants have evolved sophisticated detection and defense systems to protect themselves from pathogen invasion. When plants perceive a pathogen attack, an increase of transcription of ethylene response genes is generally observed. This over-production of ethylene is usually associated with induction of defence reaction. However, depending on the type of pathogen and plant species, the role of ethylene can be dramatically different, as ethylene has been demonstrated to stimulate, as well as to counteract disease development. Ethylene can enhance resistance against various pathogens (Thomma et al., 2001;[START_REF] Díaz | The role of ethylene and wound signaling in resistance of tomato to Botrytis cinerea[END_REF], but it can also increase disease severity, probably by promoting chlorosis, senescence, and cell necrosis [START_REF] Abeles | Ethylene in plant biology 2nd edn[END_REF]. Taking advantage of the availability of plant mutants and transgenic lines that are affected in their response to ethylene, the reactions of these mutant and transgenic plants to different types of attackers were compared, either enhanced or reduced disease development were observed (van Loon et al., 2006; Table 1). It is shown that as a result of increased symptom severity in non-responsive mutants, ethylene was found to reduce diseases caused by several fungi and bacteria that kill their hosts (necrotrophs), or have a mixed biotrophicnecrotrophic lifestyle (in which they start exploiting the living host before killing it). By contrast, the occurrence of less severe symptoms indicated that ethylene stimulated diseases caused by various other fungi and bacteria with varying lifestyles, as well as infection by a cyst nematode and insect attack. [START_REF] Bent | Disease development in ethylene-insensitive Arabidopsis thaliana infected with virulent and avirulent Pseudomonas and Xanthomonas pathogens[END_REF] showed that the ethylene-insensitive Arabidopsis mutant ein2-1 was resistant to X. campestris pv. campestris, whereas the etr1 and etr2 mutants were reported to display more severe symptoms, indicating an enhanced susceptibility [START_REF] O'donnell | Multiple hormones act sequentially to mediate a susceptible tomato pathogen defense response[END_REF]. Moreover, tomato plants treated with 1-methylcyclopropene (MCP), an inhibitor of ethylene perception, were show to display enhanced susceptibility to B. cinerea [START_REF] Díaz | The role of ethylene and wound signaling in resistance of tomato to Botrytis cinerea[END_REF].

Nevertheless, the ethylene-insensitive tomato mutant Never ripe (Nr) seemed to be as susceptible as wild-type plants to B. cinerea [START_REF] Díaz | The role of ethylene and wound signaling in resistance of tomato to Botrytis cinerea[END_REF], and even less susceptible to the vascular wilt fungus Fusarium oxysporum f.sp. lycopersici [START_REF] Lund | Ethylene regulates the susceptible response to pathogen infection in tomato[END_REF].

Ethylene-insensitive soybean mutants displayed increased disease severity after infection with the brown-spot fungus Septoria glycines or the root rot fungus Rhizoctonia solani, but less severe symptoms upon inoculation with the root and crown rot-causing oomycete Phytophthora sojae. By contrast, it was shown that inoculation of ethylene-insensitive soybean with the bacterial blight pathogen P. syringae pv. glycinea led to reduced disease severity compared to wild-type plants [START_REF] Hoffman | Isolation of ethylene-insensitive soybean mutants that are altered in pathogen susceptibility and gene-for-gene disease resistance[END_REF]. These results indicate that altered ethylene sensitivity can result in more or less severe disease, reflecting reduced or increased pathogen resistance, respectively, depending on the plant-pathogen combination. Indeed, after systematic testing of several pathogenic fungi and bacteria on different accessions and various mutants of Arabidopsis the conclusion that, in general, ethylene contributes to resistance against necrotrophic, but not biotrophic pathogens was proposed (Thomma et al., 2001;Ton et al., 2002). In response to pathogen attack in plant, ethylene can induce certain types of pathogenesis-related (PR) proteins or phytoalexins, and, through stimulation of the phenylpropanoid pathway, can rigidify cell walls in various plant species [START_REF] Abeles | Ethylene in plant biology 2nd edn[END_REF]. (hevein-like), and PR-12 (plant defensins, PDFs) have been well demonstrated [START_REF] Broglie | Functional analysis of DNA sequences responsible for ethylene regulation of a bean chitinase gene in transgenic tobacco[END_REF][START_REF] Samac | Isolation and Characterization of the Genes Encoding Basic and Acidic Chitinase in Arabidopsis thaliana[END_REF][START_REF] Penninckx | Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway[END_REF][START_REF] Penninckx | Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis[END_REF][START_REF] Penninckx | Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis[END_REF]Thomma et al., 1999;Thomma et al., 2001;van Loon et al., 2006). Analysis of the promoters of the PR genes led to the identification of several cis-elements required for ethylene regulation, including GCC-box and the dehydration responsive element/Crepeat element (DRE/CRT). The GCC-box (11-bp sequence TAAGAGCCGCC), was

shown to be necessary, and in some cases sufficient, for the regulation by ethylene of PR genes in different plant species [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF]Solano et al., 1998;[START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF][START_REF] Gu | Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase[END_REF][START_REF] Brown | A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis[END_REF][START_REF] Chakravarthy | The tomato transcription factor Pti4 regulates defenserelated gene expression via GCC box and non-GCC box cis elements[END_REF][START_REF] Oñate-Sánchez | AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense[END_REF][START_REF] Zhou | Abscisic acid regulates TSRF1-mediated resistance to Ralstonia solanacearum by modifying the expression of GCC boxcontaining genes in tobacco[END_REF][START_REF] Anderson | The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia[END_REF]. The Ethylene Response Factors (ERFs) which involves in the ethylene signal cascade have been shown

to regulate the expression of PR genes via directly binding to the GCC-box or the dehydration responsive element/C-repeat element (DRE/CRT) located in the promoters of various pathogenesis-related (PR) genes [START_REF] Park | Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco[END_REF][START_REF] Hao | Determinants in the sequence specific binding of two plant transcription factors, CBF1 and NtERF2, to the DRE and GCC motifs[END_REF][START_REF] Gutterson | Regulation of disease resistance pathways by AP2/ERF transcription factors[END_REF][START_REF] Moffat | ERF5 and ERF6 play redundant roles as positive regulators of JA/Et-mediated defense against Botrytis cinerea in Arabidopsis[END_REF]. Indeed, Plant ERF transcription factors are widely involved in biotic stress responses and particularly in pathogen resistance.

Overexpression of ERF genes, such as Pti4, ERF1, OPBP1, TSRF1, AtERF14, ORA59, confers resistance to fungal and bacterial pathogens in transgenic plants [START_REF] Berrocal-Lobo | Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi[END_REF][START_REF] Gu | Tomato transcription factors pti4, pti5, and pti6 activate defense responses when expressed in Arabidopsis[END_REF][START_REF] Oñate-Sánchez | Identification of Arabidopsis ethyleneresponsive element binding factors with distinct induction kinetics after pathogen infection[END_REF][START_REF] Guo | Overexpression of the AP2/EREBP transcription factor OPBP1 enhances disease resistance and salt tolerance in tobacco[END_REF][START_REF] Oñate-Sánchez | AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense[END_REF][START_REF] Pré | The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense[END_REF][START_REF] Zhang | Expression of the ethylene response factor gene TSRF1 enhances abscisic acid responses during seedling development in tobacco[END_REF]. The functionality of ERF subfamily members in different species has suggested their involvement in ethylene signaling and ethylene-related defenses and the complexity of the regulation of repressor and activator-types of ERFs during pathogen challenge may explain the different role of ethylene in mediating pathogen stress.

Crosstalk between ethylene and other phytohormones

Ethylene regulates many aspects of plant developmental processes, and it is no doubt that the diversity of ethylene functions is achieved, at least in part, by its interactions with other hormones. The interactions between ethylene and other phytohormones are discussed below.

Ethylene and auxin

Ethylene and auxin interact at both the physiological and molecular levels in plant growth and development with either synergistic or antagonistic effects. Ethylene and auxin are able to regulate the synthesis of each other. Elevated levels of auxin lead to increased ethylene synthesis via increased transcription of the genes that drive ethylene synthesis, including specific members of the ACC synthase (ACS) family, which catalyze the rate-limiting step in ethylene synthesis ( Abel et al., 1995;[START_REF] Wang | Ethylene biosynthesis and signaling networks[END_REF]Tsuchisaka and Theologis, 2004;[START_REF] Stepanova | Multilevel interactions between ethylene and auxin in Arabidopsis roots[END_REF]. Reciprocally, ethylene regulates the expression of WEI2/ASA1 and WEI7/ASB1, the subunits of an anthranilate synthase that catalyzes the first step in tryptophan biosynthesis, the principal precursor of auxin biosynthesis [START_REF] Stepanova | A Link between ethylene and auxin uncovered by the characterization of two rootspecific ethylene-insensitive mutants in Arabidopsis[END_REF].

At root level, ethylene and auxin affect synergistically in the processes of root elongation and root hair formation, while in other processes, such as lateral root formation, they act antagonistically [START_REF] Muday | Auxin and ethylene: collaborators or competitors?[END_REF]. The earliest genetic evidence that ethylene and auxin may act through convergent pathways to regulate root growth came from the identification of ethylene-insensitive mutants with defects in auxin transporters: aux1 and ethylene insensitive root 1/pinformed 2 (eir1/pin2) [START_REF] Pickett | The aux1 Mutation of Arabidopsis Confers Both Auxin and Ethylene Resistance[END_REF][START_REF] Roman | Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway[END_REF][START_REF] Luschnig | EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana[END_REF]. Kinematic analyses of root growth inhibition by ethylene and auxin by high temporal and spatial resolution revealed that ethylene and auxin reduce the expansion rate of the cells in the central elongation zone [START_REF] Rahman | Auxin, actin and growth of the Arabidopsis thaliana primary root[END_REF]Swarup et al., 2007). Mutants with enhanced ethylene or auxin synthesis have reduced root elongation and wild-type plants treated with exogenous ethylene or auxin also show reduction in root elongation (Kieber et al., 1993;[START_REF] Delarue | Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis[END_REF][START_REF] Zhao | A role for flavin monooxygenase-like enzymes in auxin biosynthesis[END_REF][START_REF] Rahman | Auxin, actin and growth of the Arabidopsis thaliana primary root[END_REF]. Moreover, like in ethylene-insensitive mutants, auxin-induced root growth inhibition is lost or substantially reduced in auxin-resistant mutants such as tir1, axr2, axr3, and solitary-root (slr) (Timpte et al., 1994;[START_REF] Leyser | Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter[END_REF][START_REF] Fukaki | Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis[END_REF][START_REF] Biswas | Genetic characterization of mutants resistant to the antiauxin p-chlorophenoxyisobutyric acid reveals that AAR3, a gene encoding a DCN1-like protein, regulates responses to the synthetic auxin 2,4-dichlorophenoxyacetic acid in Arabidopsis roots[END_REF]. These results indicate that auxin and ethylene have similar effects on root elongation. Indeed, more and more evidences proved that ethylene inhibits root growth via modulation of auxin signaling, transport and synthesis is one of the mechanisms by which ethylene and auxin synergistically inhibit root elongation [START_REF] Stepanova | A Link between ethylene and auxin uncovered by the characterization of two rootspecific ethylene-insensitive mutants in Arabidopsis[END_REF][START_REF] Stepanova | Multilevel interactions between ethylene and auxin in Arabidopsis roots[END_REF].

In contrast to root elongation, which is synergistically inhibited by auxin and ethylene, these two hormones act antagonistically on lateral root initiation. Treatment with ethylene or ACC reduces lateral root initiation in both Arabidopsis and tomato. Dominant negative etr1 and Nr ethylene receptor mutants, as well as the ethylene-insensitive ein2 and Gr mutants have an enhanced number of lateral roots [START_REF] Negi | Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana[END_REF][START_REF] Negi | Genetic dissection of the role of ethylene in regulating auxin-dependent lateral and adventitious root formation in tomato[END_REF].

By contrast, auxin stimulates lateral root formation and elongation, mutants and inhibitors that reduce auxin transport reduce lateral root initiation and emergence [START_REF] Reed | Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis[END_REF][START_REF] Casimiro | Auxin transport promotes Arabidopsis lateral root initiation[END_REF][START_REF] Ivanchenko | Ethylene-auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana[END_REF][START_REF] Péret | Arabidopsis lateral root development: an emerging story[END_REF]. Recently, it was demonstrated that ethylene inhibits lateral root development by blocking changes in the abundance of local auxin transport protein needed to form local auxin maxima that drive lateral root formation [START_REF] Lewis | Ethylene inhibits lateral root development, increases IAA transport and expression of PIN3 and PIN7 auxin efflux carriers[END_REF].

Pharmacological and genetic studies have revealed that ethylene and auxin promote the processes of root hair initiation (Tanimoto et al., 1995;[START_REF] Rahman | Auxin and ethylene response interactions during Arabidopsis root hair development dissected by auxin influx modulators[END_REF]. Application of ethylene or auxin to the root hair-deficient mutant root hair defective 6 (rhd6) was found can restore the root hair initiation (Masucci and Schiefelbein, 1994b). Moreover, auxin-insensitive mutants that also show ethylene insensitivity, such as aux1, axr2 and axr3, display reduced root hair initiation [START_REF] Pickett | The aux1 Mutation of Arabidopsis Confers Both Auxin and Ethylene Resistance[END_REF][START_REF] Wilson | A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid[END_REF][START_REF] Leyser | Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR-AC1 promoter[END_REF]. It was predicted that root hair initiation is directly linked to the amount of auxin and auxin signaling, and the effect of ethylene is less direct and likely to occur through intracellular auxin levels [START_REF] Muday | Auxin and ethylene: collaborators or competitors?[END_REF].

The induction of apical hook formation in Arabidopsis represents one of the best described examples of ethylene-auxin interaction in plants ( [START_REF] Lehman | HOOKLESS1, an ethylene response gene, is required for differential cell elongation in the Arabidopsis hypocotyl[END_REF][START_REF] Raz | Regulation of differential growth in the apical hook of Arabidopsis[END_REF]. Various studies indicate that ethylene affects auxin transport, synthesis, and, perhaps, signaling to regulate the differential growth leading to the apical hook.

Combination of all of these studies, [START_REF] Muday | Auxin and ethylene: collaborators or competitors?[END_REF] proposed a working model of ethylene-auxin crosstalk in apical hook formation (Figure 16). In this model, ethylene causes enhanced apical hook formation by both increasing the levels of components important for auxin signaling and increasing auxin levels on the concave side of the apical hook. The interplay between ethylene and auxin in flower and fruit abscission is also well established [START_REF] Abeles | Regulation of Ethylene Evolution and Leaf Abscission by Auxin[END_REF][START_REF] Roberts | Abscission, dehiscence, and other cell separation processes[END_REF]. The generally accepted model is that a basipetal auxin flux through the abscission zone (AZ) prevents abscission by rendering the AZ insensitive to ethylene.

In addition, ethylene and auxin crosstalk is necessary to determine normal fruit ripening.

Indeed, the levels of auxin must decrease prior to the onset of ripening in both climacteric and non-climacteric fruits.

Ethylene and gibberellins (GAs)

Ethylene and gibberellins (GAs) control similar developmental processes in plants.

The crosstalk between GA and ethylene has been demonstrated [START_REF] Achard | Ethylene regulates arabidopsis development via the modulation of DELLA protein growth repressor function[END_REF][START_REF] Vriezen | Ethylenemediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent[END_REF][START_REF] Achard | The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristemidentity genes[END_REF][START_REF] Grauwe | Reciprocal influence of ethylene and gibberellins on response-gene expression in Arabidopsis thaliana[END_REF]. DELLA proteins, which act as nuclear repressors of GA signaling, appear to be key integrators in the ethylene-GA crosstalk. It was shown that ethylene controls the maintenance and exaggeration of the apical hook via modifying DELLA degradation [START_REF] Achard | Ethylene regulates arabidopsis development via the modulation of DELLA protein growth repressor function[END_REF][START_REF] Vriezen | Ethylenemediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent[END_REF]. In addition, [START_REF] Achard | The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristemidentity genes[END_REF] reported that ethylene controls floral transition via DELLA-dependent regulation of floral meristem identity genes. It was reported that regulatory crosstalk involving ethylene and GA affects the transition from seed dormancy to germination in common beech (Fagus sylvatica L.) seeds where a drastic increase in FsACO1 expression when seeds were treated with GA3 or ethephon, but the stimulatory effect of ethephon could be reversed by paclobutrazol, a GA biosynthesis inhibitor, suggesting that GA positively regulates the expression of FsACO1 gene [START_REF] Calvo | Evidence of a cross-talk regulation of a GA 20-oxidase (FsGA20ox1) by gibberellins and ethylene during the breaking of dormancy in Fagus sylvatica seeds[END_REF]. De [START_REF] Grauwe | Reciprocal influence of ethylene and gibberellins on response-gene expression in Arabidopsis thaliana[END_REF] also demonstrated that the absence of an active GA-signaling cascade suppresses the higher ethylene biosynthesis observed in eto2-1 while the responsiveness to ethylene is slightly enhanced. The suppression of ethylene biosynthesis in the double mutant suggests that the absence of active GA signaling may affect the stability of ethylene-biosynthesis enzymes in a negative feedback mechanism. The enhanced sensitivity to GA in the gai eto2-1 double mutant suggests a reciprocal influence of the two pathways on one another and this also was corroborated by earlier data demonstrating that ACC enhances the activity of the GAbiosynthesis gene GA1 [START_REF] Vriezen | Ethylenemediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent[END_REF]. [START_REF] Pierik | Interactions between ethylene and gibberellins in phytochrome-mediated shade avoidance responses in tobacco[END_REF] reported that the involvement of ethylene in phytochrome-mediated shade avoidance responses can at least partly be attributed to interactions between ethylene and GA action, and it is likely that GA acts downstream of ethylene in regulating shade avoidance responses. In addition, [START_REF] Dubois | Ethylene Response Factor6 acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis[END_REF] also reported that upon exposure to osmotic stress, ACC accumulates in the actively growing leaves, where it is converted to ethylene. Ethylene further activates the signaling pathway involving MPK3 and MPK6. These kinases phosphorylate the basal amount of ERF5 and ERF6

proteins present in the cell prior to stress exposure. The activated ERF5 and ERF6 then activation of leaf growth inhibition via the transcriptional activation of the gene encoding the GA-inactivating enzyme GA2-OX6, thereby decreasing the bioactive GA concentration and stabilizing the DELLA proteins [START_REF] Dubois | Ethylene Response Factor6 acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis[END_REF]. Recently, in tomato, it was found that dominant repression of an ethylene response factor, Sl-ERF.B3, confers ethylene hypersensitivity with reduced plant size and delayed flowering time. The reduced expression of GA oxidase genes in the transgenic lines sustains the idea of altered GA metabolism and suggests that ERFs may represent a potential molecular link between ethylene and GA [START_REF] Liu | A dominant repressor version of the tomato Sl-ERF.B3 gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components[END_REF].

Ethylene and abscisic acid (ABA)

Abscisic acid (ABA) is a classic phytohormone that plays an important role in various aspects of plant growth and development. It has been shown that a subset of the functions of ABA overlaps with those of ethylene including in seed germination and early seedling establishment, albeit with antagonistic effects [START_REF] Zhou | Glucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucose-insensitive mutant[END_REF]. In Arabidopsis, ethylene counteracts the inhibitory effects of ABA on endosperm cap weakening and endosperm rupture [START_REF] Linkies | Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana[END_REF]. ABA increases the ethylene requirement to release primary and secondary dormancies. Inhibition of seed germination by ABA was shown to be associated with a reduction in ethylene synthesis (reviewed in [START_REF] Arc | ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination[END_REF]. In Arabidopsis, ABA inhibited the accumulation of ACO1 transcripts in both the embryo and endosperm during seed germination and the high levels of ACO1 transcripts in ABA-insensitive mutants also suggests the regulation of ACO expression by ABA [START_REF] Penfield | Arabidopsis ABA INSENSITIVE4 regulates lipid mobilization in the embryo and reveals repression of seed germination by the endosperm[END_REF][START_REF] Linkies | Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana[END_REF]. Interestingly, it was also reported that ABA-deficient mutants of Arabidopsis aba2 and tomato flacca and notabilis reveal inhibition of shoot growth, largely because of high ethylene production in these mutants [START_REF] Sharp | Endogenous ABA maintains shoot growth in tomato independently of effects on plant water balance: evidence for an interaction with ethylene[END_REF][START_REF] Lenoble | Maintenance of shoot growth by endogenous ABA: genetic assessment of the involvement of ethylene suppression[END_REF].

