Julien Pirrello
email: julien.pirrello@cirad.fr

Narasimha Bc

Prasad

Wangshu Zhang

Kunsong Chen

Isabelle Mila
email: mila@ensat.fr

Mohamed Zouine
email: mohamed.zouine@ensat.fr

Alain Latché
email: latche@ensat.fr

Jean Claude Pech
email: pech@ensat.fr

Masaru Ohme-Takagi
email: m-takagi@aist.go.jp

Farid Regad
email: regad@ensat.fr

Mondher Bouzayen
email: bouzayen@ensat.fr

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

Keywords: Ethylene, ERF, Transcriptional regulation, cis-regulatory elements, tomato

Background

The phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Ethylene Response Factor genes (ERF) which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins.

Results

A set of 28 cDNA clones encoding ERFs in the tomato (Solanum lycopersicon) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis-acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters.

Based on their expression pattern, ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones.

Conclusions

The data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner.

Background

The gaseous plant hormone ethylene is reported to play an active 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 abiotic stresses and pathogen attacks [START_REF] Bleecker | Ethylene: a gaseous signal molecule in plants[END_REF][START_REF] Johnson | The ethylene gas signal transduction pathway: a molecular perspective[END_REF][START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF]. Components of ethylene signalling have been extensively studied mainly in Arabidopsis model plant [START_REF] Benavente | Molecular mechanisms of ethylene signaling in Arabidopsis[END_REF] revealing a linear transduction pathway that leads to the activation of transcriptional regulators belonging to the Ethylene Response Factor (ERF) type. These last components of the ethylene signalling pathway are responsible for modulating the transcription of ethylene-regulated genes. Whereas the apparent simplicity of the ethylene transduction pathway cannot account for the wide diversity of plant responses to ethylene, the molecular mechanisms that enable this hormone to drive different physiological and developmental processes in an appropriate way are yet to be elucidated. Being encoded by one of the largest family of plant transcription factors, ERF proteins are the most suited step of ethylene signalling where the diversity and specificity of ethylene responses may originate.

ERFs are trans-acting factors unique to plants shown to bind specifically to GCC box cisacting elements found in the promoter regions of ethylene-responsive genes [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF][START_REF] Solano | Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1[END_REF]. The ERF family is part of the AP2-containing domain superfamily which also contains the AP2 and RAV families of transcription factors [START_REF] Riechmann | Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[END_REF]. The AP2 family is characterized by the presence of two copies of the AP2 domain initially described in the Arabidopsis homeotic gene APETALA2 [START_REF] Jofuku | Control of Arabidopsis flower and seed development by the homeotic gene APETALA2[END_REF]. The RAV family contains two DNA binding domains, an AP2-like domain that binds the CAACA motif and a B3-like domain that binds the CACCTG motif [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]. The ERF type family is further divided into two major subfamilies, the ERF and the CBF/DREB (C-repeat binding factor/dehydration responsive element binding factor) subfamilies of transcription factors [START_REF] Sakuma | DNAbinding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible gene expression[END_REF] both containing a single AP2 domain. DREB subfamily is characterized by the presence of a valine and glutamic acid respectively at position 14 and 19 in the AP2 domain, whereas alanine and aspartic acid are conserved in the corresponding positions for ERFs [START_REF] Sakuma | DNAbinding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible gene expression[END_REF]. It has been demonstrated that the amino acid residues involved in DNA binding are not conserved between AP2 and ERFs [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][START_REF] Nole-Wilson | DNA binding properties of the Arabidopsis floral development protein AINTEGUMENTA[END_REF]. ERF type proteins have been first isolated in the context of plant responses to biotic stress while DREB are associated with abiotic stress. ERFs have been shown to bind the GCC-box found in ethylene-responsive genes and DREBs to the DRE cis-regulatory element. In Arabidopsis, ERFs account for up to 53 % of the total ERF/DREB proteins [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF]. In poplar and rice ERF proteins represent 54 % of the ERF/DREB family [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF][START_REF] Zhuang | Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa[END_REF] whereas it represents 51 % in soybean [START_REF] Zhang | Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.)[END_REF]. Comparative structural analysis of Arabidopsis and rice ERFs, have been performed in silico using either the entire protein sequence for phylogenetic analysis [START_REF] Fujimoto | Arabidopsis ethyleneresponsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF][START_REF] Tournier | New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element[END_REF], or the conserved AP2 domain [START_REF] Sakuma | DNAbinding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible gene expression[END_REF][START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF] to infer relationship between ERF family members. In Arabidopsis the ERF subfamily contains 65 members and is divided into 5 subclasses based on the conservation of the AP2 domain [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF]. The ERF domain, first identified as a conserved motif of 59 amino acids in four DNA-binding proteins from Nicotiana tabacum [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF] is characterized by 3 β-sheets and 1 α-helix and allows binding of the ERFs to the GCC-box [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF][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]. In addition to the requirement for a perfectly conserved GCC motif, it has been suggested that the binding affinity may also depend on the nucleotide environment of the GCC box as well as on the nature of some amino acid residues within the ERF biding domain [START_REF] Tournier | New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element[END_REF]. Moreover, in line with the data reporting that Pti4, an ERF type transcription factor, is able to bind promoters lacking a GCC box cis-element [START_REF] Chakravarthy | The tomato transcription factor Pti4 regulates defense-related gene expression via GCC box and non-GCC box cis elements[END_REF], recent studies demonstrated that ERFs can also bind different cis-elements such as VWRE (Vascular Wounding Responsive Element) [START_REF] Sasaki | Two novel AP2/ERF domain proteins interact with cis-element VWRE for wound-induced expression of the Tobacco tpoxN1 gene[END_REF].