The intertwining nature of ethylene and ABA biosynthesis and signaling pathways in germination has been well studied [START_REF] Beaudoin | Interactions between abscisic acid and ethylene signaling cascades[END_REF][START_REF] Ghassemian | Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis[END_REF][START_REF] Cutler | Abscisic acid: emergence of a core signaling network[END_REF]. [START_REF] Beaudoin | Interactions between abscisic acid and ethylene signaling cascades[END_REF] reported that genetic screening of the enhancer and repressor of ABA-insensitive germination of the abi1-1 mutant were allelic to ctr1 and ein2, respectively. [START_REF] Ghassemian | Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis[END_REF] also found that the enhanced response to ABA3 (era3) mutant was a new allele of ein2 that shows hypersensitivity to ABA in seed germination. Seeds of Arabidopsis ethylene-insensitive mutants, etr1 and ein2, exhibit higher ABA content than wild type and consistently germinate more slowly [START_REF] Chiwocha | The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination[END_REF][START_REF] Wang | Arabidopsis EIN2 modulates stress response through abscisic acid response pathway[END_REF]. Moreover, [START_REF] Cheng | Antagonism between abscisic acid and ethylene in Arabidopsis acts in parallel with the reciprocal regulation of their metabolism and signaling pathways[END_REF] showed that the expression of 9-CIS-EP-OXYCAROTENOID DIOXYGENASE 3 (NCED3), which encodes the key enzyme in ABA biosynthesis, is up-regulated in the ein2-1 mutant, and CYP707A2, a cytochrome P450 gene which encodes the key component of ABA catabolism, is down-regulated in etr1-1, suggesting that when ethylene signaling is impaired, ABA biosynthesis may be enhanced. Mutations that reduce ethylene sensitivity, such as etr1, ein2, and ein6, result in an increase in ABA sensitivity, while increased ethylene sensitivity in ctr1 and eto1 reduces ABA sensitivity [START_REF] Beaudoin | Interactions between abscisic acid and ethylene signaling cascades[END_REF][START_REF] Ghassemian | Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis[END_REF][START_REF] Chiwocha | The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination[END_REF][START_REF] Linkies | Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana[END_REF]Subbiah and Reddy, 2010). These results suggest that ethylene not only acts on ABA metabolism to reduce ABA levels, but also negatively regulates ABA signaling.

The interactions between ethylene and ABA are known not only in developmental processes but also in adaptive stress responses of plants. Exogenous ABA suppresses ethylene-responsive defense genes such as PDF1.2 and b-CHI, while mutations in the ABA biosynthesis pathway have the opposite effect. Accordingly, aba2-1 mutants with enhanced levels of these PR proteins exhibited improved resistance against F. oxysporum.

An ethylene response factor gene, AtERF4, has been shown to modulate the antagonistic ethylene-ABA crosstalk [START_REF] Yang | Arabidopsis ERF4 is a transcriptional repressor capable of modulating ethylene and abscisic acid responses[END_REF]. Moreover, it was reported that ethylene biosynthesis gene ACS7 acts as a negative regulator of ABA sensitivity and accumulation under stress and appears as a node in the cross-talk between ethylene and ABA [START_REF] Dong | Loss of ACS7 confers abiotic stress tolerance by modulating ABA sensitivity and accumulation in Arabidopsis[END_REF].

Based on the timing of ABA accumulation, changes to ethylene production and the expression of ABA and ethylene biosynthesis genes, (Zhang et al., 2009) concluded that the two hormones may also play a coordinating role in tomato ripening. Treatment of fruit with ABA increased the expression of three ethylene biosynthetic genes, promoting ethylene synthesis and ripening, while inhibitors of ABA synthesis prevented this increase.

Ethylene and jasmonates (JAs)

Interactions between ethylene and JAs have been shown to contribute to a variety of responses of plants to biotic and abiotic stresses or developmental cues. Studies have indicated that ethylene-and JA-signaling often operate synergistically to induce the expression of a number of defense related genes including PR1b, PR5 (osmitin), PDF1.2, the basic chitinase gene CHI-B, a hevein-like protein gene, and proteinase inhibitors (PIN)

genes after pathogen inoculation [START_REF] Xu | Plant Defense Genes Are Synergistically Induced by Ethylene and Methyl Jasmonate[END_REF][START_REF] O'donnell | Ethylene as a Signal Mediating the Wound Response of Tomato Plants[END_REF][START_REF] Penninckx | Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis[END_REF][START_REF] Ellis | The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens[END_REF]Thomma et al., 2001). Moreover, the Arabidopsis cev1 mutant, that is defective in the cellulose synthase gene CesA3, displays constitutively active ethylene and JA responses indicating that CEV1 acts as a negative regulator of ethylene and JA signaling in Arabidopsis [START_REF] Ellis | The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses[END_REF]. A convergence point between ethylene and JAs pathways was represented by the transcriptional activation of ETHYLENE TRANSCRIPTION FACTOR1 (ERF1), a transcription factor that regulates the expression of pathogen response genes that prevent disease progression [START_REF] Lorenzo | ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense[END_REF]. The expression of ERF1 was induced rapidly by ethylene or JAs and could be activated synergistically by both hormones. Moreover, constitutive expression of ERF1 could rescue the defense response defects of coi1 (coronatine insensitive1) and ein2

(ethylene insensitive2) by restoring PR gene expression, suggesting that ERF1 is a key downstream element of both ethylene and JAs signaling pathways for the regulation of defense response genes [START_REF] Lorenzo | ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense[END_REF]; Figure 18). Indeed, several members of In the wound response, the oligosaccharide-mediated repression of the JA-dependent signaling pathway was exerted through the production and perception of ethylene in the locally damaged tissue. This negative interaction between ethylene and JA allows the establishment of the correct spatial pattern of systemically induced genes in plants reacting to injury [START_REF] Rojo | Cross-talk between wound signalling pathways determines local versus systemic gene expression in Arabidopsis thaliana[END_REF]. Furthermore, by using JA-deficient (asLOX3), ethylene-insensitive (mETR1) Nicotiana attenuata plants, and their genetic cross, it was proposed that in N. attenuata, the crosstalk between ethylene and JA restrains local cell expansion and growth after herbivore attack, allowing more resources to be allocated to induced defenses against herbivores [START_REF] Onkokesung | Jasmonic acid and ethylene modulate local responses to wounding and simulated herbivory in Nicotiana attenuata leaves[END_REF].

It was reported that the effects of JAs on root hair development were abolished in the ethylene-insensitive mutants etr1-1 and etr1-3, or by ethylene action (Ag + ) or biosynthesis inhibitors (AVG). Moreover, it was found that JA biosynthesis inhibitors, ibuprofen and SHAM, also repressed ACC-driven or eto1-1-induced root hair formation [START_REF] Zhu | Interactions between jasmonates and ethylene in the regulation of root hair development in Arabidopsis[END_REF]. These results support a role for the interaction between ethylene and JAs in the regulation of root hair development. In addition, the triple-response that includes an exaggerated apical hook of Arabidopsis seedlings germinated in the dark in the presence of ethylene can be suppressed by JA in a COI1-dependent manner is an example of interaction of ethylene and JAs in development process [START_REF] Ellis | The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens[END_REF].

Ethylene and brassinosteroids (BRs)

Brassinosteroids (BRs) are a family of poly-hydroxylated steroid hormones that are involved in many aspects of plant growth and development. Studies have shown that BRs and ethylene have overlapping functions in hypocotyl elongation and apical hook formation (De [START_REF] Grauwe | Auxin, ethylene and brassinosteroids: tripartite control of growth in the Arabidopsis hypocotyl[END_REF]. It was suggested that ethylene controls the biosynthesis of BRs and establishes a gradient of BR in the apical hook region that contributes to the hook formation. Furthermore, [START_REF] Gendron | Chemical genetic dissection of brassinosteroid-ethylene interaction[END_REF] reported that ethylene functions partly through BR to regulate both hypocotyl length and apical hook formation, and it is likely that ethylene functions through the BES1 dependent branch of the BR signaling pathway to control hypocotyl elongation.

BRs are known for a long time to stimulate the production of ethylene in shoots and roots [START_REF] Yi | Auxin and brassinosteroid differentially regulate the expression of three members of the 1aminocyclopropane-1-carboxylate synthase gene family in mung bean (Vigna radiata L.)[END_REF][START_REF] Arteca | Brassinosteroid-induced exaggerated growth in hydroponically grown Arabidopsis plants[END_REF], and this ethylene probably plays an important role in the many effects of BR on plant growth and development. In mung bean (Vigna radiata), the brassinosteroid (BR) 2, 4-epibrassinolide has been shown to specifically enhance the expression of VrACS7 in hypocotyls, and BR also synergistically increased the IAA-induced VRACS6 and VrACS7 transcript levels [START_REF] Yi | Auxin and brassinosteroid differentially regulate the expression of three members of the 1aminocyclopropane-1-carboxylate synthase gene family in mung bean (Vigna radiata L.)[END_REF]. [START_REF] Müssig | Brassinosteroids promote root growth in Arabidopsis[END_REF] also reported that BR has a positive effect on genes involved in ethylene biosynthesis and ethylene response based on the expression data. Interestingly, it was found that brassinopride (BRP), an inhibitor of BR biosynthesis, also causes exaggerated apical hooks in dark-grown seedlings, an effect similar to that of ethylene [START_REF] Gendron | Chemical genetic dissection of brassinosteroid-ethylene interaction[END_REF]. Physiological experiments using ethylene mutants and treatment with ethylene (ACC) and an ethylene perception inhibitor suggest that BRP promotes ethylene action at a step of or upstream of ethylene perception, possibly ethylene synthesis.

It was shown that ACS5 protein was stabilized in response to BR [START_REF] Hansen | Regulation of ACS protein stability by cytokinin and brassinosteroid[END_REF], suggesting that BR increases ethylene synthesis by regulation of ethylene biosynthesis gene at post-transcriptional levels. However, studies in mung bean have indicated that

VrACS7 is regulated transcriptionally by BR [START_REF] Yi | Auxin and brassinosteroid differentially regulate the expression of three members of the 1aminocyclopropane-1-carboxylate synthase gene family in mung bean (Vigna radiata L.)[END_REF][START_REF] Zimmermann | GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox[END_REF].

Moreover, transcriptome data indicated that both ACS5 and ACS6 transcripts are elevated in response to BR [START_REF] Hansen | Regulation of ACS protein stability by cytokinin and brassinosteroid[END_REF], and in etiolated seedlings, BR treatment also resulted in increase of ACS5 transcript levels. Taken together, BRs have an effect on the transcription of ACS genes, but also act by increasing ACS protein stability.

Tomato as model plant

The Solanaceae family comprises many agriculturally valuable crops, including eggplant, potato, pepper, tobacco, and tomato. Among them, tomato is one of the most important crops in the fresh vegetable market and the food-processing industry [START_REF] Matsukura | Comprehensive resources for tomato functional genomics based on the miniature model tomato micro-tom[END_REF]. Tomato (Solanum lycopersicum) originated in South America and was brought to Europe in the early 16th century. After its introduction in Europe the tomato has gone a long way. Dedicated breeding has resulted in numerous cultivars grown all over the world, differing in all kind of aspects such as yield, shape, resistance, taste and quality.

Up to now, there are over 7500 tomato varieties cultivated throughout the world presenting a huge variability in fruit color or size. Although original tomatoes were small fruit, now most cultivars produce large red fruit, a number of cultivars with yellow, orange, pink, purple, green, black, or white fruit are also available. Tomato fruit size varies from 5mm of diameter in cherry tomatoes to more than 10 centimeters in beefsteak tomatoes (http://en.wikipedia.org/wiki/Tomato). In addition, tomatoes are loaded with phytochemicals, plant-derived chemical compounds, which work in concert with the body to protect against cancer, clogged arteries and skin ailments. The lycopene in tomatoes is one of the most powerful anti-oxidants and helps in the fight against cancerous cell formation.

In addition to its agronomical and economic importance, tomato is an excellent model analyses, and plant-pathogen interactions [START_REF] Robinson | Sink metabolism in tomato fruit : I. Developmental changes in carbohydrate metabolizing enzymes[END_REF][START_REF] Wilkinson | An ethylene-inducible component of signal transduction encoded by never-ripe[END_REF][START_REF] Frary | fw2.2: a quantitative trait locus key to the evolution of tomato fruit size[END_REF][START_REF] Giovannoni | MOLECULAR BIOLOGY OF FRUIT MATURATION AND RIPENING[END_REF][START_REF] Bramley | Regulation of carotenoid formation during tomato fruit ripening and development[END_REF][START_REF] Pedley | Molecular basis of Pto-mediated resistance to bacterial speck disease in tomato[END_REF]Carrari et al., 2006;[START_REF] Giovannoni | Fruit ripening mutants yield insights into ripening control[END_REF]. Indeed, the adaptation of a range of technological tools (e.g. Once the fruit has reached its final size it starts to ripen [START_REF] Mounet | Quantitative metabolic profiles of tomato flesh and seeds during fruit development: complementary analysis with ANN and PCA[END_REF].

Ripening in the cultivated tomato comprises a series of biochemical and physiological events, including softening, pigment change, development of flavor components, autocatalytic ethylene production, and climacteric respiratory behavior, which together make ripe fruits. Several naturally occurring ripening mutations have been characterized.

These include for example ripening-inhibitor (rin, [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF], non-ripening (nor, [START_REF] Giovannoni | Genetic regulation of fruit development and ripening[END_REF], Colorless non-ripening (Cnr, [START_REF] Manning | A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening[END_REF], Green-ripe (Gr, [START_REF] Barry | Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling[END_REF], Never-ripe (Nr, [START_REF] Wilkinson | An ethylene-inducible component of signal transduction encoded by never-ripe[END_REF], high-pigment 1 [START_REF] Liu | Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato[END_REF] and Never-ripe 2 (Nr-2, [START_REF] Barry | Ethylene insensitivity conferred by the Green-ripe and Never-ripe 2 ripening mutants of tomato[END_REF]. The availability of these well characterized ripening mutants is indeed an important reason for tomato being taken as a model for study fruit ripening (Figure 20).

Through investigation of these tomato mutants in fruit ripening, many of the underlying genes control the ripening processes were isolated. The RIN, CNR, and NOR genes have been shown to encode transcriptional regulators and act to regulate the expression of other genes responsible for fruit ripening processes, including ethylene biosynthesis [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF][START_REF] Giovannoni | Genetic regulation of fruit development and ripening[END_REF][START_REF] Manning | A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening[END_REF]. The Gr gene encodes a still poorly defined component of ethylene signal transduction while Nr encodes an ethylene receptor important for fruit and additional non-fruit ethylene responses (Lanahan et al., 1994;[START_REF] Barry | Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling[END_REF]. Moreover, other ripening transcriptional regulators have also been demonstrated via functional studies in transgenic plants, including LeHB1 which directly regulates ACC oxidase expression [START_REF] Lin | SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development[END_REF] and TAGL1, a MADS box transcription factor, which links early fruit fleshy expansion with downstream ripening [START_REF] Lin | SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development[END_REF][START_REF] Itkin | TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network[END_REF][START_REF] Vrebalov | Fleshy fruit expansion and ripening are regulated by the Tomato SHATTERPROOF gene TAGL1[END_REF][START_REF] Pan | Ectopic expression of an EAR motif deletion mutant of SlERF3 enhances tolerance to salt stress and Ralstonia solanacearum in tomato[END_REF]. The putative transcription factor, Sl-AP2a, a member of the AP2/ERF superfamily gene was also recently described as a negative regulator of fruit ripening and of ethylene production [START_REF] Chung | A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening[END_REF][START_REF] Karlova | Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening[END_REF]. These discoveries have further facilitated the demonstration of regulatory mechanisms for fruit ripening.

Figure 20. Tomato ripening mutants [START_REF] Giovannoni | Genetic regulation of fruit development and ripening[END_REF][START_REF] Barry | Ethylene insensitivity conferred by the Green-ripe and Never-ripe 2 ripening mutants of tomato[END_REF][START_REF] Giovannoni | Fruit ripening mutants yield insights into ripening control[END_REF].

Like Arabidopsis, the convenient small size and amenability to large-scale cultivation are also found in tomato. Micro-Tom (MT), a dwarf cultivar of tomato, has been proposed as a preferred variety to carry out molecular research in tomato. MT was initially created for ornamental purposes by crossing Florida Basket and Ohio 4013-3 cultivars [START_REF] Martí | Genetic and physiological characterization of tomato cv[END_REF]. MT cultivar displays a very dwarf phenotype with a bushy appearance and its leaves are small, with deformed leaflets, and a deep green color compared with other commonly used wild-type cultivars (Figure 21). It was confirmed that Micro-Tom phenotype results from mutations in the SELF PRUNING (SP) and DWARF (D) genes [START_REF] Martí | Genetic and physiological characterization of tomato cv[END_REF]. SP belongs to the CETS family of regulatory genes encoding modulator proteins that determine the potential for continuous growth of the shoot apical meristem, while, the DWARF (D) gene encodes a P450 protein involved in brassinosteroid (BR) biosynthesis [START_REF] Bishop | The tomato Dwarf gene isolated by heterologous transposon tagging encodes the first member of a new cytochrome P450 family[END_REF][START_REF] Pnueli | Tomato SP-interacting proteins define a conserved signaling system that regulates shoot architecture and flowering[END_REF]. Regardless of the presence of mutations that cause the MT's dwarf size, it has been proven to be suitable as a standard genotype in tomato research, including the study of novel hormonal interactions [START_REF] Wang | Regulatory features underlying pollination-dependent and -independent tomato fruit set revealed by transcript and primary metabolite profiling[END_REF][START_REF] Campos | Small and remarkable: The Micro-Tom model system as a tool to discover novel hormonal functions and interactions[END_REF][START_REF] Serrani | Inhibition of auxin transport from the ovary or from the apical shoot induces parthenocarpic fruit-set in tomato mediated by gibberellins[END_REF]. Due to its

INTRODUCTION

The plant hormone ethylene is involved in many developmental processes and plays a critical role in a wide range of physiological responses, including seed germination, cell elongation, flowering, fruit ripening, organ senescence, abscission, root nodulation, programmed cell death, and response to abiotic stresses and pathogen attacks [START_REF] Johnson | THE ETHYLENE GAS SIGNAL TRANSDUCTION PATHWAY: A Molecular Perspective[END_REF][START_REF] Bleecker | Ethylene: a gaseous signal molecule in plants[END_REF][START_REF] Lin | Recent advances in ethylene research[END_REF]. Ethylene Response Factors (ERFs) are known to be the last downstream components of the ethylene transduction pathway and signal transmission cascade has been linked to the transcriptional activation of some ERF genes (Solano et al., 1998;[START_REF] Benavente | Molecular mechanisms of ethylene signaling in Arabidopsis[END_REF]. According to the currently accepted model, ethylene is perceived by specific receptors, which have been shown to activate the hormone transduction pathway through releasing the block exerted by CTR1 on EIN2 (Solano and Ecker, 1998;Ju et al., 2012). The release of EIN2 then activates EIN3/EIL1 primary transcription factors, resulting in the expression of secondary transcription factors, namely ERFs, which regulate the expression of downstream ethylene-responsive genes (Solano et al., 1998;[START_REF] Alonso | Genome-wide insertional mutagenesis of Arabidopsis thaliana[END_REF]. The receptors act as redundant negative regulators of ethylene signaling to suppress ethylene responses (Hua and Meyerowitz, 1998;[START_REF] Hall | Analysis of combinatorial loss-of-function mutants in the Arabidopsis ethylene receptors reveals that the ers1 etr1 double mutant has severe developmental defects that are EIN2 dependent[END_REF]. In the absence of the hormone, the receptor actively suppresses ethylene responses and ethylene binding removes this suppression. EIN3/EILs type of transcription factors are positive regulators of the ethylene signaling that function as trans-activating factors to trigger ethylene responses [START_REF] Chao | Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins[END_REF]Solano et al., 1998). In Arabidopsis, overexpression of EIN3 or EIL1 results in a constitutive ethylene phenotype and reduced expression of multiple LeEIL genes in the tomato results in decreased ethylene sensitivity [START_REF] Chao | Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins[END_REF]Tieman et al., 2001).

ERFs are plant specific transcription factors, belonging to the large AP2/ERF multi-gene family [START_REF] Riechmann | Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[END_REF]. Proteins encoded by this gene family have a highly conserved DNA-binding domain known as AP2 domain made of 58-59 amino acids involved in the binding to the target DNA sequences [START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF]. ERFs from different plant species have been reported to be involved in a variety of processes such as responses to biotic and abiotic stresses, metabolic pathways, fruit ripening and ethylene response [START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF][START_REF] Fits | ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[END_REF][START_REF] Li | LeERF1 positively modulated ethylene triple response on etiolated seedling, plant development and fruit ripening and softening in tomato[END_REF]Trujillo et al., 2008;[START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF]. ERF proteins are known to interact with multiple cis-acting elements found in the promoter regions of ethylene-responsive genes, including the GCC box and DRE/CRT dehydration-responsive element/C-repeat (Ohme- [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF][START_REF] Hao | Determinants in the sequence specific binding of two plant transcription factors, CBF1 and NtERF2, to the DRE and GCC motifs[END_REF][START_REF] Oñate-Sánchez | AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense[END_REF]. It was also shown that Pti4, an ERF type transcription factor, regulates gene expression by directly interacting with a non-GCC element [START_REF] Chakravarthy | The tomato transcription factor Pti4 regulates defenserelated gene expression via GCC box and non-GCC box cis elements[END_REF]. Moreover, in addition to regulating the expression of ethylene-responsive genes, ERFs can regulate jasmonic acid and salicylic acid-responsive genes [START_REF] Gu | Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase[END_REF][START_REF] Brown | A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis[END_REF]. ERFs can also bind the Vascular Wounding Responsive Element (VWRE) in tobacco [START_REF] Sasaki | Two novel AP2/ERF domain proteins interact with cis-element VWRE for woundinduced expression of the Tobacco tpoxN1 gene[END_REF] further demonstrating their capacity to bind a wide range of cis-regulatory elements beside the GCC and DRE/CRT boxes.

ERFs have been associated with ethylene-regulated growth control, with either a positive or a negative regulatory function [START_REF] Alonso | Genome-wide insertional mutagenesis of Arabidopsis thaliana[END_REF][START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF][START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF]. Strikingly, in Arabidopsis little has been reported [START_REF] Mcgrath | Repressor-and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression[END_REF] on ethylene-responsive phenotypes caused by silencing, mutation, or knockout of ERFs probably due to the high level of functional redundancy among family members. Indeed, the ERF family is composed of up to 65 members in Arabidopsis [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF], many of which are regulated by the same stimuli and can potentially bind the same target promoter. Chimeric Repressor Silencing Technology (CRES-T), consisting in the expression of a dominant repressor version of a transcription factor encoding gene proved to be an efficient mean to overcome experimental limitations caused by functional redundancy and this strategy has been developed to study the consequences of silencing target genes of single transcription factors [START_REF] Hiratsu | Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis[END_REF][START_REF] Matsui | A chimeric AtMYB23 repressor induces hairy roots, elongation of leaves and stems, and inhibition of the deposition of mucilage on seed coats in Arabidopsis[END_REF][START_REF] Heyl | The transcriptional repressor ARR1-SRDX suppresses pleiotropic cytokinin activities in Arabidopsis[END_REF]. Fusing the so-called SRDX repression domain to a transcription factor suppresses the expression of its target genes dominantly over the activity of endogenous and functionally redundant transcription factors and as a result, the transgenic plants expressing the chimeric repressor version exhibit phenotypes similar to loss-of-function of the alleles of the gene encoding the transcription factor [START_REF] Hiratsu | Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis[END_REF][START_REF] Matsui | Detection of protein-protein interactions in plants using the transrepressive activity of the EAR motif repression domain[END_REF].

Genome-wide study recently showed that the tomato ERF gene family comprises 9 subclasses defined by distinct structural features and a new nomenclature for tomato ERFs was proposed [START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF] which complies with the most complete classification available in Arabidopsis and clarifies the correspondence between ERF subclasses in different species [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF]. In the tomato, only few ERF genes have been functionally characterized so far, most of these have been shown to participate in stress and/or hormonal responses [START_REF] Gu | Tomato transcription factors pti4, pti5, and pti6 activate defense responses when expressed in Arabidopsis[END_REF][START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF][START_REF] Li | LeERF1 positively modulated ethylene triple response on etiolated seedling, plant development and fruit ripening and softening in tomato[END_REF]Zhang et al., 2009;[START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF][START_REF] Pan | An ethylene response factor (ERF5) promoting adaptation to drought and salt tolerance in tomato[END_REF]. The tomato Sl-ERF.B3 is related to

Arabidopsis factors ERF106 and ERF107, which are members of group IX according to [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF]. This group has been implicated in the regulation of defense responses and knock-out analysis of ORA59 [START_REF] Pré | The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense[END_REF]

RESULTS

Classical down-and up-regulation approaches failed to provide clear clues on Sl-ERF.B3 functional significance

To address the physiological significance of Sl-ERF.B3 and its potential role in mediating ethylene responses, tomato lines under-and over-expressing Sl-ERF.B3 gene were generated by stably transforming tomato plants with either sense or antisense constructs under the control of the constitutive 35S promoter. A number of homozygous transgenic lines corresponding to independent transformation events were obtained for both antisense and sense construct. Overall, 10 antisense and 12 sense independent lines were examined and the evidence for the expression of the transgene and for its ability to alter the levels of endogenous Sl-ERF.B3 transcripts in the transgenic lines was provided by qRT-PCR analysis (Figure S1a). No consistent phenotypes could be revealed in antisense lines whereas close examination of Sl-ERF.B3 over-expressing plants revealed slightly but significantly higher plants at early development stages (4-week-old) though the plant size returned to normal at 8-week-old plants (Figure S1b). No other consistent growth or reproductive phenotypes could be detected in these Sl-ERF.B3 over-expressing lines.

ERF.B3-SRDX suppresses the transactivation capacity of Sl-ERF.B3

In (Figure 1). These data confirm that ERF.B3-SRDX retains the capacity to bind the same target promoters than Sl-ERF.B3 and to dominantly repress its transcriptional activity.

These data support the hypothesis that the ERF.B3-SRDX chimeric protein can potentially be used as transcriptional repressor of Sl-ERF.B3 target genes in planta.

Dark-grown 35S:ERF.B3-SRDX seedlings display enhanced triple response

To gain insight on the physiological function of Sl-ERF.B3, transgenic tomato lines (Microtom cv) expressing the ERF.B3-SRDX dominant repressor construct were produced. Ten independent homozygous 35S:ERF.B3-SRDX lines were generated, all of them displayed similar pleiotropic alterations. Three representative lines, SR1, SR2 and SR3, showing a characteristic phenotype with different expressivity, were selected for further studies. The relative expression level of ERF.B3-SRDX transcript in these three lines was assessed using primers specific for ERF.B3-SRDX (Figure S2). The accumulation of the endogenous Sl-ERF.B3 assessed by qRT-PCR was similar in the transformed and non-transformed plants ruling out the eventuality of a feedback regulation of Sl-ERF.B3 in the transgenic lines (Figure S2).

Dark-grown ERF.B3-SRDX seedlings exhibited exaggerated apical hook formation and inhibited hypocotyl elongation in the absence of exogenous ethylene treatment (Figure 2a). Hypocotyl length of 7-day-old etiolated seedlings was 50% lower in ERF.B3-SRDX lines compared to wild type (Figure 2b). Interestingly, application of 1-MCP, the ethylene perception inhibitor, reversed the triple response phenotype of ERF.B3-SRDX dominant repressor lines (Figure 2a) leading to a complete loss of the exaggerated apical hook and a recovery of hypocotyl length similar to that of wild type (Figure 2a,b).

Treatment with 10 μL L -1 ethylene resulted in a more pronounced ethylene triple response in ERF.B3-SRDX lines than in wild type (Figure 2a, b), suggesting a higher sensitivity to the hormone for the transgenic lines. Because Sl-ERF.B3 over-expressing plants displayed some, though very mild, growth phenotype at early stages (4-week-old) of plant development, these lines have been tested

for the ethylene response phenotype. While the over-expressing lines cannot be discriminated from wild-type plants when dark-grown in air, upon exogenous ethylene treatment some of the transgenic lines show a slightly lower reduction in hypocotyl length than in wild type thus suggesting a reduced response to the hormone (Figure S3).

35S:ERF.B3-SRDX plants show a suite of ethylene hypersensitive phenotypes

Several developmental processes known to be regulated by ethylene were altered in the dominant repressor lines among which leaf and petiole epinasty (Figure 3). Additional ethylene-related phenotypes displayed by ERF.B3-SRDX plants included premature flower senescence and early fruit abscission (Figure 3). The majority of flowers in ERF.B3-SRDX plants undergo premature senescence and abscission before full opening of the petals (Figure 3). Moreover, the ERF.B3-SRDX fruits display early abscission compared to wild-type fruit (Figure 3). Approximately two weeks after the breaker stage, the fruit abscission zone starts to dehisce in the ERF.B3-SRDX lines, whereas this occurs at later stages in wild-type lines (Figure 3). Collectively, these ethylene-related phenotypes are consistent with an ethylene hypersensitivity of the ERF.B3-SRDX dominant repressor lines. and the size of adult plants was severely reduced (Figure 4a) with an average height being less than one third of that of wild-type plants after 80 days (Figure 4b).

Noteworthy, the transcript level of two GA oxidase biosynthetic genes, Sl-GA20ox1 and Sl-GA20ox2, was found to be significantly lower than the transgenic plants (Figure 4c). A reduced GA synthesis may therefore account for the dramatic dwarf phenotype displayed by ERF.B3-SRDX plants. Consistent with this hypothesis, application of GA 3 to 10-dayold transgenic plants partially rescued the dwarf phenotype (Figure 4d). Nevertheless, in silico analysis of the promoter region of the two GA biosynthesis genes did not reveal the presence of any canonical ethylene-response elements.

Leaf morphology is remarkably altered in the transgenic lines (Figure S4a) with a severe reduction in leaflet size, ranging from 51% to 32% in length and 47% to 22% in width (Figure S4b). The leaf margins of the ERF.B3-SRDX plants are twisted and the lamina is often wrinkled (Figure S4a). Scanning electron microscopy revealed smaller epidermal cells in the transgenic leaves (Figure S4c) with the strongest ERF.B3-SRDX expressing line showing epidermal cell size less than one third of that in wild type (Figure S4d). ERF.B3-SRDX plants also showed severely delayed reproductive growth (Figure 5a). The time from germination to flower bud setting was delayed by 14 to 20 days in transgenic lines compared to the reference wild-type lines (Figure 5b). Likewise, flower anthesis in ERF.B3-SRDX plants occurred 29 to 34 days later than in WT (Figure 5b). Moreover, compared to wild type, transgenic plants produced significantly smaller flowers (Figure S5a) with up to 30% reduction in anther length. A reduction in fruit size was also observed in the ERF.B3 dominant repressor lines which produced heart-like shaped fruit (Figure S5b) and small seeds with aberrant shape (Figure S5c). The ERF.B3-SRDX lines also displayed dramatic reduction in fruit set, leading to markedly lower fruit number per plant at maturity (Figure 5d). Up to 91% of successful fruit set was achieved in wild type while in the same growing condition, the fruit set rate reached 10-18% in the ERF.B3-SRDX lines (Figure 5d). Cross-fertilization assay was performed to examine fertility of transgenic flower. Using wild-type flowers as female recipient and ERF.B3-SRDX plants as pollen donor, 87% of successful fruit set was achieved. Notably, all the developed fruits were seeded, and when germinated, all the seeds were hygromycin resistant (Table 1) indicating that ERF.B3-SRDX pollen is viable and fertile. Using wild type as pollen donor, pollinated ERF.B3-SRDX flowers also showed 80% success of fruit set (Table 1).

The reciprocal crossing indicated that both ovule and pollen are fertile in the ERF.B3-SRDX dominant repressor lines (Table 1). Pollen viability of transgenic lines was further confirmed by Alexander's staining assay (Figure S5d). A closer examination of the flower organ structure revealed that ERF.B3-SRDX flowers display exerted stigma positioned beyond the tip of the anther cone, in contrast to wild-type flowers where the stigma is slightly inserted within the anther cone (Figure 5c). The stigma to anther length ratio is significantly higher in the transgenic lines (Figure 5e) which may consequently prevent efficient self-pollination thus resulting in poor fruit set. (e) Stigma to anther length ratio in ERF.B3-SRDX lines compared to WT.

Values are means ± SD (n ≥ 30) of three replicates. *, 0.01< P < 0.05, **, 0.001 < P < 0.01, ***, P < 0.001 (Student's test). SR1, SR2 and SR3 are three independent 35S:ERF.B3-SRDX lines. pollen and the number of fruit was assessed at the ripe stage. Conversely, tomato pollen from wild-type flowers was used to fertilize emasculated ERF.B3-SRDX flowers. In the control assay, wild-type emasculated flowers were fertilized with wild-type pollen. For each cross-fertilization assay, the capacity of the F1 seeds to grow on hygromycin-containing medium was assessed. Results are representative of data from three independent ERF.B3-SRDX lines (SR1, SR2, and SR3).

Expression of ERF.B3-SRDX leads to reduced ethylene production

To investigate the role of Sl-ERF.B3 in regulating ethylene biosynthesis, the level of ethylene production was assessed in etiolated seedlings revealing that ERF.B3-SRDX seedlings produce significantly less ethylene than wild type (Figure 6a). Accordingly the dominant repressor lines displayed reduced accumulation of transcripts corresponding to

Sl-ACS and Sl-ACO ethylene biosynthesis genes (Figure 6b) which account for the decreased ethylene production in the ERF.B3-SRDX lines. In silico analysis of the promoter regions of Sl-ACS and Sl-ACO genes using three software packages (PLACE, PlantCARE and PlantPAN) revealed the presence of cis-acting elements that can serve as putative targets for ERFs, including a GCC box (GCCGCC) and DRE/CRT (CCGAC) in Sl-ACO3 promoter and a conserved DRE/CRT (CCGAC) motif in Sl-ACS1 promoter (Table S1).

Ethylene receptor levels are down-regulated in ERF.B3-SRDX plants

In order to determine whether the expression of ethylene receptor genes may contribute to the ethylene hypersensitivity of the 35S:ERF.B3-SRDX lines, we assessed the transcript accumulation of six tomato ethylene receptor genes in the leaves of transgenic plants. While no significant change was found for the expression of Sl-ETR1 and Sl-ETR4, the four remaining ethylene receptor genes (Sl-ETR2, Sl-ETR5, Sl-ETR6 and NR)

were substantially down-regulated in the ERF.B3-SRDX lines (Figure 7a). Notably, the expression of Sl-ETR5 was decreased by 84% in the strongest ERF.B3-SRDX line (Figure 7a). The expression of Sl-ETR2 was reduced by 52-65% in three independent lines (Figure 7a) while that of NR was decreased by 46-61% (Figure 7a). The transcript levels of Sl-ETR6 showed 35-50% reduction compared to wild type (Figure 7a). In silico search revealed the absence of conserved GCC-box in the promoter regions of all four ethylene receptor genes displaying altered expression in the transgenic lines (Table S1), in contrast to NR and Sl-ETR5 promoters which contain GCC-box-like and DRE/CRT consensus sequences. However, because Sl-ETR6 receptor has been shown to play a prominent role in regulating ethylene response (Tieman et al., 2000;Kevany et al., 2007b), the ability of the native Sl-ERF.B3 and the chimeric ERF.B3-SRDX proteins to regulate the Sl-ETR6

promoter activity was tested. Transactivation assays show that Sl-ERF.B3 induced more than 2-fold increase of the Sl-ETR6 promoter activity whereas ERF.B3-SRDX strongly suppressed this activity (Figure 7b) indicating that Sl-ERF.B3 and its dominant repressor version can both regulate the expression of Sl-ETR6 in despite of the absence of a typical ethylene-responsive element in its promoter region. Given that ERF.B3-SRDX downregulates the expression of the ethylene receptor genes in vivo and that both Sl-ERF.B3

and its repressor version strongly impact the transcriptional activity of Sl-ETR6 in the transactivation assay, we then looked at the expression of ethylene receptor genes in tomato over-expressing lines. Among all six receptor genes present in the tomato genome, ETR1, NR and ETR6 are up-regulated in the Sl-ERF.B3 over-expressing lines consistent with the activator function of the Sl-ERF.B3 protein (Figure 7c). as demonstrated by its repressing activity on the promoters activated by Sl-ERF.B3

(Figure 9b). By contrast, neither Sl-ERF.B3 nor ERF.B3-SRDX proteins were able to modulate the activity of the Sl-ERF.G1 promoter. Taking advantage of the available Sl-ERF.B3 up-regulated lines we also examined the expression level of Sl-ERF genes in these over-expressing lines. Opposite to the situation prevailing in the ERF.B3-SRDX lines most ERF genes are up-regulated in the Sl-ERF.B3 over-expressing lines (Figure 9c) with the most significant up-regulation found in the lines displaying a reduced ethylene response (Figure S3). Of particular note, Sl-ERF genes (Sl-ERF.C3, Sl-ERF.D2, Sl-ERF.F5 and Sl-ERF.F4) shown to be direct target for Sl-ERF.B3 in the transactivation assay are all up-regulated in the Sl-ERF.B3 over-expressing lines. Moreover, ERF genes that show regulation by Sl-ERF.B3 in the single cell system (Figure 9b) harbor cis-acting elements (GCC-box and DRE/CRT) known to be putative binding site for ERFs whereas the Sl-ERF.G1 promoter lacks any of these typical cis-elements (Figure 9d and Table S2). The relative mRNA level of each gene in WT was standardized to 1.0, referring to Sl-Actin as internal control.

(d) The presence of putative ERF binding sites in the promoters of Sl-ERFs genes. The cis-acting elements identified are represented by black bars.

Values are means ± SD of three replicates *, 0.01 < P < 0.05, **, 0.001 < P < 0.01, ***, P < 0.001 (Student's test).

DISCUSSION

Although ERFs are generally considered as important components of the ethylene response mechanism, direct evidences for the involvement of these transcription factors in this process are still scarce. So far, classical approaches of forward and reverse genetics aiming at up-or down-regulating the expression of ERF genes failed to provide sufficient clues on the physiological significance of different members of this gene family likely owing to functional redundancy among family members. In the present study, the ectopic expression of a dominant repressor form of the Sl-ERF.B3 protein provided a mean towards altering the activity of the native Sl-ERF.B3 protein. This strategy allowed revealing vegetative and reproductive growth phenotypes that could not be uncovered by the expression of neither sense nor antisense constructs of Sl-ERF.B3. Notably, the ERF.B3-SRDX plants display enhanced ethylene responses that tend to phenocopy the Arabidopsis ctr1 mutant as well as the transgenic tomato lines deficient in receptors, exhibiting all of the hallmarks of exposure to ethylene (Kieber et al., 1993;Tieman et al., 2000). Although, the opposite effect would have been intuitively expected from blocking the action of an ERF, the physiological and molecular characterization clearly indicated that the phenotypes are consistent with enhanced ethylene sensitivity due to depletion of ethylene receptor pools but not to ethylene over-production.

The 35S:ERF.B3-SRDX lines displayed enhanced ethylene responses and pleiotropic ethylene-related alterations, likely resulting from the transcriptional repression of ethylene-responsive genes that are natural targets of the native protein. Indeed, Sl-ERF.B3 and ERF.B3-SRDX are shown to modulate the activity of the same promoters harboring ethylene-responsive elements, indicating that ERF.B3-SRDX has the ability to interfere with the regulation of Sl-ERF.B3 target genes. ERF.B3-SRDX fusion protein is a strong repressor of both synthetic and native ethylene-responsive promoters whereas the native Sl-ERF.B3 protein enhances the activity of these promoters. The eventuality that the pleiotropic phenotypes displayed by the ERF.B3-SRDX dominant suppressor plants may arise from a co-suppression of the endogenous Sl-ERF.B3 is ruled out since the levels of Sl-ERF.B3 transcripts are not altered in the transgenic lines. Notably, the higher the ERF.B3-SRDX transgene expression the more severe was the phenotypic abnormality, indicating that the phenotypic effects were directly related to the expression levels of the ERF.B3-SRDX transgene. Therefore, the ERF.B3-SRDX tomato lines proved to be a valuable tool to uncover at least some of the processes controlled by Sl-ERF.B3

and to reveal roles for ERF genes that have not been described previously.