ERFs are ubiquitous in plant kingdom and their functional implications have been studied in various plant species and in a wide range of processes including hormonal signal transduction [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF], response to biotic [START_REF] Gu | Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase[END_REF][START_REF] Yamamoto | Elicitor-responsive, ethylene-independent activation of GCC box-mediated transcription that is regulated by both protein phosphorylation and dephosphorylation in cultured tobacco cells[END_REF] and abiotic stresses [START_REF] Dubouzet | OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt-and cold-responsive gene expression[END_REF][START_REF] Liu | Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and lowtemperature-responsive gene expression, respectively, in Arabidopsis[END_REF][START_REF] Stockinger | Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cisacting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[END_REF], regulation of metabolic pathways [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] Broun | WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis[END_REF][START_REF] Van Der Fits | ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[END_REF][START_REF] Zhang | Overexpression of WXP1, a putative medicago truncatula AP2 domain-containing transcription factor gene, increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa (medicago sativa)[END_REF] and developmental processes [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] Van Der Graaff | Activation tagging of the LEAFY PETIOLE gene affects leaf petiole development in Arabidopsis thaliana[END_REF]. Expression studies indicated that some ERFs are regulated by abiotic stresses such as wounding and salt stress [START_REF] Tournier | New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element[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] 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] and more recently, it was demonstrated that ERFs are also involved in seed germination [START_REF] Pirrello | Sl-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination[END_REF]. A number of studies demonstrated that in addition to regulating the expression of ethylene-responsive genes harbouring the GCC-box, ERFs can also regulate jasmonic acid and salicylic acidresponsive 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][START_REF] Lorenzo | ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense[END_REF]. The presence of distinctive structural features among ERF classes suggests that different members of this family may display different functionalities and binding activities. The differential binding activities of ERFs might represent the mean by which ethylene signalling targets a specific set of genes thus providing the basis of the observed tissue and developmental specificity of plant responses to the hormone. However, structural characterization of ERF proteins has been restricted to in silico analysis and so far the structure/function relationship has been only superficially addressed [START_REF] Wehner | Highthroughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function[END_REF].