Dark-grown ERF.B3-SRDX seedlings displayed a constitutive ethylene response-like phenotype with inhibited hypocotyl elongation and exaggerated apical hook formation in the absence of exogenous ethylene. Moreover, adult plants show typical constitutive ethylene responses including leaf epinasty, premature flower senescence and accelerated fruit abscission. These phenotypes may arise from: (i) a constitutive ethylene response, (ii) an increased sensitivity to endogenous ethylene, or (iii) an ethylene overproduction.

Noteworthy, the ethylene response phenotypes displayed by ERF.B3-SRDX etiolated seedlings can be reversed by the inhibition of ethylene perception (Figure 2a) and treatment with exogenous ethylene resulted in a more pronounced ethylene triple response compared to wild type. Taken together with the reduced ethylene production, these results indicate that the ethylene response phenotypes displayed by ERF.B3-SRDX lines are not due to constitutive activation of ethylene signaling pathway but rather to enhanced ethylene sensitivity. It is well accepted that ethylene receptors act as negative regulators and function redundantly in ethylene signaling with a decreased expression of ethylene receptor genes resulting in increased sensitivity to the hormone (Hua and Meyerowitz, 1998;Kevany and Klee, 2007). The reduced transcript levels of the receptors and the ethylene hypersensitivity of ERF.B3-SRDX lines are consistent with this model. In tomato, although gene-specific antisense reductions in Sl-ETR1, Sl-ETR2, NR or Sl-ETR5 do not affect ethylene sensitivity, transgenic lines with single reduction in Sl-ETR4 or Sl-ETR6 expression display phenotypes consistent with enhanced ethylene response (Tieman et al., 2000;Kevany et al., 2007) indicating these two receptors may act as a special component in regulating ethylene response. The down-regulation of Sl-ETR6 in the ERF.B3-SRDX lines may therefore account for the increased ethylene sensitivity. Interestingly, opposite to its down-regulation in the dominant repressor lines, ETR6 shows a net up-regulation in the Sl-ERF.B3 over-expressing plants suggesting that this receptor gene may represent a direct target for Sl-ERF.B3 protein in vivo.

The increased expression of transcription factors belonging to the EIN3 gene family may also contribute to enhanced ethylene responses. Over-expression of EIN3 or EIL1confers constitutive ethylene phenotypes in Arabidopsis, while reduced Sl-EILs expression in transgenic tomato decreases ethylene sensitivity [START_REF] Chao | Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins[END_REF]Tieman et al., 2001).

Four EIN3-like genes were isolated in tomato and designed as Sl-EIL1, Sl-EIL2, Sl-EIL3

and Sl-EIL4 (Tieman et al., 2001;[START_REF] Yokotani | Characterization of a novel tomato EIN3-like gene (LeEIL4)[END_REF]. Since it is well documented that EIN3/EIL proteins act as transactivation factors to trigger ethylene responses, upregulation of all four Sl-EIL genes in the ERF.B3-SRDX plants may contribute to their ethylene hypersensitivity. However, because the promoter of EIN-like genes are devoid of consensus ethylene-response elements and since transactivation assays indicated that Sl-ERF.B3 and ERF.B3-SRDX proteins are unable to modulate transcription driven by any of the four Sl-EILs promoters, it is likely that the up-regulation of Sl-EIL genes in the dominant repressor lines is due to intermediate factor(s) whose expression/activation is regulated by ERF.B3-SRDX.

Previous studies have already shown that ERF proteins are involved in a feedback regulation of ethylene production by modulating the expression of ethylene biosynthesis genes (Zhang et al., 2009;[START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF]. Our data show that ectopic expression of the ERF.B3-SRDX dominant repressor results in reduced ethylene production associated with the down-regulation of ACS and ACO ethylene biosynthesis genes. The presence of conserved GCC box and DRE/CRT motifs in ACS and ACO promoters that can serve as binding sites for ERF proteins supports the hypothesis that these ethylene biosynthesis genes can directly be regulated by Sl-ERF.B3. Together, the reduced ethylene production and enhanced ethylene sensitivity in the ERF.B3-SRDX lines suggest the presence of a feedback loop regulating both ethylene biosynthesis and signal transduction pathway and involving ERF proteins.

Strikingly, the expression of a considerable number of Sl-ERF genes, 15 out of 19 monitored in our study, was found to be markedly altered in ERF.B3-SRDX tomato lines suggesting intense inter-regulation among ERF family members. Consistent with the dominant repressor function of the ERF.B3-SRDX protein, most of the ERF genes were down-regulated while solely Sl-ERF.G1 displayed higher transcript levels in the dominant repressor lines. By contrast, in Sl-ERF.B3 over-expressing lines, most ERF genes tested displayed enhanced transcript levels. In particular, Sl-ERF.C3, Sl-ERF.D2, Sl-ERF.F5 and Sl-ERF.F4, shown to be direct target for Sl-ERF.B3 in the transactivation assay, display enhanced expression in the Sl-ERF.B3 sense lines. While these data support the idea that these ERFs can serve as direct target for both the native and chimeric Sl-ERF.B3 proteins, the up-regulation of Sl-ERF.G1 in the dominant repressor lines likely requires an additional mediating factor. In silico search revealed that all ERF genes down-regulated in the transgenic lines harbor cis-acting elements known to be putative binding targets for ERFs. The down-regulation of such a high number of Sl-ERFs supports a model implying that a single ERF can impact the expression of other members of the gene family. This inter-connected regulation among ERF genes may therefore account for the pleiotropic alterations in the ERF.B3-SRDX lines and for the diversity of responses displayed by the dominant repressor lines.

Phenotypes such as stunted plant development, reduced leaf size and late flowering time are reminiscent not only of constitutive ethylene-response mutants but also of GA deficient Arabidopsis plants ( Kieber et al., 1993;Hua and Meyerowitz, 1998;[START_REF] Hall | Analysis of combinatorial loss-of-function mutants in the Arabidopsis ethylene receptors reveals that the ers1 etr1 double mutant has severe developmental defects that are EIN2 dependent[END_REF][START_REF] Magome | dwarf and delayed-flowering 1, a novel Arabidopsis mutant deficient in gibberellin biosynthesis because of overexpression of a putative AP2 transcription factor[END_REF][START_REF] Qu | A strong constitutive ethyleneresponse phenotype conferred on Arabidopsis plants containing null mutations in the ethylene receptors ETR1 and ERS1[END_REF]. The partial rescue of the dwarf phenotype in the ERF.B3-SRDX lines by exogenous application of GA suggests that these alterations are partly due to GA deficiency. In line with the model supporting that ethylene regulates plant growth and floral organ differentiation via modulating GA levels [START_REF] Achard | The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristemidentity genes[END_REF], ethylene hypersensitivity in the ERF.B3-SRDX dominant suppressor lines is associated with reduced plant size and substantially delayed flowering time. The reduced expression of GA oxidase genes in the transgenic lines sustains the idea of altered GA metabolism and suggests that ERFs may represent a potential molecular link between ethylene and GA. In agreement with this, it has been recently

reported that transcriptional activation of some genes involved in GA metabolism is mediated by ERF6 in Arabidopsis leaves [START_REF] Dubois | Ethylene Response Factor6 acts as a central regulator of leaf growth under water-limiting conditions in Arabidopsis[END_REF]. Because the study has been carried out with Micro-Tom, a dwarf genotype, it is important to mention that the dwarfing mutations in this genotype do not seem to impact the phenotype displayed by ERF.B3-SRDX plants since the dwarf phenotype is well reproduced in Ailsa Craig tomato, a non-dwarf variety (data not shown). Altogether, the data suggest that ethylene hypersensitivity is likely to be the fundamental cause of the severe dwarf and lateflowering phenotypes in the ERF.B3-SRDX plants.

Since ectopic expression of transcription factors might influence target genes that are normally not under the control of this regulator, it cannot be totally ruled out that at least part of gene regulations caused by ERF.B3-SRDX are off-target effects due to interference with other related transcription factors. However, the data support the idea that Sl-ERF.B3 is part of an intricate web of regulation in which multiple transcription factors are competing for promoters to control the expression of genes that are essential for a wide range of plant responses to ethylene. As depicted in the tentative regulation model presented in Figure 10, Sl-ERF.B3 is shown to modulate ethylene responses at four different levels: (i) ethylene biosynthesis, (ii) ethylene receptor, (iii) primary ethylene transcription factors (EIL genes), and (iv) downstream ERF genes. The high number of ERF genes regulated by Sl-ERF.B3 is consistent with the pleiotropic phenotypes displayed by the dominant repressor lines and suggests that ERFs form a complex network with a subset of the family members functioning in an interconnected manner. Such level of complexity matches the high level of plasticity needed for the implementation of plant growth and developmental processes which require continuous fine-tuning through the integration of different cues and signaling pathways. This scheme is validated by transactivation assays showing direct regulation of the target ERFs and ETR6

genes by the native form of Sl-ERF.B3 protein and by the enhanced transcript levels of these target genes in the Sl-ERF.B3 over-expressing lines.

protoplasts transformed with the reporter vector in combination with the vector used as effector but lacking the Sl-ERF.B3 coding sequence.

RNA isolation and qRT-PCR

Total RNA from 4-week-old plants was extracted using a Plant RNA Purification Reagent (Invitrogen, Cat. No. 12322-012). Total RNA was DNase-treated (Invitrogen, Cat. No. AM1906) and first-strand cDNA was reverse transcribed from 2 μg of total RNA using an Omniscript Reverse Transcription kit (Qiagen, Cat. No. 74904). Genespecific primers were designed by Primer Express software (PE-Applied Biosystems) and were further checked using BLAST against all tomato unigenes (Tomato unigene database). qRT-PCR analyses were performed as described previously [START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF]. The primer sequences used in this study are listed in Table S3.

Gibberellin treatment

For application of gibberellin to young plants growing on soil, 10 -5 M of Gibberellic Acid (GA 3 ) was sprayed twice a week starting on the 10th day post-germination. After 2 weeks of treatment, the treated plants were compared with the control ones.

Triple-response assay

Sterilized seeds were first put on MS/2 medium plates and placed at 4°C for 3 days and then transferred to 25°C for germination in the dark for another 5 days. The seedling triple response was scored by assessing hypocotyl length and apical curvature. At least 50 seedlings were scored for each measurement. For ethylene treatment, Petri dishes were enclosed in wide mouth Mason jars sealed with a lid containing a rubber syringe cap.

Ethylene (10 μL L -1 ) was then injected into the Mason jars using a syringe. For 1-MCP treatment, 1 μL L -1 was applied into the Mason jars and kept in the dark for one week. At least 50 seedlings were used for each experiment and three independent biological replicates were performed.

Ethylene production

Ethylene production was assayed on 7 day-old dark-grown seedlings for 12 h by withdrawing 1-mL gas samples from sealed jars. Gas samples were analyzed via gas chromatography (7820A GC system Agilent Technologies). Ethylene was identified via co-migration with an ethylene standard and quantified with reference to a standard curve for ethylene concentration. S3. List of the primers used in the study.

Gene Name Primer Sequence

Sl-Actin

INTRODUCTION

The maturation and ripening of fleshy fruits is a developmental process unique to plants.

Although specific fruit-ripening characteristics vary among species, fruit ripening can be generally described as a complex, genetically programmed process that culminates in dramatic changes in color, texture, flavor, aroma and nutritional characteristics [START_REF] Carrari | Metabolic regulation underlying tomato fruit development[END_REF]. In the case of fleshy fruits these changes not only make fruit attractive for seed dispersal organisms, but also provide essential vitamins, minerals and antioxidants (phenolics, folate, lycopene, and β-carotene) for human diet [START_REF] Seymour | Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct[END_REF][START_REF] Fraser | Genetic engineering of carotenoid formation in tomato fruit and the potential application of systems and synthetic biology approaches[END_REF][START_REF] Chung | A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening[END_REF].

Fruits have been classically categorized into climacteric and non-climacteric based on increased ethylene synthesis and a concomitant rise in the rate of respiration during ripening. Climacteric fruits display a burst in respiration at the onset of ripening, in contrast to non-climacteric ones. Climacteric fruits, such as tomatoes, bananas and apples, also show increased biosynthesis of the gaseous hormone ethylene, which is a fundamental signal in climacteric fruit ripening [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF][START_REF] Alba | Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development[END_REF].

In the ethylene biosynthetic pathway, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) catalyze the conversion of S-adenosyl-Lmethionine (SAM) to ACC and of ACC to ethylene, respectively [START_REF] Adams | Ethylene biosynthesis: Identification of 1aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene[END_REF][START_REF] Bleecker | Ethylene: a gaseous signal molecule in plants[END_REF]. Autocatalytic ethylene synthesis at the onset of tomato fruit ripening is mainly mediated through ethylene-stimulated expression of ACS2, ACS4, ACO1 and ACO4 [START_REF] Barry | Ethylene and fruit ripening[END_REF]. Unraveling the regulation of the ethylene signaling pathway is important to understanding the processes of fruit ripening.

Tomato possesses many favorable genetic characteristics such as simple diploid genetics, relatively small genome size, short generation time, efficient genetic transformation and distinct ripening phenotypes, making it a primary model system for studying the molecular basis of fleshy fruit development and the role of ethylene in climacteric fruit ripening. The adaptation of a range of technological tools and the generation of new biological resources on the tomato (e.g. EST database, TILLING resources, genetic and physical maps) have led to a large progress in our understanding of the molecular mechanisms underlying the ripening process through the identification of the associated key regulatory genes (Pirrello et al., 2009). Moreover, the ripening process of tomato has been well characterized in terms of metabolic changes impacting softening, accumulation of sugars and acids, chlorophyll degradation, lycopene accumulation, and dramatic increases in ethylene and flavor volatiles [START_REF] Chung | A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening[END_REF][START_REF] Karlova | Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening[END_REF].

Through investigation of tomato mutants affected in fruit development and ripening (mainly ripening-deficient mutants), such as ripening inhibitor (rin; [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF], Colorless non-ripening (Cnr; [START_REF] Manning | A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening[END_REF], non-ripening (nor; [START_REF] Giovannoni | Genetic regulation of fruit development and ripening[END_REF], Green-ripe (Gr; [START_REF] Barry | Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling[END_REF], and Never-ripe (Nr; [START_REF] Wilkinson | An ethylene-inducible component of signal transduction encoded by never-ripe[END_REF], many genes were isolated and shown to act upstream of ethylene in the ripening cascade, determining the competence of the fruit to ripen [START_REF] Barry | Ethylene and fruit ripening[END_REF]. The RIN, CNR, and NOR genes encode transcriptional regulators regulating the expression of other genes responsible for fruit ripening processes, including ethylene and carotenoid biosynthesis [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF][START_REF] Giovannoni | Genetic regulation of fruit development and ripening[END_REF][START_REF] Manning | A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening[END_REF][START_REF] Martel | The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENINGdependent manner[END_REF][START_REF] Fujisawa | A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening[END_REF]. The Gr gene encodes a still poorly defined component of ethylene signal transduction, while Nr encodes an ethylene receptor important for both fruit and non-fruit ethylene responses (Lanahan et al., 1994;[START_REF] Barry | Ripening in the tomato Green-ripe mutant is inhibited by ectopic expression of a protein that disrupts ethylene signaling[END_REF]. Other ripening transcriptional regulators have recently been characterized via functional studies in transgenic plants, including LeHB1, which directly regulates ACC oxidase expression [START_REF] Lin | SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development[END_REF] and TAGL1, a MADS box transcription factor, which links early fruit fleshy expansion with downstream ripening [START_REF] Itkin | TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network[END_REF][START_REF] Vrebalov | Fleshy fruit expansion and ripening are regulated by the Tomato SHATTERPROOF gene TAGL1[END_REF]. The putative transcription factor, Sl-AP2a, a member of the AP2/ERF superfamily was also recently described as a negative regulator of fruit ripening and of ethylene production [START_REF] Chung | A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening[END_REF][START_REF] Karlova | Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening[END_REF]. Unraveling the transcriptional networks that regulate fruit ripening is crucial for the understanding of this complex process. The present study describes the critical roles of Sl-ERF.B3 in fruit ripening, a member of Sl-ERF multi-family genes.

ERFs are plant specific transcription factors, belonging to the large AP2/ERF superfamily [START_REF] Riechmann | Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[END_REF]. Proteins encoded by this family have a highly conserved DNAbinding domain known as the AP2 domain, containing 58-59 amino acids involved in the high affinity binding to target DNA sequences [START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF]. A growing number of investigations suggest that through interacting with multiple cis-acting elements found in 106 the promoter regions of ethylene-responsive genes, including the GCC box and dehydration-responsive element/C-repeat (DRE/CRT), ERF proteins play a critical role during plant development and adaptation to stress conditions [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF]Wu et al., 2002;[START_REF] Wan | Transcriptional activation of OsDERF1 in OsERF3 and OsAP2-39 negatively modulates ethylene synthesis and drought tolerance in rice[END_REF]. Generally, in different plant species ERFs have been shown to be involved in hormonal signaling, responses to biotic and abiotic stresses, developmental processes, metabolic regulation, ethylene biosynthesis and fruit ripening [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF][START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF][START_REF] Fits | ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[END_REF]Wu et al., 2002;Dubouzet et al., 2003;Zhang et al., 2009;[START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF].

Although Sl-ERF6 was reported to play an important role in fruit ripening by integrating ethylene and carotenoid pathways in tomato [START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF], the role of most ERF proteins in the ripening process awaits elucidation.

To date, no ERF-like mutants have been identified in tomato. As we described in our previous article [START_REF] Liu | A dominant repressor version of the tomato Sl-ERF.B3 gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components[END_REF], classical reverse genetics approach based on down-and up-regulation of ERF.B3 failed to provide a clue regarding its functional significance. In an attempt to overcome the experimental limitations due to the functional redundancy among members of the ERF gene family, we generated a dominant repressor version of ERF.B3 (ERF.B3-SRDX) using the Chimeric Repressor Silencing Technology (CRES-T).

Through the CRES-T strategy, we show here that ERF.B3 plays a critical role in fruit ripening. This gene was previously described as an important regulator of ethylene response and plant development [START_REF] Liu | A dominant repressor version of the tomato Sl-ERF.B3 gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components[END_REF]. We found here that Sl-ERF.B3 also

Sl-ERF.B3 shows fruit development-and ripening-related expression pattern

The Sl-ERF.B3 transcript is induced at the breaker stage and maintained a high levels at all later stages of ripening, suggesting that its expression might be continuously required along the ripening process. This observation motivated the further assessment of Sl-ERF.B3 transcript accumulation in vegetative and reproductive tissues by quantitative RT-PCR (Figure 2A). Analysis of stem, root, leaf, flower and in a series of fruit developmental stages, indicated that the accumulation of Sl-ERF.B3 transcripts is relatively high in both vegetative and reproductive tissues (Figure 2A).

To address the functional significance of this tomato ERF family member, we first attempted to alter its expression using antisense or overexpression strategies. However, both approaches failed to provide significant clues on the physiological role of Sl-ERF.B3, which prompted the use of a dominant repressor version of the gene (ERF.B3-SRDX), relying on the so-called Chimeric Repressor Silencing Technology (CRES-T).

This technology has been developed to study the consequences of silencing of the target genes of single transcription factors and has also been used to overcome the experimental limitations caused by functional redundancy of transcription factor families [START_REF] Hiratsu | Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis[END_REF]. Three out of ten transgenic ERF.B3-SRDX lines (SR1, SR2 and SR3) showed a characteristic phenotype with different expressivity and were selected for further studies.

The relative expression levels of the ERF.B3-SRDX transcript in fruit tissues of the three independent lines was assessed by qRT-PCR using primers specific for the transgene (Figure 2B).