The present study shows that the ERF gene family in the tomato is organised into 9 subclasses defined by distinct structural features. Based on functional analysis of 28 tomato ERFs and through testing their ability to activate or repress transcriptional activity of target genes, the data suggest that functional activity is conserved among ERF proteins sharing the same structural features. Moreover, data demonstrate that flanking regions of the core GCCbox sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. The data also show that ERF genes display tissue-specific patterns of transcript accumulation and uncover their regulation by auxin.

Results

Isolation and phylogenetic analysis of tomato ERFs

Tomato ERF cDNA clones were initially identified by TBLASTN search [START_REF] Altschul | Basic local alignment search tool[END_REF] in the tomato unigene database (http://solgenomics.net/) using a consensus sequence within the AP2/ERF domain as a query sequence. Forty nine unigenes were found from which AP2, RAV and DREB sequences were discarded based on their distinctive features regarding the number of AP2 domains, the presence of a B3-like domain and the presence of conserved amino acid residues, respectively. Using RACE-PCR extension approach, complete CDSs were obtained for 28 ERF unigenes that are representative of the main ERF sub-groups. Subsequently, building on the annotated whole tomato genome sequence recently released [START_REF] Sato | The tomato genome sequence provides insights into fleshy fruit evolution[END_REF], a genome wide in silico screening allowed the identification of up to 146 genes encoding putative AP2containg proteins distributed into 77 ERFs, 48 DREBs, 18 AP2 and 3 RAVs (Table 1). Since Arabidopsis ERFs have been classified so far using the AP2/ERF domain exclusively [START_REF] Sakuma | DNAbinding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible gene expression[END_REF], we constructed the ERF phylogenetic trees using either the whole protein sequences or only the AP2/ERF domain. Due to the weak homology among ERF proteins outside the AP2/ERF domain, identical classification patterns were obtained with the two clustering methods (Figure 1). However, while 10 subclasses (A to J) define the ERF family in Arabidopsis, only nine are represented in tomato, which lacks subclass I (Figure 1, Table 2). The distribution of the tomato ERF proteins into nine individual subclasses is further supported by the presence within the AP2 domain of distinctive motifs and amino acid signatures specific for each subclass previously described in Arabidopsis [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF] . In the absence of a consensual nomenclature and due to a lack of rational naming of ERF genes across plant species, we attempted to rename the tomato genes by giving a letter (A to J) to discriminate between different subclasses and a number to distinguish between members within the same subclass (Table 3). While complying with the most complete classification available in Arabidopsis [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF] the proposed nomenclature better clarifies the correspondence between ERF subclasses in different species. The correspondence between the classification adopted here and those previously proposed for tomato ERFs [START_REF] Tournier | New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element[END_REF][START_REF] Sharma | Identification, phylogeny, and transcript profiling of ERF family genes during development and abiotic stress treatments in tomato[END_REF] and for Arabidopsis ERFs [START_REF] Sakuma | DNAbinding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible gene expression[END_REF][START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF] is given in Table 2. In this newly proposed classification class XI, defined by Sharma et al. 2010, splits into two subclasses I and J as proposed by [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF] [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF] clearly demonstrating that Arabidopsis subclass I has no representatives in the tomato. Of particular note, members of subclass I harbour imperfect AP2 domain and comparatively to other subclasses is underrepresented in Arabidopsis while it is missing in the tomato. Like in Arabidopsis, the overwhelming majority of tomato ERF genes (59 out of 77 genes) are intronless (Table 2). However, while Arabidopsis ERFs can bear at most a single intron, among the 18 tomato intronic genes, 5 have two introns. ERF intronic genes are found in four subclasses in Arabidopsis while they are spread across 6 subclasses in the tomato (Table 4). Although tomato ERF genes could be localized on 12 chromosomes, they present an uneven distribution with Chromosome 1 and 3 bearing 13 and 11 genes, respectively (Table 5). Fine mapping of ERFs on the tomato genome revealed that these genes could be distributed individually or in clusters. In particular, chromosome1 contains a cluster of 5 ERF genes (Solyc01g09300, Solyc01g09310, Solyc01g09320, Solyc01g09340, Solyc01g09370) that likely result from tandem duplication events (Table 5) as suggested by their position within sub-group B in the neighbourhood phylogenetic tree (Figure 1).