Altered fruit development in ERF.B3-SRDX dominant repressor lines

One of the most evident phenotypes displayed by ERF.B3-SRDX transgenic lines is the altered fruit shape and reduced size (Figure 3A). Wild-type tomato fruits (Figure 3A) are round shaped in contrast to the ERF.B3-SRDX fruits which heart shaped with bumpy areas present intermittently on the surface of the fruit (Figure 3A). Changes in fruit anatomy also include thicker pericarp and decreased jelly formation with enhanced pericarp to total fruit volume ratio (Figure 3A, lower panel). As a consequence of the smaller size, the mean weight of ERF.B3-SRDX fruits is significantly reduced (Figure 3B). The number of seeds is dramatically reduced in ERF.B3-SRDX fruits compared to wild type and the average seed number drops from 25 per fruit in wild-type to 6 in ERF.B3-SRDX lines (Figure S1A). In addition to their reduced number, the seeds show reduced size in dominant repressor lines (Figure S1B). In the ERF.B3-SRDX line showing Moreover, once the ripening process starts at breaker stage, when a visible color change just begins to occur, the ripening of ERF.B3-SRDX fruits seems much slower than wild type (Figure 4A). In contrast to wild type which reaches the red-ripe stage 5 days postbreaker (Br+5), ERF.B3-SRDX fruits remain orange at breaker+10 stage (Br+10; Figure 4A). The assessment of color change via measuring the evolution of hue angle values, indicative of color saturation, further emphasized the difference between wild type and dominant repressor lines throughout the ripening process (Figure 4B). The value of hue angle is even higher for ERF.B3-SRDX fruit at Br+10 than for wild type at Br+5, thus confirming the orange-ripe phenotype observed visually (Figure 4A, 4B). though transcript of phytoene synthase (PSY1) in ERF.B3-SRDX lines showed similar ripening-regulated accumulation pattern than in wild type, PSY1 levels were dramatically reduced in ERF.B3-SRDX fruits at the breaker stages (Figure 6). PSY1 is a key regulator of flux through the carotenoid pathway and its repression is consistent with the reduction of lycopene and total carotenoids at the breaker stage fruits (Figure 5). A decrease in phytoene desaturase (PDS) expression levels was also observed in ERF.B3-SRDX fruits 114

ERF.B3-SRDX fruits show fast softening and elevated ethylene production

To uncover whether the failure to reach the red-ripe stage in dominant repressor fruits results from the incapacity to ripen, other major ripening-associated features, like softening and climacteric rise of ethylene production, were investigated. The evolution of firmness determined from breaker stage to 20 days post-breaker (Br+20) showed that ERF.B3-SRDX transgenic fruits undergo significantly faster softening than control fruit (Figure 7A). Given that fruit softening is highly regulated by ethylene, we therefore assessed the production of ripening-associated ethylene in the ERF.B3-SRDX fruits. As shown in Figure 7B, the accelerated softening observed in transgenic fruits was associated with a dramatic increase in climacteric ethylene production (Figure 7B) which reached a maximum 3 to 4 times higher than in wild type fruit. Altogether these data indicate that once triggered, the ripening process is accelerated in the ERF.B3-SRDX repressor lines. The expression of key regulatory genes of the ripening process like RIN, NOR and CNR was increased at post-breaker stages compared to wild type, even though their induction took place later than in control fruit (Figure 9). The altered expression pattern of these genes in the ERF.B3-SRDX fruits is consistent with the dramatically delayed attainment of competence to ripen in transgenic fruits. Moreover, the mRNA levels of LeHB-1, another ripening regulator gene, were higher in ERF.B3-SRDX lines at all ripening stages (Figure 9). By contrast, the expression of TAGL1, a tomato SHATTERPROOF gene, and

AP2a, an AP2/ERF family gene acting as a negative regulator of fruit ripening did not display significant changes in ERF.B3-SRDX dominant repressor fruits compared to wild type (Figure 9). signaling and ERF genes [START_REF] Liu | A dominant repressor version of the tomato Sl-ERF.B3 gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components[END_REF]. Here we show that dominant repression of the Sl-ERF.B3 transcription factor in tomato also broadly impacts fruit development and ripening. Indeed, ERF genes have been shown to be involved in fruit ripening in tomato [START_REF] Li | LeERF1 positively modulated ethylene triple response on etiolated seedling, plant development and fruit ripening and softening in tomato[END_REF][START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF] the breaker stage and the ERF.B3-SRDX fruits retained its orange color, failing to turn red as in wild type at the final ripening stage. Data from the time course for fruit firmness also showed an early fruit softening phenotype in the ERF.B3-SRDX lines. Moreover, ripening fruit anatomy showed that ERF.B3-SRDX fruits have a thicker pericarp, a smaller volume of jelly, a dry and crumbly appearance of the pericarp, suggesting a defect in the expansion or elasticity of the epidermis. Since fruit ripening is a complex process with dramatic changes in color, texture, flavor, and aroma of the fruit flesh [START_REF] Seymour | Down-regulation of two non-homologous endogenous tomato genes with a single chimaeric sense gene construct[END_REF][START_REF] Alexander | Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening[END_REF][START_REF] Carrari | Metabolic regulation underlying tomato fruit development[END_REF], the changes in the ERF.B3-SRDX lines related to fruit ripening indicate the involvement of Sl-ERF.B3

gene in regulating fruit ripening in tomato.

The dramatic development of red pigmentation of ripening fruits is one of the most notable features of tomato and the principal carotenoids that accumulate in ripening tomato are lycopene and β-carotene, which confer the red and orange colors to ripe fruits, respectively [START_REF] Fraser | Carotenoid Biosynthesis during Tomato Fruit Development (Evidence for Tissue-Specific Gene Expression)[END_REF]Burns et al., 2003;[START_REF] Alba | Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development[END_REF]. Changes in carotenoid accumulation have been demonstrated to correspond with alterations in expression of genes encoding the carotenoid pathway enzymes (Giuliano et al., 1993;[START_REF] Ronen | An alternative pathway to beta -carotene formation in plant chromoplasts discovered by mapbased cloning of beta and old-gold color mutations in tomato[END_REF][START_REF] Galpaz | A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus[END_REF][START_REF] Chung | A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening[END_REF][START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF]Luo et al., 2013). Dominant repression of Sl-ERF.B3 in tomato resulted in orange fruit color with decreased levels of lycopene and elevated accumulation of β-carotene. This suggests a role of Sl-ERF.B3 in regulating carotenoid biosynthesis in tomato. The altered carotenoid levels in ERF.B3-SRDX lines are tightly correlated with the altered mRNA accumulation of the carotenoid biosynthesis genes. The transcript accumulation of PSY1 which acts as a major regulator of metabolic flux toward downstream carotenoids during fruit maturation [START_REF] Fray | Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression[END_REF], was markedly reduced and its repression is consistent with the substantial reduction in total carotenoid levels at the breaker stage (due to a precipitous decline in lycopene) observed in the ERF.B3-SRDX dominant repressor lines. Moreover, PDS, another carotenoid biosynthesis gene which encodes phytoene desaturase catalyzing the conversion of phytoene to ζ-carotene upstream of lycopene synthesis [START_REF] Pecker | A single polypeptide catalyzing the conversion of phytoene to zeta-carotene is transcriptionally regulated during tomato fruit ripening[END_REF], was also down regulated in the ERF.B3-SRDX fruits during ripening and thus may also contribute to the decreased carotenoid levels. In addition, a significant induction of all three lycopene beta cyclases (β-LCY1, β-LCY2, CYC-β) genes was observed in ERF.B3-SRDX lines, most probably accounting for the elevated levels of β-carotene. In line with this hypothesis, overexpression of β-LCY and CYC-β has been previously shown to cause β-carotene accumulation in fruits (Rosati et al., 2000;[START_REF] Ronen | An alternative pathway to beta -carotene formation in plant chromoplasts discovered by mapbased cloning of beta and old-gold color mutations in tomato[END_REF]. In addition, positive correlations between β-LCY and CYC-β expression and β-carotene levels were revealed by correlation analysis of fruit metabolome and transcriptome data from S. pennellii x S. lycopersicum introgression lines [START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF]. Ethylene, light and some transcription factors are known putative regulators of carotenoid accumulation (Mustilli et al., 1999;[START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF][START_REF] Vrebalov | Fleshy fruit expansion and ripening are regulated by the Tomato SHATTERPROOF gene TAGL1[END_REF][START_REF] Giovannoni | Genetic regulation of fruit development and ripening[END_REF][START_REF] Liu | Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato[END_REF][START_REF] Alba | Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development[END_REF]. It is known that ethylene regulates carotenoid accumulation during fruit ripening by regulating the expression of carotenoid biosynthesis genes controlling final carotenoid profiles such as PSY1, β-LCY and CYC-β [START_REF] Fraser | Carotenoid Biosynthesis during Tomato Fruit Development (Evidence for Tissue-Specific Gene Expression)[END_REF][START_REF] Ronen | An alternative pathway to beta -carotene formation in plant chromoplasts discovered by mapbased cloning of beta and old-gold color mutations in tomato[END_REF][START_REF] Alba | Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development[END_REF]. It is noteworthy that dominant repression of Sl-ERF.B3 in tomato resulted in decreased total carotenoid levels and elevated ethylene production.

This phenotype at least partially resembles that of the SlAP2a repressed lines, in which significantly elevated ethylene levels are associated with altered total carotenoids and a shift to β-carotene rather than lycopene [START_REF] Chung | A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening[END_REF][START_REF] Karlova | Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening[END_REF].

Moreover, the phenotype also recalls that of the tomato never ripe mutant, in which lycopene biosynthesis and PDS gene expression are repressed and ethylene production is increased [START_REF] Alba | Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development[END_REF]. Furthermore, assessing the relative mRNA accumulation of SlAP2a failed to show significant difference in its expression levels in ERF.B3-SRDX fruits compared to wild type, suggesting that the regulation of dominant repression of Sl-ERF.B3 in fruit ripening is likely to be independent of SlAP2a. It is possible that dominant repression of Sl-ERF.B3 in tomato results in complex alterations in carotenoid accumulation network and impacts carotenoid biosynthesis genes through mechanisms beyond the influence of ethylene. Transcription factors impacting carotenoid accumulation in tomato include RIN [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF], CNR [START_REF] Manning | A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening[END_REF], TAGL1 [START_REF] Vrebalov | Fleshy fruit expansion and ripening are regulated by the Tomato SHATTERPROOF gene TAGL1[END_REF], SlAP2a [START_REF] Chung | A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening[END_REF][START_REF] Karlova | Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening[END_REF], SlERF6 [START_REF] Lee | Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening and carotenoid accumulation[END_REF] and SlMADS1 (Dong et al., 2013). The expression data during fruit ripening revealed altered expression of RIN, CNR and SlERF6 (Sl-ERF.E4) genes in ERF.B3-SRDX lines compared with wild-type controls, suggesting that these transcription factors may be involved in the regulation networks of carotenoid accumulation in the ERF.B3-SRDX lines. Interestingly, an Arabidopsis ERF transcription factor, RAP2.2, via binding to the ATCTA cis-element in the promoter regions of PSY and PDS, regulates the expression of carotenoid biosynthesis genes [START_REF] Welsch | Transcription factor RAP2.2 and its interacting partner SINAT2: stable elements in the carotenogenesis of Arabidopsis leaves[END_REF].

Given that the Sl-ERF.B3 fused to the SRDX motif strongly suppresses the expression of the putative target genes [START_REF] Liu | A dominant repressor version of the tomato Sl-ERF.B3 gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components[END_REF] together with the presence of the ATCTA motif in the promoter regions of both tomato PSY1 and PDS genes, it is possible that ERF.B3-SRDX represses the expression of PSY1 and PDS in ERF.B3-SRDX lines by directly binding to their promoters.

A hallmark of climacteric fruit ripening such as tomato is the dramatically induced respiration and ethylene production at the onset of ripening. Dominant repression of Sl-ERF.B3 in tomato resulted in substantially elevated levels of ethylene production (Figure .   7B), indicating that altered fruit ripening in ERF.B3-SRDX dominant repressor lines was at least partly through influencing ethylene synthesis. ERF.B3-SRDX fruits produced up to four-fold more ethylene than the wild-type fruits during ripening with correspondingly elevated transcript accumulation of ethylene biosynthesis genes, including ACS2, ACS4

and ACO1 (Figure. 8). Ethylene biosynthesis is tightly controlled by ACS and ACO multigene families during fruit development and ripening [START_REF] Nakatsuka | Differential expression and internal feedback regulation of 1aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening[END_REF][START_REF] Barry | The regulation of 1aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[END_REF][START_REF] Barry | Ethylene and fruit ripening[END_REF]. Based on the level of ethylene production during fruit development, two systems of ethylene regulation have been proposed [START_REF] Mcmurchie | Treatment of fruit with propylene gives information about the biogenesis of ethylene[END_REF]. System 1 represents the basal level of ethylene in immature fruit and vegetative tissues, whereas system 2 represents a high level of ethylene production associated with fruit ripening (Oetiker & Yang, 1995). Tomato ACS1 and ACS6 have been shown to mediate the system 1 ethylene production in immature fruit in tomato, and the autocatalytic ethylene biosynthesis in system 2 is mediated through ethylene-stimulated expression of ACS2, ACS4, ACO1 and ACO4 genes [START_REF] Nakatsuka | Differential expression and internal feedback regulation of 1aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening[END_REF][START_REF] Barry | The regulation of 1aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[END_REF][START_REF] Barry | Ethylene and fruit ripening[END_REF]. The mRNA accumulation of ACS2 is induced at the onset of ripening and this induction is ethylene-dependent but is independent of rin [START_REF] Nakatsuka | Differential expression and internal feedback regulation of 1aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening[END_REF][START_REF] Barry | The regulation of 1aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[END_REF]. Moreover, repression of the ACS2 gene could block fruit ripening in tomato [START_REF] Oeller | Reversible inhibition of tomato fruit senescence by antisense RNA[END_REF]. ACS4 is also induced during ripening in a rin-dependent fashion. Indeed, the tomato ACS2 and ACS4

are the predominant ACS mRNAs in ripening fruit and both genes are under additional developmental controls [START_REF] Barry | The regulation of 1aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[END_REF]Yokotani et al., 2004). The transcript of ACO1 increases at the onset of ripening and is sustained in high expression during tomato fruit ripening [START_REF] Nakatsuka | Differential expression and internal feedback regulation of 1aminocyclopropane-1-carboxylate synthase, 1-aminocyclopropane-1-carboxylate oxidase, and ethylene receptor genes in tomato fruit during development and ripening[END_REF], suggesting a role of this gene in controlling ethylene synthesis in fruit ripening. Since ACS and ACO catalyze the rate limiting and final steps in ethylene biosynthesis, the significantly elevated mRNA levels of ACS2, ACS4 and ACO1 are probably responsible for the elevated ethylene levels in the ERF.B3-SRDX dominant repressor lines. Dominant repression of Sl-ERF.B3 results in high levels of ethylene, suggesting that regulators (either directly or indirectly regulated by ERF.B3-SRDX) for regulation of these key ethylene synthesis genes may lie upstream of ethylene synthesis control and remain to be discovered. It is noteworthy that dominant repression of Sl-ERF.B3 leads to reduced ethylene production in dark-grown seedlings with reduced mRNA accumulation of ACS1A and ACO1 genes [START_REF] Liu | A dominant repressor version of the tomato Sl-ERF.B3 gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components[END_REF] indicating that the mechanisms of controlling ethylene production in ERF.B3-SRDX lines by the regulation of ethylene biosynthesis genes in vegetative tissue and ripening fruits are distinct.

Fruit ripening is a complex developmental process and is genetically controlled by intricate transcriptional cascades through ethylene-and non-ethylene-mediated regulation.

The MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene-and non-ethylene-mediated controls in tomato [START_REF] Vrebalov | A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus[END_REF][START_REF] Ito | DNA-binding specificity, transcriptional activation potential, and the rin mutation effect for the tomato fruit-ripening regulator RIN[END_REF][START_REF] Fujisawa | Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation[END_REF][START_REF] Martel | The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENINGdependent manner[END_REF][START_REF] Fujisawa | A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening[END_REF]. Using a chromatin immunoprecipitation (CHIP) approach, RIN was proved to be a master regulator of ripening that directly influences many ripening-associated processes in a developmental specific pattern [START_REF] Fujisawa | Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation[END_REF][START_REF] Martel | The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENINGdependent manner[END_REF][START_REF] Fujisawa | A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening[END_REF]. Indeed, RIN interacts with the promoters (CArG motif) of genes involved in the major pathways associated with observed and well-studied ripening phenotypes and phenomena, including the transcriptional control network involved in overall ripening regulation (CNR, NOR and HB1), ethylene biosynthesis (ACS2 and ACS4), downstream ethylene response (E4 and E8), cell wall metabolism (PG2a), and carotenoid biosynthesis (PSY1) [START_REF] Fujisawa | Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation[END_REF][START_REF] Martel | The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENINGdependent manner[END_REF][START_REF] Fujisawa | A large-scale identification of direct targets of the tomato MADS box transcription factor RIPENING INHIBITOR reveals the regulation of fruit ripening[END_REF]. ACO1 is influenced by RIN via the homebox protein HB1, which interacts with the promoter of ACO1 [START_REF] Lin | SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development[END_REF][START_REF] Martel | The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENINGdependent manner[END_REF]. In ERF.B3-SRDX mutants, although the induction of RIN is delayed at the breaker stage, the transcript accumulation is significantly increased compared to the wild-type controls during the later development stages (Br+3 and Br+7).

MATERIAL AND METHODS

Plant materials and growth conditions

Tomato (Solanum lycopersicum cv. MicroTom) plants were transferred to soil and grown under standard greenhouse conditions. Conditions in the culture chamber room were set as follows: 14h-day/10h-night cycle, 25/20°C day/ night temperature, 80% hygrometry, 250µmol.m -2 .s -1 intense luminosity. For measuring time to ripening, flowers were tagged at anthesis and number of days from anthesis to breaker stage was counted. More than 15 fruits of each genotype were used for this analysis.

Plant transformation

To generate the ERF.B3-SRDX transgenic plants, the coding sequence of Sl-ERF.B3

missing the stop codon was amplified by PCR from a tomato fruit cDNA library. This coding region was cloned via blunt-end ligation into the SmaI site of p35SSRDXG in frame to the region that encodes the SRDX repression domain (LDLDLELRLGFA) from SUPERMAN [START_REF] Hiratsu | Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis[END_REF][START_REF] Mitsuda | Efficient production of male and female sterile plants by expression of a chimeric repressor in Arabidopsis and rice[END_REF]. The transgene cassette was transferred into the destination vector pBCKH, which was derived from the plant transformation vector pBIG-HYG (Becker, 1990) using the gateway LR reaction (Invitrogen Corp.) Agrobacterium tumefaciens-mediated transformation of tomato plants was carried out according to [START_REF] Wang | The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis[END_REF], and transformed lines were selected on a hygromycin-containing medium. All experiments were carried out using homozygous lines from F3 or later generations.

gradient was: 95%A:5%B for 1.3 minutes, followed by 80%A:5%B:15%C for 2.0 minutes and by a linear gradient to 30%A:5%B:65%C over 9.2 minutes. UV-Vis detection was performed continuously from 220 to 700 nm with an online Accela Surveyor photodiode array detector (PDA, Thermo Fischer Scientific, Waltham, MA).

All solvents used were LC-MS grade quality (CHROMASOLV® from Sigma-Aldrich).

Carotenoids were quantified on the basis of the internal standard amounts, obtained by through comparison with peak areas of known amounts of external standard LC-MS runs;

data were then normalized on spectrophotometric chlorophyll/carotenoid contents. For APCI-MS ionization of xanthophylls (0-6 minutes of LC-MS run), nitrogen was used as sheath and auxiliary gas which were set to 40 and 20 units, respectively while the vaporizer temperature was 300 °C, the capillary temperature was 250 °C, the discharge current was set to 4.0 µA, the capillary voltage and tube lens settings were 27 V and 90

V, respectively. APCI-MS ionization of carotenes (6-14 minutes of LC-MS runs) was performed with the following parameters: 30 and 10 unites of, respectively, nitrogen sheath and auxiliary gas; 300 °C and 250 °C for, respectively, vaporizer and capillary temperatures, 5.0 µA as discharge current, 0 and 95 V as, respectively, capillary voltage and tube lens settings. Identification was performed as previously reported [START_REF] Fantini | Dissection of Tomato Lycopene Biosynthesis through Virus-Induced Gene Silencing[END_REF], and on the basis of the m/z accurate masses, as reported on Pubchem database Overall, Sl-ERF.B3 is shown to modulate ethylene responses at four different levels: (i) ethylene biosynthesis, (ii) ethylene receptor, (iii) primary ethylene transcription factors (EIL genes), and (iv) downstream ERF genes.

Expression dynamics of most ERF genes also suggest their involvement in the ripening process, although no link was established between their repressor or activator function and their pattern of expression. The transcript accumulation of Sl-ERF.B3 is induced at the breaker stage and maintained a high level all stages along the ripening process, suggesting that its expression might be continuously required for the modulation of the ripening-regulated genes all along the ripening process. Indeed, over-expression of Sl-ERF.B3 as a chimeric repressor (ERF.B3-SRDX) in tomato results in alterations in both fruit morphology and ripening process. Transgenic lines produce significantly smaller fruit with heart shape and raised bumpy areas present intermittently on the epidermal surface. In addition to the altered fruit morphology, ERF.B3-SRDX lines exhibited a distinct ripening process. The attainment of competence to ripen is dramatically delayed in the transgenic lines, however, once the ripening process starts at breaker stage, when a visible color change just begins to occur, the softening of ERF.B3-SRDX fruits is actually much faster than wild type. These results indicate the involvement of Sl-ERF.B3 in fruit development and ripening. Although after the breaker stage, the ERF.B3-SRDX fruit soften faster with a significantly higher ethylene production, these transgenic fruits fail to display a red-ripe phenotype with a shift of carotenoid accumulation from lycopene to βcarotene at the ripening stages. Moreover, genes involved in different metabolic pathways, such as carotenoid biosynthesis pathway, ethylene synthesis, and cell wall metabolism exhibit altered mRNA accumulation patterns in transgenic lines during fruit ripening process, indicating that Sl-ERF.B3 impacts fruit ripening through mediating fruit ripening-associated genes.

In this study, the ectopic expression of a dominant repressor form of the Sl-ERF.B3

protein provided a mean towards altering the activity of the native Sl-ERF.B3 protein.

This Chimeric Repressor Silencing Technology (CRES-T) strategy allowed revealing vegetative and reproductive growth phenotypes that could not be uncovered by the expression of neither sense nor antisense constructs of Sl-ERF.B3. The eventuality that the pleiotropic phenotypes displayed by the ERF.B3-SRDX dominant suppressor plants may arise from a co-suppression of the endogenous Sl-ERF.B3 is ruled out since the levels of Sl-ERF.B3 transcripts are not altered in the transgenic lines. Therefore, the ERF.B3-SRDX tomato lines proved to be a valuable tool to uncover at least some of the processes controlled by Sl-ERF.B3 and to reveal roles for ERF genes that have not been described previously. Moreover, since the study has been carried out with Micro-Tom, a dwarf genotype, it is important to mention that the dwarfing mutations in this genotype do not seem to impact the phenotype displayed by ERF.B3-SRDX plants since the dwarf phenotype is well reproduced in Ailsa Craig tomato, a non-dwarf variety.

Sl-ERF.B3 is positively regulated by both ethylene and auxin. This suggests that Sl-ERF.B3 could a suitable candidate gene to analyze the cross-talk between ethylene and auxin during plant growth and fruit development. Indeed, ERF.B3-SRDX transgenic lines show several auxin-related phenotypes, such as altered auxin sensitivity with modified root development and reduced auxin responsiveness. These results indicate that Sl-ERF.B3 may act as a regulator at the crossroads between ethylene and auxin signaling.

Moreover, exogenous application of GA partially rescued the dwarf phenotype of the transgenic lines together with the significantly decreased transcript levels of GA biosynthesis genes (Sl-GA20ox1 and Sl-GA20ox2) also indicates a crosstalk between ethylene and GA. Future work will study the putative role of Sl-ERF.B3 in the crosstalk network between ethylene and other hormones.

The tomato Sl-ERF.B3 is related to Arabidopsis factors ERF106 and ERF107, which are members of group IX according to [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF]. This group has been implicated

in the regulation of defense responses and knock-out analysis of ORA59 [START_REF] Pré | The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense[END_REF] and AtERF14 [START_REF] Oñate-Sánchez | AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense[END_REF], prominent representatives of group IX, has revealed disease susceptibility phenotypes. Consistently, overexpression of ERF1, another member of the group, has led to enhanced resistance to necrotrophic pathogens [START_REF] Berrocal-Lobo | Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi[END_REF]. As one member of this group, Sl-ERF.B3 may also play an important role in plant response to various stresses. In the future, we will focus on the investigation of the specific role of Sl-ERF.B3 in plant immunity.

ERFs act as the last known downstream components of ethylene signal pathway, the investigation of their roles in ethylene-dependent processes is important for better

  system specific and Wound Responsive cis-Element WT: Wild Type

  ripening it is important to study specific function of the molecular components involved in ethylene signaling pathway. Ethylene Response Factors (ERFs) are the last known downstream components of the ethylene signal transduction pathway. Being encoded by one of the largest plant families of transcription factors, ERF proteins are the suited step where the diversity and specificity of ethylene responses may originate. Using tomato (Solanum lycopersicum) as a model plant, my Ph.D research project aimed at deciphering the role of Sl-ERF.B3, a tomato Ethylene Response Factor, during plant growth and fruit development using advanced reverse genetics and genomics methodologies.

Figure 1 .

 1 Figure 1. Main steps of ethylene biosynthesis pathway.S-adenosylmethionine (S-AdoMet) is synthesized from the methionine by the S-adenosylmethionine synthetase (SAM synthetase) with one ATP molecule expensed per S-AdoMet synthesized. S-AdoMet is then converted to 1-aminocyclopropane-1carboxylicacid (ACC) by ACC synthase (ACS), 5'-methylthioadenosine (MTA) being a by-product. MTA is recycled to methionine by successive enzymatic reactions involving various intermediates (MTR, 5-methylthioribose; KMB, 2-keto-4-methylthiobutyrate), which constitute the methionine (Yang) cycle. Ethylene production is catalyzed by the ACC oxidase using ACC as substrate, and generates carbon dioxide and hydrogen cyanide[START_REF] Arc | ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination[END_REF].

Figure 2 .

 2 Figure 2. Phenotypes of dark-grown three-dayold seedlings of Arabidopsis thaliana. The plant on the left was grown without hormonal treatment, whereas the plant on the right was exposed to 10 μM ethylene precursor ACC and thus shows a typical triple response[START_REF] Benavente | Molecular mechanisms of ethylene signaling in Arabidopsis[END_REF].

Figure 3 .

 3 Figure 3. Model of ethylene signaling. In the absence of ethylene (Left), the ethylene receptors (e.g., ETR1) at the ER membrane activate the CTR1 protein kinase, a dimer, which phosphorylates the Cterminal domain of EIN2, preventing its nuclear localization. Without ethylene, EIN2 is targeted for 26S proteasomal degradation by F-box proteins ETP1/2. Transcription factors EIN3/EIL1 are also targeted for degradation by F-box proteins EBF1/2. In the presence of ethylene (Right), the receptors are inactivated and therefore the CTR1 kinase is no longer active. The absence of phosphorylation on EIN2 results in EIN2

Figure. 4

 4 Figure. 4 Ethylene receptor family of Arabidopsis. The ethylene receptor family of Arabidopsis is divided into subfamilies 1 and 2 based on phylogenetic analysis and structural features. Receptors are shown as homo-dimers. The ethylene-binding domain (EBD) is found within the conserved transmembrane domains (white rectangles), and includes a copper cofactor (Cu); subfamily 2 receptors have an additional predicted transmembrane domain (grey rectangle) that may function as a signal sequence. All five members of the ethylene receptor family have a GAF domain (yellow diamond) implicated in protein-protein interactions. His kinase domains are indicated by green or red rectangles, green indicating a functional His kinase domain and red indicating a diverged His kinase domain. The receiver domains (ovals) have the conserved residues required for function and are therefore colored green. Conserved His (H) and Asp (D) phosphorylation sites are indicated if present[START_REF] Shakeel | Mechanisms of signal transduction by ethylene: overlapping and non-overlapping signalling roles in a receptor family[END_REF].

Figure 5 .

 5 Figure 5. The binding of ethylene to the receptors results in ethylene response through inactivation of CTR1. When ethylene is not bound (A), the receptors interact and activate CTR1, which results in the inhibition of ethylene responses. When ethylene is bound (B), the receptors are inactive with respect to activating the downstream CTR1 protein (Chang and Stadler 2001).

Figure 6 .

 6 Figure 6. Model of CTR1-mediated receptor oligomerization and cross talk. Ethylene receptor dimers are shown as cartoons with the endoplasmic-reticulum-membrane embedded domains in red and the cytosolic domains in orange. Also shown as cartoons are the N-terminal domains of CTR1 (gray and blue), which interact with the ethylene receptors. The connection between the N-terminal CTR1 domains and their C-terminal kinase domains are indicated as dotted lines. Three consecutive CTR1 kinase dimers are depicted as ribbons as they form across the back-to-back and front-to-front interfaces in the crystal. The back-to-back interface dimer was placed at a receptor dimer and the activation interface connects neighboring receptors. Active CTR1-kd is depicted in gray and activation loops are in red. Inactive dimers of CTR1-D676N (colored blue) are positioned across the front-to-front interface, as observed in the CTR1-D676N crystals. The disordered activation loop is indicated by a dotted line[START_REF] Mayerhofer | Protein kinase domain of CTR1 from Arabidopsis thaliana promotes ethylene receptor cross talk[END_REF].

Figure 7 .

 7 Figure 7. Cartoon of EIN2 protein domain structure. EIN2 consists of an N-terminal integral membrane domain of 12 transmembrane helices followed by a hydrophilic C-terminal domain containing a conserved nuclear localization signal (NLS; Ju et al., 2012).

Figure 8 .

 8 Figure 8. Model of EIN2 action in the ethylene signaling pathway. Ethylene is perceived by ER-located receptors, and the signal acts to promote the cleavage of EIN2 in an unknown mechanism. The cleaved Cterminal fragment (CEND) can be transported into the nucleus, preventing EIN3 from EBF1/2-mediated

Figure 9 .

 9 Figure 9. Model for the regulation of SCF/26S proteasome to the level of EIN3/EIL1.

Figure 10 .

 10 Figure 10. Phylogenetic tree of Arabidopsis and Tomato ERFs. Different subclasses are named by

Figure 11 :

 11 Figure 11: Outline of some of the stress responses and/or signals linked to ERF transcription factors.

Figure 12 .

 12 Figure 12. Model for the regulation of ethylene and ABA in endosperm cap weakening and rupture. ABA delays ACO activity in the radicle and inhibits ACO1 transcript accumulation, but not ACO2 transcript accumulation. The later increase in ACO activity in the radicle of ABA-treated seeds is therefore due to ACO2, and the ethylene produced promotes endosperm cap weakening by antagonizing the ABA inhibition. In the endosperm cap, ABA inhibits ACO2 and ACO1 transcript accumulation.

Figure 13 .

 13 Figure 13. Ethylene inhibits root elongation and lateral root development.Five-day-old Arabidopsis seedlings were transferred to medium containing 1 μM ACC and the tip of the roots at the time of transfer was marked by a black dot. When their roots were imaged five days later, ethylene had decreased the rate of root elongation relative to an untreated control, as judged by the length of root that had formed below the black dots. By contrast, ethylene treatment prevented lateral root formation in the region formed after transfer[START_REF] Muday | Auxin and ethylene: collaborators or competitors?[END_REF]

Figure 14 .

 14 Figure 14. Differential expression of ACS and ACO genes associated with system 1 and system 2 ethylene synthesis during fruit development and ripening in tomato. Auto-inhibition of ethylene synthesis during system 1 ethylene production is mediated by a reduced expression of LeACS1A and 6.

Figure 15 .

 15 Figure 15. A schematic representation of the proposed model for a regulatory mechanism of tomato fruit ripening. Bold line arrows indicate an ethylene-mediated positive feedback loop that enhances RIN expression. It is unclear whether the loop regulates the expression of the other ripening regulators (such as NOR and TDR4) affected by ethylene during ripening directly or indirectly (via RIN). Arrows indicate the direction of the transcriptional regulatory pathways. Blunt-ended lines indicate repression. Circle arrows on RIN and TAGL1 indicate auto-regulation and on ethylene indicate autocatalytic ethylene production

  Pathogenesis-related (PR) proteins which are constituted by a broad class of inducible defense-related proteins expressed either locally or systemically in response to pathogen stress are the most extensively studied set of defense molecules in relation to ethylene. The extensive role of ethylene in the regulation of expression of different classes of PR genes, such as PR-2 (β-1,3-glucanases), PR-3 (basic-chitinases), PR-4

Figure 16 .

 16 Figure 16. Model of the control of apical hook curvature by auxin and ethylene. Auxin is transported in at rootward direction from the cotyledons predominantly through the action of PIN1 and PIN3. The apical hook is formed because of the asymmetric distribution of auxin (shown in blue) that arises through differential auxin synthesis, auxin transport and auxin signaling. This is reflected by an asymmetrical distribution of proteins regulating these processes in the region of the apical hook. When ethylene is added (right-hand image), auxin levels rise on the concave side of the hook to cause an exaggerated curvature due to increases in PIN3, AUX1, IAA3, IAA12, IAA13 and TAR2 on the concave side of the hook (Muday et al., 2012).

  Enhanced ethylene response reduces bioactive GA levels, thus promoting the accumulation of DELLA proteins. DELLA accumulation in turn slows the plant life cycle and delays flowering via repression of floral meristem identity LFY and SOC1 genes[START_REF] Achard | The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristemidentity genes[END_REF] Figure 17).

Figure 17 .

 17 Figure 17. Model for integration of the ethylene and GA-DELLA signaling pathways in the regulation of floral transition. Activation of ethylene signaling reduces bioactive GA levels, thus promoting the accumulation of DELLAs. DELLA accumulation in turn slows the plant life cycle and delays flowering. Accumulation of DELLAs delays floral transition (via regulation of LFY and SOC1 transcript levels) and increases the abundance of GA-biosynthesis gene transcripts via a negative feedback loop[START_REF] Achard | The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristemidentity genes[END_REF].

  ERF family have been shown to play important role in mediating defense responses inArabidopsis[START_REF] Mcgrath | Repressor-and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression[END_REF]. The Arabidopsis transcription factor MYC2 has also been shown to regulate the crosstalk between ethylene-and JA-mediated defense signaling[START_REF] Lorenzo | Molecular players regulating the jasmonate signalling network[END_REF][START_REF] Dombrecht | MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis[END_REF].

Figure

  Figure 18. Ethylene/Jasmonate-dependent pathway of the Arabidopsis response to pathogens. Infection by some types of pathogens induces the synthesis and subsequent activation of the ethylene and jasmonate pathways simultaneously (black arrows). As a consequence, ERF1 is transcriptionally activated; in turn, it activates the expression of defense-related genes that prevent disease progression. Other types of stress or pathogens (white arrows) induce the activation of only one of these signaling pathways and, therefore,ethylene-or jasmonate-specific responses[START_REF] Lorenzo | ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense[END_REF].

  plant for genomic research of solanaceous plants, as well as for studying the development, ripening, and metabolism of fruit. Indeed, tomato has been recognized as a model system for studying the molecular basis of fleshy fruit development and unravelling the role of ethylene in controlling the ripening of climacteric fruit since the early 1980s. For genetic and genomic studies, tomato has many advantages over other species of agronomical interest, such as simple diploid genetics (n = 12), a relatively compact genome (900 Mb) that has recently been sequenced, numerous mapped traits, developed DNA markers, rich collections of germplasm and mutants, available RNAseq data, highly efficient transformation protocols and a relatively short reproductive cycle (3-4 generation per year)(Tanksley et al., 1992;[START_REF] Hoeven | Deductions about the number, organization, and evolution of genes in the tomato genome based on analysis of a large expressed sequence tag collection and selective genomic sequencing[END_REF] Tomato Genome Consortium, 2012). Moreover, the tomato plant has many interesting features such as fleshy fruit, a sympodial shoot, compound leaves, photoperiod-independent sympodial flowering, glandular trichomes and the formation of fleshy climacteric fruits, which other model plants (such as Arabidopsis and rice) do not have. Most of these traits are agronomically important and cannot be studied using other model plant systems. These advantages have made tomato an excellent model organism for investigating fruit development, ripening processes, sugar metabolism, carotenoid biosynthesis, quantitative trait locus (QTL)

  microarray) and the generation of new biological resources on the tomato (e.g. EST database, TILLING resources, genetic and physical maps) have led to a step forward on the understanding of the molecular mechanisms underlying plant development and fruit ripening. Since the genome structures of most of the solanaceous plants are relatively well conserved, the genomic and molecular studies of tomato can serve as a reference to understanding of other Solanaceae species, which then allows researchers to investigate molecular mechanisms underlying fruit development and ripening in different species. Fleshy fruits are important worldwide crops because they are important sources of useful and functional compounds for human diet. Tomato has proved to be an excellent model system for the research on fleshy fruit development and ripening. Fruit development starts after the ovules in the ovary have been successfully fertilized and the ovary begins to develop into the fruit. Generally, fruit development can be divided into essentially three stages which are depicted in Figure 19. These are (i) a period of intensive cell division that begins at anthesis and continues approximately for 2 weeks after fertilization;(ii) a period of rapid cell expansion that begins toward the end of the cell division stage and continues until one week before the onset of ripening; (iii) a ripening phase that initiates after growth has ceased and involves rapid chemical and structural changes that determine fruit aroma, color, texture and biochemical composition but not fruit size and shape.

Figure 19 .

 19 Figure 19. Overview of tomato fruit development.Fruit set is the initiation of fruit growth after the flower has been successfully pollinated and fertilized. After fertilization, cell division takes place, which lasts up to 12 d. This period is followed cell expansion, during which the volume of the fruit rapidly increases. Once the fruit has reached its final size it starts to ripen[START_REF] Mounet | Quantitative metabolic profiles of tomato flesh and seeds during fruit development: complementary analysis with ANN and PCA[END_REF].

  Sl-ERF.B3 was previously shown to act as strong transcriptional activator on GCC-boxcontaining promoters and its transcripts accumulate upon ethylene treatment, suggesting a putative involvement in ethylene-regulated processes(Tournier et al., 2003;[START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF]. Because overexpressing and down-regulated lines failed to reveal the functional significance of Sl-ERF.B3, a dominant chimeric repressor version was used which resulted in phenotypes consistent with Sl-ERF.B3 being involved in both ethylene biosynthesis and signaling pathway. The ERF.B3-SRDX lines displayed constitutive ethylene-responses in the absence of ethylene and the data identified a set of ERFs among the target genes regulated by Sl-ERF.B3 supporting the idea that the alteration of such a high number of ERFs may account for the pleiotropic phenotypes displayed by the transgenic lines.

  an attempt to overcome the experimental limitations likely owing to functional redundancy among members of the ERF gene family, we generated a dominant repressor version of Sl-ERF.B3 (ERF.B3-SRDX) using the Chimeric Repressor Silencing Technology (CRES-T). The Sl-ERF.B3 coding sequence lacking the Stop Codon was fused to the SRDX repression domain LDLDLELRLGFA, known as the EAR motif (Mitsuda et al., 2006) and cloned downstream of the Cauliflower Mosaic Virus 35S promoter. The capacity of the ERF.B3-SRDX chimeric protein to function as a transcriptional repressor on ethylene-responsive genes was assessed in a transient transformation assay via co-transfection of protoplasts with reporter and effector constructs. The reporter construct was obtained by fusing the GFP coding sequence either to a synthetic promoter containing the ethylene inducible GCC box, or to a native osmotin promoter containing the canonical GCC cis-acting element. The effector constructs allow the expression of either the Sl-ERF.B3 protein or its repressor version fused to the SRDX motif (ERF.B3-SRDX). Transactivation assays indicated that Sl-ERF.B3 enhances the expression of the reporter gene driven by both the synthetic and native promoter, clearly indicating that Sl-ERF.B3 acts as a transcriptional activator of GCC-box containing promoters (Figure 1). By contrast, co-transfection of the reporter constructs with the ERF.B3-SRDX results in 8-fold and 15-fold suppression of the activity of the synthetic and the native ethylene-responsive promoters, respectively

Figure 1 .

 1 Figure 1. Transactivation assay in a single cell system. Protoplasts were co-transfected with a reporter construct consisting of the GFP gene driven by a GCC-rich synthetic promoter or a native osmotin GCCcontaining promoter and an effector plasmid expressing either ERF.B3 or ERF.B3-SRDX protein. The basal fluorescence obtained in the assay transfected with the reporter construct and an empty effector construct was standardized to 100 and is taken as reference. Values are means ± SD of three independent biological replicates.