A I IX IXa B3 B III IX IXb B3 C IX IXc B3 D X X B4 E IV VII VII B2 F II VIII VIII B1 G VI VI B5 H V V B6 I XI Xb-L B6 J XI VI-L B6

Table 3 Correspondence between the proposed nomenclature of tomato ERFs and previous nomenclatures

New name Previous name Reference

Sl-ERF.A.1 - Sl-ERF.A.2 LeERF1 [17] Sl-ERF.A.3 Pti4 [40] Sl-ERF.B.1 - Sl-ERF.B.2 - Sl-ERF.B.3 LeERF4 [17] Sl-ERF.C.1 JERF2/TERF1 [41] Sl-ERF.C.2 - Sl-ERF.C.3 - Sl-ERF.C.4 TSRF1 [42] Sl-ERF.C.5 Pti5 [40] Sl-ERF.C.6 ERF5 [43] Sl-ERF.D.1 - Sl-ERF.D.2 - Sl-ERF.D.3 - Sl-ERF.D.4 - Sl-ERF.E.1 LeERF2 [17] Sl-ERF.E.2 JERF1 [42] Sl-ERF.E.3 JERF3 [44] Sl-ERF.E.4 - Sl-ERF.F.1 - Sl-ERF.F.2 - Sl-ERF.F.3 - Sl-ERF.F.4 - Sl-ERF.F.5 LeERF3 [17] Sl-ERF.G.1 - Sl-ERF.G.2 Pti6 [40] Sl-ERF.H.1
-Reference to the first paper describing the gene is given in the last column

H 2 Solyc01g065980 E 1 Solyc03g116610 H 1 Solyc03g118190 D 1 Solyc03g123500 C 2 Solyc04g012050 D 1 Solyc04g051360 D 1 Solyc04g054910 H 2 Solyc04g071770 D 1 Solyc06g063070 A 2 Solyc06g065820 H 1 Solyc06g068360 H 1 Solyc06g068830 D 2 Solyc09g075420 E 1 Solyc12g042210 D 1 Solyc12g049560 E 1 Solyc12g056590 D 1 Solyc12g056980 H 1

Subcellular localization of tomato ERFs

All

Relationship between structural features and functional activity of ERFs

To address whether specific structural features affect the ability of ERFs from different subclasses to drive the transcriptional activity of target promoters, a transient expression assay in a single cell system was used. Tobacco BY-2 protoplasts were co-transformed with effectors constructs carrying the ERF coding sequences driven by the 35S constitutive promoter and reporter constructs consisting of the GFP coding sequence driven either by a GCC-rich synthetic promoter or a native ethylene-responsive promoter. To discriminate between the situation where the absence of activity is due to the lack of binding to the target promoter from those where the ERF binds but remains neutral on the promoter activity, we used chimerical constructs as effectors consisting of ERF coding sequences fused to the SRDX repressor motif [START_REF] Hiratsu | Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis[END_REF]. Since the dominant repression activity of the chimerical construct is mediated by epigenetics mechanisms [START_REF] Kagale | EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression[END_REF], the absence of repression with any of the ERF-SRDX constructs can be interpreted as resulting from the incapacity of the ERF to recognise the target promoter. Figure 3A shows that ERF proteins can not only act as activators or repressors, but can also be neutral on the ethylene-responsive promoters.