Figure 2 .

 2 Figure 2. Ethylene hypersensitivity of 35S:ERF.B3-SRDX lines. (a) Etiolated 35S:ERF.B3-SRDX seedlings display partial constitutive ethylene response in the absence of exogenous ethylene that can be removed by 1-MCP application (1.0 mg L -1 ) or exaggerated upon exogenous ethylene (10 μL L -1 ) treatment. (b) Hypocotyl elongation in ERF.B3-SRDX etiolated seedlings and WT treated or untreated with ethylene and 1-MCP. Values are means ± SD (n ≥ 30) of three replicates. *, 0.01 < P < 0.05, ***, P < 0.001 (Student's test). SR1, SR2 and SR3 are three independent 35S:ERF.B3-SRDX lines.

Figure 3 .

 3 Figure 3. Ethylene hypersensitive phenotypes of adult 35S:ERF.B3-SRDX plants showing petioles and leaves epinasty (upper panel) enhanced premature flower senescence (middle panel) and accelerated fruit abscission (lower panel). The white arrows point to the abscission zone.

Figure 4 .

 4 Figure 4. Dwarf phenotype of 35S:ERF.B3-SRDX plants. (a) Dwarf phenotype of 35S:ERF.B3-SRDX plants. Photographs were taken at 7 days (upper panel) and 80 days (lower panel) after germination. (b) Reduced plant size of 80-day-old ERF.B3-SRDX plants. Values are means ± SD (n ≥ 15) of three replicates. (c) Relative mRNA levels of two GA oxidase genes in wild-type and ERF.B3-SRDX lines assessed by qRT-PCR. The relative mRNA levels of each gene in the wild type were standardized to 1.0, referring to Sl-Actin gene as internal control. (d) ERF.B3-SRDX dwarfism partially rescued by exogenous gibberellic acid (GA) application. Ten-day-old wild-type and ERF.B3-SRDX plants were sprayed with GA (10 -5 M) twice a week for three weeks. *, 0.01< P < 0.05, **, 0.001 < P < 0.01, ***, P < 0.001 (Student's test). SR1, SR2 and SR3 are three independent 35S:ERF.B3-SRDX lines.

Figure 5 .

 5 Figure 5. Delayed reproductive development and reduced fruit set in 35S:ERF.B3-SRDX plants. (a) Late flower bud setting and flowering time in ERF.B3-SRDX plants compared to WT. DAG, Day After Germination. (b) Assessing the time of flower bud setting and flower opening in ERF.B3-SRDX and WT plants. (c) Abnormal flowers with short anther and exerted stigma in ERF.B3-SRDX lines. (d) Reduced fruit set rate in ERF.B3-SRDX lines.

Figure 6 .

 6 Figure 6. Down-regulation of ethylene production and ethylene biosynthesis genes in 35S:ERF.B3-SRDX plants. (a) Ethylene production of etiolated seedlings in WT and ERF.B3-SRDX lines. (b) ACS and ACO transcript accumulation in WT and ERF.B3-SRDX plants assessed by qRT-PCR. The relative mRNA levels of each gene in the wild type were standardized to 1.0, referring to Sl-Actin gene as internal control.Values are means ± SD of three replicates. *, 0.01 < P < 0.05, **, 0.001 < P < 0.01 (Student's test). SR1, SR2 and SR3 are three independent 35S:ERF.B3-SRDX lines.

Figure 7 .

 7 Figure 7. Expression of ethylene receptor genes in 35:ERF.B3-SRDX and ERF.B3 overexpression lines. (a) Relative mRNA levels of ETR2, NR, ETR5 and ETR6 receptor genes assessed by qRT-PCR in 4-weekold WT and ERF.B3-SRDX lines. (b) The transcriptional activity of ETR6 promoter is regulated by both ERF.B3 and ERF.B3-SRDX in a protoplast transactivation assay. Protoplasts were co-transfected with GFP reporters fused to the ETR6 promoter and with an effector plasmid expressing either ERF.B3 or ERF.B3-SRDX proteins. (c) Relative mRNA levels of ETR1, NR, and ETR6 assessed by qRT-PCR in 4-week-old WT and ERF.B3 overexpression lines. *, 0.01< P < 0.05, **, 0.001 < P < 0.01, ***, P < 0.001 (Student's test). SR1, SR2 and SR3 are three independent ERF.B3-SRDX lines. OX1, OX2 and OX3 are three independent Sl-ERF.B3 overexpressing lines.

Figure 9 .

 9 Figure 9. Sl-ERFs are among the target genes of ERF.B3 and ERF.B3-SRDX. (a) Accumulation of Sl-ERFs transcripts in WT and ERF.B3-SRDX lines assessed by qRT-PCR in 4-weekold plants. The relative mRNA level of each gene in WT was standardized to 1.0, referring to Sl-Actin as internal control. (b) Transactivation of Sl-ERF promoters by ERF.B3 and ERF.B3-SRDX. Protoplasts were co-transfected with GFP reporter fused to the promoters of Sl-ERFs (ERF.C3, ERF.D2, ERF.F4, ERF.F5 and ERF.G1) and an effector plasmid expressing ERF.B3 or ERF.B3-SRDX. (c) Sl-ERFs transcript levels in ERF.B3 overexpression lines assessed by qRT-PCR in 4-week-old plants.

Figure 10 .

 10 Figure 10. Tentative model proposing the involvement of Sl-ERF.B3 in the control of ethylene responses. Sl-ERF.B3 modulates ethylene responses at different levels including ethylene biosynthesis (ACO/ACS), receptors, and ERF genes. ERF.B3-mediated ethylene response occurs partly via direct transcriptional regulation of specific ethylene receptor genes (ETR6) and selected members of the ERF gene family (ERF.C3, ERF.D2, ERF.F4 and ERF.F5). Ectopic expression of Sl-ERF.B3 decreases ethylene responses in vegetative tissues through up-regulation of ethylene receptor genes and down-regulation of EIN3-like genes (panel a). By contrast, ectopic expression of Sl-ERF.B3-SRDX repressor version, leads to enhanced ethylene responses via down-regulation of receptor genes and repression of some ERF genes (panel b).

Figure S2 .

 S2 Figure S2. Transcript accumulation of the chimeric ERF.B3-SRDX and the endogenous ERF.B3 genes in transgenic lines. Transcript levels were assessed by qRT-PCR in 4-week-old plants. The relative mRNA levels of Sl-ERF.B3 endogenous (Endo) in the wild type were standardized to 1.0, referring to Sl-Actin gene as internal control. Values are means ± SD of three replicates. SR1, SR2 and SR3 are three independent 35S:ERF.B3-SRDX lines.

Figure S3 .

 S3 Figure S3. Triple response phenotype of Sl-ERF.B3 overexpression lines. (a) Responsiveness of dark-grown WT and Sl-ERF.B3 overexpressing lines treated with exogenous ethylene (10 μL L -1 ). (b) Inhibition of hypocotyl length in WT and ERF.B3 overexpressing lines treated with ethylene (10 μL L - 1 ). Values are expressed as % of the initial hypocotyl length prior to hormone treatment of dark-grown seedlings. Values are means ± SD (n ≥ 30) of three replicates. *, 0.01 < P < 0.05 (Student's test). OX1, OX2 and OX3 are three independent Sl-ERF.B3 overexpression lines.

Figure S4 .

 S4 Figure S4. Altered leaf morphology in ERF.B3-SRDX plants. (a) Leaf margins are twisted and the lamina is often wrinkled. (b) Leaflet length and width assessed in 30-day-old ERF.B3-SRDX and WT plants. Values are means ± SD (n ≥ 15) of three replicates. (c) Scanning electron microscopy observation of leaf epidermal cells in WT and ERF.B3-SRDX lines. (d) Monitoring epidermal cell size in WT and ERF.B3-SRDX leaves. **, 0.001 < P < 0.01, ***, P < 0.001 (Student's test). SR1, SR2 and SR3 are three independent 35S:ERF.B3-SRDX lines.

Figure S5 .

 S5 Figure S5. Phenotypes of reproductive organs (a) ERF.B3-SRDX plants show reduced flower size. (b) Reduced fruit size with altered fruit shape in ERF.B3-SRDX plants. (c) Reduced seed size in ERF.B3-SRDX lines. (d) ERF.B3-SRDX pollen viability is similar to that of WT as revealed by Alexander's staining test.

Figure S6 .

 S6 Figure S6. ERF.B3 and ERF.B3-SRDX are unable to modulate the transcriptional activity of Sl-EIL promoters in a protoplast transactivation assay Protoplasts were co-transfected with a reporter construct consisting of the GFP gene driven by the promoters of Sl-EILs (EIL1, EIL2, EIL3 and EIL4) and an effector plasmid expressing either ERF.B3 or ERF.B3-SRDX. GFP fluorescence was measured by flow cytometry 16 h after transfection. The basal fluorescence obtained by co-transformation with the promoter fused to the reporter gene and the empty vector was standardized to 100 and is taken as reference. The results are mean of 3 independent biological repetitions.

  function in fruit development and plays a critical role in the fruit ripening process, In addition, we show that Sl-ERF.B3 primarily regulates genes involved in both carotenoid and ethylene biosynthesis. Moreover, by the analysis of the expression levels of the tomato ripening regulators including RIN, CNR, NOR, HB-1, TAGL1 and AP2a during fruit ripening process in the ERF.B3-SRDX lines, we demonstrated that Sl-ERF.B3 is a new regulator involved in the tomato regulatory network controlling ripening.107RESULTSExpression patterns of tomato ERF genes during fruit ripeningEthylene is known to play a critical role in fruit development and ripening, and Ethylene Response Factors (ERFs) are considered as the primary actors in mediating responses to the hormone. To gain further insight on the expression of members of the tomato ERF gene family during the ripening process, the accumulation of Sl-ERFs transcripts was assessed by quantitative RT-PCR at different stages of fruit ripening. The data indicate (Figure1) that out of the 25 ERF genes tested, eleven (Sl-ERF.A2, A3, B1, B2, C1, C3, D1, D2, F3, F5 and G2) show an increase in their expression peaking 3 days post-breaker (Br+3) and then decline at later ripening stages. The expression of 5 genes (Sl-ERF.B3, C2, E2, E4 and F2) peaks at the breaker stage (Br) and then decreases. A distinct expression pattern is displayed by a group of ERF genes (Sl-ERF.A1, C6, D3, D4, E1, F1, F4 and H1) that undergo steady increase in transcript accumulation throughout ripening.Sl-ERF.E3, is the only gene showing no ripening-induced expression but rather a decline of its transcript levels from breaker to late ripening stages. While the expression dynamics of most ERF genes suggests their involvement in the ripening process, no link was established between their repressor or activator function and their pattern of expression.

Figure 1

 1 Figure 1. Relative expression profiles of tomato ERF genes in different ripening stages obtained by quantitative RT-PCR. MG, mature green fruit; Br, breaker stage fruit; Br+3, 3 days after breaker stage; Br+7, 7 days after breaker stage. Values represent means of three biological replicates, and vertical bars represent SD of the means.

Figure 2 .

 2 Figure 2. Sl-ERF.B3 gene expression during development and in ERF.B3-SRDX dominant repressor lines. A, Total RNA was extracted from different developmental stages; St (stem), R (root), L (leaf), Fl (flower), IMG (immature fruit), MG (mature green fruit), Br (breaker stage fruit), Br+3 (3 days after breaker stage), Br+7 (7 days after breaker stage). The relative mRNA levels of Sl-ERF.B3 at immature green stage were standardized to 1.0, referring to Sl-Actin gene as internal control. Values are means ± SD of three biological replicates. B, Transcript accumulation of the chimeric ERF.B3-SRDX gene in wild-type and transgenic lines (SR1, SR2 and SR3) in ripening fruit. Total RNA was extracted from fruit at 3 days after breaker stage (Br+3).

Figure. 4

 4 Figure. 4 Fruit ripening in wild-type and ERF.B3-SRDX lines. A, Different stages of fruit ripening for wild-type (WT) and ERF.B3-SRDX lines. Fruits from independent transformant lines are shown, which are delayed in color development, never developing full red color. Br, breaker stage , Br+2, tuning stage (2 days after breaker stage), Br+5, pink stage (5 days after breaker stage), and Br+10, red ripe stage (10 days after breaker stage). B, Changes in hue angle in WT and ERF.B3-SRDX lines during different ripening stages. SR1, SR2 and SR3 are three independent ERF.B3-SRDX lines.

(

  Figure. 6). It is also noteworthy that transcript accumulation of all three lycopene beta cyclases (β-LCY1, β-LCY2, CYC-β) was markedly elevated in ERF.B3-SRDX fruits compared to wild-type (Figure6) accounting for the significantly increased β-carotene content in ERF.B3-SRDX lines (Figure5). The data indicate that the dominant repressor version of ERF.B3 leads to decreased expression of PSY1 and PDS and increased expression of lycopene beta cyclases, thus resulting in a modified lycopene to β-carotene ratio.

Figure 6 .

 6 Figure 6. Expression of carotenoid biosynthesis genes in wild-type and ERF.B3-SRDX lines. Total RNA was extracted from the indicated developmental stages of fruit (MG, Br, Br+3 and Br+7). The relative mRNA levels of each gene at breaker (Br) stage were standardized to 1.0, referring to Sl-Actin gene as internal control. Values are means ± SD of three biological replicates. *, 0.01< P < 0.05, **, 0.001 < P < 0.01, ***, P < 0.001 (Student's test). SR1 and SR2 are two independent ERF.B3-SRDX lines.

Figure 7 .

 7 Figure 7. Firmness and ethylene production in wild-type and ERF.B3-SRDX fruits. A, Firmness analysis of control and ERF.B3-SRDX fruits. Fruits were harvested at breaker stage, kept at room temperate and measured for firmness at different stages (Br, Be+3, Br+7, Br+10, Br+15 and Br+20 as defined in the methods). 15 fruit were used for each measurement and values shown are the means ± SD. B, Production of ethylene in control and ERF.B3-SRDX lines. Fruits of different ripening stages representative by Days Post Anthesis (DPA). Values represent means of at least ten individual fruits. Vertical bars represent SD. 35 DPA represents mature green (MG) stage in WT; 40 DPA, breaker (Br) stage in WT. 50 DPA, mature green (MG) stage in ERF.B3-SRDX lines; 55 DPA, breaker (Br) stage in transgenic lines. SR1, SR2 and SR3 are three independent ERF.B3-SRDX lines.

Figure 9 .

 9 Figure 9. Ripening regulator genes expression in wild-type and ERF.B3-SRDX lines during fruit ripening. Total RNA was extracted from the indicated developmental stages of fruit ( MG, Br, Br+3 and Br+7) as defined in the methods. The relative mRNA levels of each gene at breaker (Br) stage were standardized to 1.0, referring to Sl-Actin gene as internal control. Values are means ± SD of three biological replicates. *, 0.01< P < 0.05, **, 0.001 < P < 0.01, ***, P < 0.001 (Student's test). SR1 and SR2 are two independent ERF.B3-SRDX lines.

  CNR and NOR show the same transcript accumulation patterns with RIN in ERF.B3-SRDX mutants during fruit ripening in consistent with the regulation of RIN to CNR and NOR. The transcript of HB1 also shows higher accumulation in ERF.B3-SRDX lines during fruit ripening. Moreover, genes involving in ethylene biosynthesis, ethylene downstream response and cell wall metabolism including ACS2, ACS4, ACO1, E4, E8 and PG2a display significant induction in ERF.B3-SRDX mutants during fruit ripening process. These data are in line with the model that RIN acts as a master regulator of the ripening cascade by influencing numerous molecular pathways. However, since ERF.B3-SRDX is a dominant repressor, the regulation mechanism between ERF.B3 or its chimeric protein and RIN remains to be revealed.The expression dynamics of most Sl-ERF genes during fruit ripening suggests their putative involvement in the ripening process. Moreover, the expression of a number of Sl-ERF genes was found to be markedly altered in ERF.B3-SRDX fruit at the breaker + 3 stage. The alteration of Sl-ERF transcript levels in ERF.B3-SRDX ripening fruit may 123 account for diversity of ripening phenotype displayed by the dominant repressor fruits.Notably, ERF genes (Sl-ERF. C3, Sl-ERF. D2, Sl-ERF. F5 and Sl-ERF. F4) which shown to be the putative target of Sl-ERF.B3 and ERF.B3-SRDX[START_REF] Liu | A dominant repressor version of the tomato Sl-ERF.B3 gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components[END_REF] are downregulated in ERF.B3-SRDX fruits, further supports the model that a single ERF can impact the expression of other members of the gene family and this inter-connected regulation among ERF genes may therefore account for the pleiotropic alterations in the ERF.B3-SRDX lines.

(

  http://pubchem.ncbi.nlm.nih.gov/) for monoisotopic masses identification, //fiehnlab.ucdavis.edu/staff/kind/Metabolomics/MS-Adduct-Calculator/) in case of adduct ion detection. Color measurement L, a and b values were measured with a Minolta chromameter (CR-200, 78903131) on fruit at different stages during fruit ripening. The chromameter was calibrated against a standard white tile. Hue angle values were calculated according to the following equation: Hue angle = tan -1 (b/a) if a>0 or 180+ tan -1 (b/a) if a<0. ethylene plays a critical role in a wide range of developmental processes, including germination, flower and leaf senescence, fruit ripening, leaf abscission, root nodulation, programmed cell death, and responses to biotic and abiotic stresses. After its synthesis, the perception and signaling of ethylene rely on the cooperative action of several components among which Ethylene Response Factors (ERFs) play a poorly understood role. However, being encoded by one of the largest family of plant transcription factors, ERF proteins are the most suited step of ethylene signaling where the diversity and specificity of ethylene responses may originate. My Ph.D project mainly dealt with the functional characterization of ERF genes during plant growth and fruit development using tomato as the plant model. More particularly, my work aimed to decipher the role of Sl-ERF.B.3, a member of ERF family gene, in mediating ethylene response and tomato plant development and fruit ripening using advanced reverse genetics and genomics methodologies. Sl-ERF.B3 was previously shown to act as strong transcriptional activator on GCC-boxcontaining promoters and its transcripts accumulate upon ethylene treatment, suggesting a putative involvement in ethylene-regulated processes. To address the physiological significance of Sl-ERF.B3 and its potential role in mediating ethylene responses, tomato lines under-and over-expressing Sl-ERF.B3 gene were generated by stably transforming tomato plants with either sense or antisense constructs under the control of the constitutive 35S promoter. Since classical down-and up-regulation approaches failed to provide clear clues on Sl-ERF.B3 functional significance in tomato, we generated a dominant repressor version of Sl-ERF.B3 (ERF.B3-SRDX) using the Chimeric Repressor Silencing Technology (CRES-T) to overcome the functional redundancy among ERF family members. The capacity of ERF.B3-SRDX retaining to bind the same target genes than Sl-ERF.B3 protein and to dominantly repress its transcriptional activity was confirmed by a transient transformation assay. This indicates the usefulness of this strategy in functional studies by overcoming redundancy among members of a multigene family. Using the dominant repression version of Sl-ERF.B3 (ERF.B3-SRDX), the involvement of Sl-ERF.B3 in ethylene response and a wide range of development processes was demonstrated. Expression of a dominant repressor ERF.B3-SRDX version of Sl-ERF.B3 in the tomato resulted in pleiotropic ethylene responses and vegetative and reproductive growth phenotypes. The dominant repressor etiolated seedlings displayed partial constitutive ethylene-response in the absence of ethylene and adult plants exhibited typical ethylene-related alterations such as leaf epinasty, premature flower senescence and accelerated fruit abscission. The multiple symptoms related to enhanced ethylene sensitivity correlate with the altered expression of ethylene biosynthesis and signaling genes, suggesting the involvement of Sl-ERF.B3 in a feedback mechanism regulating components of ethylene production and response. Moreover, Sl-ERF.B3 is shown to modulate the transcription of a set of ERFs revealing the existence of a complex network in which multiple transcription factors are competing for promoters to control the expression of genes that are essential for a wide range of plant responses to ethylene.

  understanding the distinct regulation mechanisms of the ethylene responses. The demonstration of the specific role of Sl-ERF.B3, a member of ERF gene family in tomato, in ethylene-mediated developmental processes such as plant growth and fruit ripening provided a clue of the functional significance of ERF genes in ethylene response processes. The data showing that most of ERF genes display altered expression patterns in the ERF.B3-SRDX dominant repressor lines which resulted in pleiotropic ethylene responses and vegetative and reproductive growth phenotypes further, stress the importance of ERF genes in a wide range of ethylene-dependent developmental processes. To further decipher the function of ERF genes in both ethylene-dependent and ethylene-independent processes, it is important to continue to generate mutant lines altered in the expression of these genes. For better understanding of the mechanisms by which these transcription factors control plant developmental processes, it is crucial to identify the target genes of ERFs. This can be achieved by comparative transcriptomic profiling of the lines altered in specific ERFs or by a ChIP-seq (Chromatin Immunoprecipitation coupled to deep sequencing) approach. Both strategies are being carried out within the GBF lab.