Experiments carried out with the SRDX fusion demonstrate that all ERFs have the ability to bind the GCC box containing promoter except ERF.A.1 and ERF.E.2. Members of subclass C display the strongest activation activity whereas ERFs from subclass F show the highest repression activity (Figure 3A). ERFs from subclass A, B and E are weak activators on the GCC box whereas those from class G and H are neutral (Figure 3A). To test whether the regulation of the ethylene-responsive promoters by ERF proteins is strictly dependent on the presence of a canonical GCC motif, tomato osmotin and E4 native promoters containing or lacking a conserved GCC motif, respectively, were fused to the GFP coding sequence and used as reporter constructs. The data obtained with the fused SRDX repressor motif suggest that up to 16 ERFs out of the 28 tested in this study (Figure 3B) are capable of binding the osmotin promoter (p<0.05). In line with the results obtained with the synthetic GCC promoter, the osmotin promoter is strongly activated by ERFs from subclass C and repressed by ERFs from subclass F. By contrast, the transcriptional regulation of the native osmotin promoter by several ERFs from other subclasses did not match their behaviour with the synthetic promoters. Notably, the native osmotin promoter contains a number of cis-elements that are likely to drive the transcriptional activity in a more complex way than that displayed by the synthetic promoter containing simply the GCC-box. Overall, ERFs showing the strongest effect on the synthetic GCC promoter also display the most significant effect on the osmotin complex promoter. E4 promoter is a well described ethylene-inducible promoter lacking the conserved GCC motif that represent a potential target for ERFs. The data presented in Figure 3C indicate that ERF.C.5, ERF.D.3, ERF.F.3, ERF.F.4 and ERF.F.5, are capable to bind the E4 promoter and to repress its activity, suggesting that some ERFs can be active on ethylene-responsive promoters lacking the canonical GCC box cis-element.

The data reveal that ERF proteins are more active on the synthetic GCC promoter than on native complex promoters. The activity of the ERFs on the GCC box (Figure 3A) indicate that members of the same sub-class tend to modulate the activity of the target promoters in the same way (ANOVA p<10 -5 ). Half of the ERFs tested (14 out of 28) have significant effect on the synthetic promoter while only 8 ERFs are active on the native osmotin GCCcontaining promoter and 4 on the native E4 lacking a conserved GCC motif.

The impact of the GCC box flanking regions on the binding affinity of ERFs

We have previously demonstrated [START_REF] Tournier | New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element[END_REF] that ERF proteins can display differential affinity to various promoters containing the highly conserved GCC-box. Hence, the hypothesis that the nucleotide environment of the GCC box may impact the binding affinity of the ERFs was tested. A total of 11 ERFs representing all subfamily types were challenged with the native Sl-Chitinase GCC box (5′-A 1 A 2 G 3 A 4 GCCGCCA 11 C 12 T 13 A 14 -3′) or with mutated versions of this motif in gel shift assay experiments (Figure 4A). The four nucleotides flanking the GCC motif (G 3, A 4 , A 11 and C 12 ) were mutated and the binding of the ERFs was tested by EMSA and assessed by phosphoimaging. Mutation of A at position 4 (A 4 ) into T or G dramatically decreased the affinity to the GCC box of all ERFs except that of ERF.F.5 (Figure 4B) while the substitution of A 4 by a C increases the affinity of all tested ERFs. Strikingly, mutation of A 11 to any of the three other nucleotides resulted in a dramatic loss of affinity of ERFs to the GCC box. These data suggest that nucleotides upstream (position 4) and downstream (position 11) to the GCC box impact the binding affinity in the same way for all the ERFs tested. By contrast, substitution of G 3 or C 12 by another nucleotide resulted in different effect on the binding affinity depending on the ERF tested (figure 4B).