Table 1 . Ethylene-related mutant and transgenic plants with altered sensitivity to pathogens

 1 (van Loon et al., 2006).

	Plant species	Mutant or transgenic	Pathogen	Lifestyle	Disease severity a
	Arabidopsis ein2-1	Botrytis cinerea	Necrotrophic +
	Arabidopsis ein2-5, ein3-1	Botrytis cinerea	Necrotrophic +
	Arabidopsis etr1-1, ein2-1	Chalara elegans	Necrotrophic +
	Arabidopsis ein2-1	Erwinia carotovora pv. carotovora	Necrotrophic +
	Arabidopsis ein2-5	Fusarium oxysporum f.sp. conglutinans	Necrotrophic +
	Arabidopsis ein2-5	Fusarium oxysporum f.sp. conglutinans	Necrotrophic +
	Arabidopsis etr1-1, ein2-1	Fusarium oxysporum f.sp. matthiolae	Necrotrophic +
	Arabidopsis etr1-1, ein2-1	Fusarium oxysporum f.sp. raphani	Mixed	-
	Arabidopsis eto1 -eto3	Heterodera schachtii	Biotrophic	+
	Arabidopsis etr1-1, ein2-1	Heterodera schachtii	Biotrophic	-
	Arabidopsis ein2-5	Plectosphaerella cucumerina	Necrotrophic +
	Arabidopsis ein2-1	Pseudomonas syringae pv. maculicola	Mixed	-
	Arabidopsis ein2-1,-3,-4,-5	Pseudomonas syringae pv. tomato	Mixed	-
	Arabidopsis etr1-1, ein2-1	Pythium spp.	Necrotrophic +
	Arabidopsis ein2-1, eto3	Ralstonia solanacearum	Necrotrophic -
	Arabidopsis etr1	Spodoptera exigua	Herbivore	-
	Arabidopsis ein2-1, hls1-1	Spodoptera littoralis	Herbivore	-
	Arabidopsis etr1-1	Verticillium dahliae	Necrotrophic -
	Arabidopsis etr1-1, etr2-1	Xanthomonas campestris pv. campestris Mixed	+
	Arabidopsis ein2-1	Xanthomonas campestris pv. campestris Mixed	-
	Arabidopsis eto1-1	Xanthomonas campestris pv. campestris Mixed	+
	Tomato	ACD	Botrytis cinerea	Necrotrophic +
	Tomato	Epi	Botrytis cinerea	Necrotrophic -
	Tomato	ACD	Verticillium dahliae	Necrotrophic -(tolerant)
	Tomato	ACD	Xanthomonas campestris pv. vesicatoria Mixed	-(tolerant)
	Tomato	NR, Nr	Xanthomonas campestris pv. vesicatoria Mixed	-(tolerant)
	Tomato	Nr	Fusarium oxysporum f.sp. lycopersici	Necrotrophic -
	Tomato	Nr	Pseudomonas syringae pv. tomato	Mixed	-(tolerant)
	Tomato	Nr	Xanthomonas campestris pv. vesicatoria Mixed	-(tolerant)
	Tomato	Atetr1-1-LeEtr3	Xanthomonas campestris pv. vesicatoria Mixed	-

a Disease severity: + increased; -, decreased.

Table 1 Cross-Fertilization Assay

 1

. Emasculated wild-type flowers were fertilized with ERF.B3-SRDX

Table S1 . Putative ERF binding cis-elements present in the promoter regions of ethylene receptor and ethylene biosynthesis genes.

 S1 For each gene, the genomic sequence corresponding to 2.0 kb upstream of the predicted translation start codon (ATG)

	was analyzed for the presence of known ethylene response cis-acting elements using
	three different softwares: (i) PLACE (http://www.dna.affrc.go.jp/ PLACE/), (ii)
	PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html), and (iii)
	PlantPAN (http://plantpan.mbc.nctu.edu.tw/seq_analysis.php).
	Gene	Motif	Position	Sequence
	Ethylene receptor genes
	Sl-ETR2	None	-	-
	NR	DRE/CRT	-700	CCGAC
		DRE/CRT	-1503	CCGAC
	Sl-ETR5	GCC-box-like	-1558(-)	TCCGCC
		DRE/CRT	-1707(-)	CCGAC
	Sl-ETR6	None	-	-
	Ethylene biosynthesis genes
	Sl-ACS1	GCC-box-like	-1685	TCCGCC
		DRE/CRT	-999	CCGAC
		DRE/CRT	-1188	CCGAC
		DRE/CRT	-1752	CCGAC
	Sl-ACS3	GCC-box-like	-1455	TCCGCC
		GCC-box-like	-1586	CCCGCC
		DRE/CRT	-718	CCGAC
		DRE/CRT	-804	CCGAC
	Sl-ACO1	None	-	-
	Sl-ACO3	GCC-box	-1851	GCCGCC
		DRE/CRT	-541	CCGAC
		DRE/CRT	-505	CCGAC
		DRE/CRT	-810	TCGAC
	Sl-ACO4	DRE/CRT	-1136	CCGAC
		GCC-box	-1218(-)	GCCGCC
		GCC-box -like	-1215(-)	CCCGCC

Table S2 . Putative cis-acting ethylene-response elements present in the promoter regions of Sl-ERF genes.

 S2 The genomic sequence corresponding to 2.0 kb upstream of the predicted translation start codon (ATG) was analyzed for the presence of known

	ethylene response cis-acting elements using three softwares, PLACE
	(http://www.dna.affrc.go.jp/	PLACE/),		PlantCARE
	(http://bioinformatics.psb.ugent.be/webtools/plantcare/html)	and	PlantPAN
	(http://plantpan.mbc.nctu.edu.tw/seq_analysis.php).
	Gene	Motif	Position	Sequence
	Down regulated genes
	Sl-ERF.C3	GCC-box	-458	GCCGCC
	Sl-ERF.D2	DRE/CRT	-475	CCGAC
		GCC-box	-70(-)	GCCGCC
		DRE/CRT	-388(-)	CCGAC
	Sl-ERF.F5	GCC-box	-173	GCCGCC
		DRE/CRT	-969	CCGAC
		DRE/CRT	-1068	CCGAC
		GCC-box	-87(-)	GCCGCC
	Sl-ERF.F4	DRE/CRT	-431	CCGAC
		GCC-box	-139(-)	GCCGCC
	Up regulated genes
	Sl-ERF.G1	None	-	-

Table

Table 1 Days from anthesis to breaker stage for control and ERF.B3-SRDX lines.

 1

	Lines

  . The highly induced mRNA accumulation of the tomato Sl-ERF.B3 gene in fruit at the breaker stage suggested a putative role of this gene in fruit ripening. The dramatically delayed time from anthesis to the breaker stage was an apparent effect of dominant repression of Sl-ERF.B3 on early fruit development. The time from anthesis to the breaker stage was delayed by approximately 16 days in the dominant repressor (ERF.B3-SRDX) lines compared with the wild type (Table1), indicating a strong influence of dominant repression of Sl-ERF.B3 on tomato early fruit development. Another obvious effect of dominant repression of Sl-ERF.B3 on fruit development is the significantly reduced fruit size with a bumpy appearance from the young green fruit stage, which is probably caused by a reduction in epidermal cell size and a defect in the normal or coordinated expansion of the pericarp. The difference in color development between wild-type and the ERF.B3-SRDX fruits became obvious after

Acknowledgements

First of all, I would like to thank my PhD supervisors, Prof. Mondher Bouzayen and Dr.

Farid Regad, for supporting me during these past four years. Words cannot express how much I appreciate Prof. Bouzayen for guiding, motivating, supporting and training me throughout these years of my studies in the GBF laboratory. Dr. Regad has always been supportive and has given me enough freedom to pursue independent work. I would have never succeeded in my project without his help and support.

I am also very grateful to Dr. Julien Pirrello for his supervision at the beginning of my Ph.D. research project in plant molecular biology. His advices helped me all the time. I would like to express my gratitude and respect to Prof. Zhengguo Li, Prof. Jean-Claude

I gratefully acknowledge the funding sources that made my Ph.D. work possible. I was funded by the China Scholarship Council (CSC).

small size, rapid life cycle, high-throughput capabilities and easy transformation, Micro-Tom was chosen as the main tomato cultivar during my Ph.D. studies. [START_REF] Martí | Genetic and physiological characterization of tomato cv[END_REF].

Chapter II A dominant repressor version of the tomato Sl-ERF.B3

gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components (The Plant Journal, 2013, 76, 406-419) A dominant repressor version of the tomato Sl-ERF.B3 gene confers ethylene hypersensitivity via feedback regulation of ethylene signaling and response components Mingchun Liu 1,2,a , Julien Pirrello 1,2,a , Ravi Kesari 1,2 , Isabelle Mila 1,2 , Jean-Paul Roustan 1,2 , Zhengguo Li 3 , Alain Latché 1,2 , Jean-Claude Pech 1,2 , Mondher Bouzayen 1,2* , Farid Regad 1,2 1 Université de Toulouse, INP-ENSA Toulouse, Génomique et Biotechnologie des Fruits, Avenue de l'Agrobiopole BP 32607, Castanet-Tolosan F-31326, France 2 INRA, Génomique et Biotechnologie des Fruits, Chemin de Borde Rouge, Castanet-Tolosan, F-31326, France 3 School of Life Sciences, Chongqing University, Chongqing 400044, China a These authors contributed equally to this work.

SUMMARY

Ethylene Response Factors (ERFs) are downstream components of the ethylene signal transduction pathway although their role in ethylene-dependent developmental processes remains poorly understood. Since the ethylene-inducible tomato Sl-ERF.B3 has been previously shown to display a strong binding affinity to GCC-box-containing promoters, its physiological significance was addressed here by reverse genetics approach. However, classical up-and down-regulation strategies failed to give clear clue on its roles in planta, Overall, the study indicates that Sl-ERF.B3 has a critical role in regulating multiple genes and identifies a number of ERFs among its primary targets, consistent with the pleiotropic phenotypes displayed by the dominant repression lines.

showing a two-fold increase in transcript accumulation for all four Sl-EIL genes in the ERF.B3-SRDX lines (Figure 8). However, none of the EIN3-like genes gather a consensus ethylene-response element in the promoter. Transactivation assays performed revealed that neither Sl-ERF.B3 nor ERF.B3-SRDX proteins are capable to modulate transcription driven by any of the four Sl-EILs promoters (Figure S6) suggesting that Sl-EILs do not serve as direct target genes for Sl-ERF.B3.

Sl-ERFs are among the target genes of Sl-ERF.B3

Considering the putative role of ERFs in mediating ethylene responses, we examined the transcript levels of Sl-ERF genes in both wild-type and the ERF.B3-SRDX lines. A dramatic change in the transcript levels for a number of ERF genes was revealed in the dominant repressor lines (Figure 9a). That is, among the 19 Sl-ERFs that showed

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

Tomato plants (Solanum lycopersicum cv. Micro Tom) were grown under standard greenhouse culture conditions. The culture chamber rooms were set as follows: 14 hday/10 h-night cycle, 25/20°C day/night temperature, 80% hygrometry, 250 µmol m-2 s-1 intense luminosity.

Constructs and plant transformation

To generate the chimeric repressor transgene, the coding sequence of Sl-ERF.B3 without the stop codon was cloned via blunt-end ligation into the SmaI site of p35SSRDXG in frame to the SRDX repression domain (LDLDLELRLGFA) from SUPERMAN [START_REF] Hiratsu | Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis[END_REF][START_REF] Mitsuda | Efficient production of male and female sterile plants by expression of a chimeric repressor in Arabidopsis and rice[END_REF]. Agrobacterium tumefaciens-mediated transformation of tomato plants was carried out according to [START_REF] Wang | The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis[END_REF] and transformed lines were selected on a hygromycin-containing medium. All experiments were carried out using homozygous lines from F3 or later generations.

Transient expression using a single cell system

Protoplasts used for transfection were isolated from suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells according to [START_REF] Leclercq | Molecular and biochemical characterization of LeCRK1, a ripening-associated tomato CDPKrelated kinase[END_REF]. The synthetic reporter construct (4xGCC-GFP) was generated by fusing the synthetic GCC-box promoter to the coding region of the GFP [START_REF] Pirrello | Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differential responses to ethylene[END_REF]. Reporter constructs were also generated with native promoters, Sl-osmotin (C08HBa0235H18.1) and Sl-ERFs

Sl-ERF.E2

F 5'-ACTTCGTGAGGAAACCCTGAAC-3' R 5'-GTTACTAATATAAGTCATGTTGGGCTGAA-3'

Chapter III

The chimeric repressor version of ERF.B3 shows contrasting effects on tomato fruit ripening

(Manuscript in preparation)

The

chimeric repressor version of Sl-ERF.B3 shows contrasting effects on tomato fruit ripening

Mingchun Liu 1,2 , Gianfranco Diretto 3 , Julien Pirrello the strongest phenotype, seed weight is less than half of the wild type (Figure S1C).

ERF.B3-SRDX lines produced hairless seed with altered morphology (Figure S1B).

ERF.B3-SRDX fruits fail to display a red-ripe phenotype

In addition to the altered fruit shape and size, ERF.B3-SRDX lines exhibited distinct ripening changes. The ripening-related phenotypes were investigated in wild-type and ERF.B3-SRDX lines using fruits at different development stages sampled from the same truss. Dramatic changes were revealed in the transgenic lines with regard to both the time at which the ripening process starts and the speed at which it proceeds. The onset of ripening occurs with at least two weeks delay in ERF.B3-SRDX lines (57 days postanthesis) than in wild type (41 days post anthesis) suggesting that the attainment of competence to ripen is dramatically delayed in the transgenic lines (Table 1).

Decreased lycopene and increased beta-carotene levels are responsible for the orange-ripe phenotype in ERF.B3-SRDX fruits

To investigate the cause of the altered pigmentation in ERF.B3-SRDX fruits, LC-PDA-MS analysis of carotenoid levels was performed on wild-type and ERF.B3-SRDX fruits at both breaker and breaker+7 stages. Notably, levels of lycopene and its precursors phytoene, phytofluene, ζ-carotene and neurosporene were significantly decreased in the ERF.B3-SRDX fruits at the breaker stage (Figure 5). At the ripe stage (Br+7), lycopene levels and its precursors were dramatically decreased, and, a sharp increase in β-carotene content was also observed in the ERF.B3-SRDX lines (Figure 5) in keeping with the orange-ripe phenotype. To uncover the molecular basis of the altered carotenoid composition in ERF.B3-SRDX lines, we examined the transcript levels of genes involved in carotenoid biosynthesis pathway at different stages of fruit ripening by quantitative RT-PCR (Figure 6). Even

Ethylene and ripening-related genes are highly induced in ERF.B3-SRDXexpressing fruits

To gain more insight on the regulation of fruit ripening in ERF.B3-SRDX lines, we examined transcript accumulation of a set of ripening-related genes. The expression of ethylene biosynthesis genes, such as ACS2, ACS4 and ACO1, was significantly higher in ERF.B3-SRDX expressing fruits than in wild type (Figure 8). Transcript accumulation of these genes was similarly low in transgenic and control fruit at mature green stage, but was more strongly induced after the breaker stage in the dominant repressor lines, concomitant to the rise in ethylene production. In addition, mRNA accumulation of ethylene-inducible genes, like E4 and E8 was also increased in ERF.B3-SRDX, consistent with the elevated ethylene production (Figure 8). The transcript accumulation of a major fruit polygalacturonase gene, PG2A, involved in ripening-related cell wall metabolism, was significantly induced in ERF.B3-SRDX fruits (Figure 8) in line with the enhanced softening phenotype.

A number of ERF gene family members show altered expression in the ERF.B3-SRDX lines

Considering the putative role of ERFs in mediating ethylene responses, and given the role of ethylene in regulating the ripening process, we examined the transcript levels of Sl-ERF genes in both wild-type and the ERF.B3-SRDX fruits. A dramatic change in the transcript levels for a number of ERF genes was revealed in the dominant repressor lines (Figure 10). That is, among the 25 Sl-ERFs that showed detectable transcript accumulation, 10 were significantly down-regulated in the ERF.B3-SRDX dominant repressor lines while 8 Sl-ERFs displayed up-regulation in transgenic lines (Figure 10).

Of particular note, accumulation of transcripts corresponding to Sl-ERF.A1, whose expression is strongly induced during ripening (Figure 1), was dramatically enhanced in the ERF.B3-SRDX expressing lines.

DISCUSSION

Our previous study has demonstrated that tomato Sl-ERF.B3 plays an important role in controlling pleiotropic ethylene responses in tomato via feedback regulation of ethylene

RNA isolation and quantitative RT-PCR

Fruits were harvested, frozen in liquid nitrogen and stored at -80°C. Total RNA from pericarp of at least five individual fruits at each developmental stage analyzed in this article was extracted using a Plant RNA Purification Reagent (Invitrogen, Cat. No.

12322-012) according to the manufacturer's instructions. Total RNA was then DNasetreated (Invitrogen, Cat. No. AM1906) to remove contaminating genomic DNA. Firststrand cDNA was reverse transcribed from 2 μg of total RNA using an Omniscript

Reverse Transcription kit (Qiagen, Cat. No. 74904) following the manufacturer's instructions. Gene-specific primers were designed by Primer Express software (PE-Applied Biosystems) and were further checked using BLAST against all tomato unigenes (Tomato unigene database). Quantitative real-time PCR analyses were performed as previously described [START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF]. The primer sequences used in this study are listed in Supplemental Table S2.

LC-MS analysis of fruit carotenoids

Carotenoid extractions have been performed as previously described [START_REF] Fantini | Dissection of Tomato Lycopene Biosynthesis through Virus-Induced Gene Silencing[END_REF]. Briefly, 5 mg of ground lyophilized tomato fruit powder were extracted with chloroform (spiked with 50 mg l -1 DL-α-tocopherol acetate as internal standard) and methanol (2:1 by volume); subsequently, 1 volume of 50 mM Tris buffer (pH 7.5, containing 1 M NaCl) was added and samples were kept 20 minutes on ice. After centrifugation (15,000 g for 10 minutes at 4°C), the organic hypophase was collected and the aqueous phase was re-extracted with the same amount of spiked chloroform.

Combined organic phases were then dried by speedvac and resuspended in 100 μl of ethyl acetate. For each genotype, at least five independent extractions were performed.

LC-MS analyses were carried out using a Discovery LTQ-Orbitrap mass spectrometry system (Thermo Fischer Scientific) operating in positive mode-atmospheric pressure chemical ionization (APCI), coupled to an Accela U-HPLC system (Thermo Fischer Scientific, Waltham, MA). LC separations were performed using a C30 reverse-phase column (100 x 3.0 mm) purchased from YMC (YMC Europe GmbH, Schermbeck, Germany). The mobile phases used were methanol (A), water/methanol (20/80 by volume), containing 0.2% ammonium acetate (B), and tert-methyl butyl ether (C). The

Fruit firmness

Fifteen fruits from each line were harvested at the breaker stage and the firmness was assessed using Harpenden calipers (British Indicators Ltd) as described by [START_REF] Ecarnot | Rapid phenotyping of the tomato fruit model, Micro-Tom, with a portable VIS-NIR spectrometer[END_REF].

Ethylene measurements

Fruits at each developmental stage were harvested and placed in open 120-ml jars for 2 h to minimize the effect of wound ethylene caused by picking. Jars were then sealed and incubated at room temperate for 35 min, and 1 ml of headspace gas was injected into an Agilent 7820A gas chromatograph equipped with a flame ionization detector. Samples were compared with reagent grade ethylene standards of known concentration and normalized for fruit weight.

Supplemental data for chapter III

Chapter IV

General conclusions and perspectives