Expression pattern of ERFs

To assist with the elucidation of the physiological function of tomato ERFs, we sought to gain more insight on the spatio-temporal expression at the transcriptional level of each member of the gene family. Transcript accumulation was assessed by qRT-PCR for 25 ERFs whereas for the remaining three, transcripts could not be detected in the nine different plant tissues tested. The heatmap representation of the global expression pattern allowed the clustering of tomato ERFs in three main clades (Figure 5), with clade I (16 ERFs) corresponding to genes preferentially expressed in reproductive tissues, clade II (4 ERFs) corresponding to genes with an ubiquitous expression without tissue preference, and clade III (5 ERFs) mainly active in vegetative tissues. Subclade Ia displays a distinctive expression pattern associated with the breaker +2 and flower stages, while subclade Ib contains genes whose expression is more pronounced at the breaker + 2 and breaker stages. These results suggest that some ERFs operate specifically in vegetative tissues (clade III) or reproductive organs (clade I), whereas few ERFs seem to be ubiquitous and might be involved in the development of various organs (clade II). Interestingly some ERFs are strongly expressed in B+2 and Flowers stage (clade Ia) suggesting a role in fruit set and during ripening while others seem to be active more specifically at the onset of ripening (Breaker and Breaker + 2, clade Ib). These data also point out to the absence of a clear relationship between the structurally defined ERF subclasses and their tissue type expression.

Figure 5 Heatmap representation of the expression of ERF genes in different tomato tissues.

The data obtained by quantitative RT-PCR correspond to the levels of ERF transcripts in total RNA samples extracted from Roots (R), Leaves (L), Stem (St), Flower (Fl), Early Immature Green (IMG), Mature Green (MG), Breaker (B), Breaker + 2 days (B+2), Breaker + 7 days (B+7). The data presented correspond to 3 independent biological repetitions. Red and white colours correspond to high and weak expression of the ERF genes, respectively. Heat map was generated using R software

Ethylene and auxin regulation of ERF genes

It has been reported that beside their regulation by ethylene, ERF genes can also be induced by other hormones among which auxin [START_REF] Gutterson | Regulation of disease resistance pathways by AP2/ERF transcription factors[END_REF]. To test the responsiveness of tomato ERF genes to ethylene and auxin, transcripts accumulation in seedlings treated with ethylene or auxin for 5 and 3 hours, respectively, was assessed by qRT-PCR. E4 and SAUR, known as ethylene and auxin-responsive genes, respectively, were used as controls to validate the hormone treatment. Transcript accumulation levels (Figure 6) indicate that 13 tomato ERFs are significantly induced (p<0.06) by ethylene and 4 significantly (p<0.06) down-regulated by this hormone. Interestingly, the expression of up to 12 ERFs is also positively regulated by auxin. Among these, 4 ERF genes (ERF.B.

Discussion

A wide range of developmental processes are known to rely at least partly on ethylene action, yet, the molecular mechanisms underlying the diversity of plant responses to this hormone are still awaiting some in depth characterization. The present study brings new insight on structural features that impact the binding affinity of Ethylene Response Factors (ERFs) to ethylene responsive promoters. Upstream of ERFs, the ethylene signal propagates via a linear transduction pathway which can hardly account for the wide range of ethylene responses. In its downstream part, ethylene signalling leads to the activation of ERFs that are encoded by one of the largest multigene family of transcription factors [START_REF] Riechmann | Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[END_REF], and these transcriptional mediators may therefore represent one of the main step where the diversity and selectivity of ethylene responses can take place. The structural and functional characterisation of tomato ERFs carried out here provides some clues to the means by which ethylene selects target genes that are required to bring into play the appropriate physiological responses in a tissuespecific and developmentally defined manner. Taking advantage of the recent release of the complete annotated tomato genome sequence [START_REF] Sato | The tomato genome sequence provides insights into fleshy fruit evolution[END_REF], a genome wide in silico screening identified 146 genes encoding putative AP2-containing proteins distributed into 77 ERFs, 48 DREBs, 18 AP2 and 3 RAVs (Table 1). Subsequently, the functional analysis concentrated on 28 members of the tomato ERF family encompassing 8 of the 9 subclasses of the gene family.

The data indicate that all ERF proteins display a clear nuclear localization, even though two members of the family lack any predictable nuclear targeting motif suggesting that their import to the nucleus may occur by a non-conventional localization signal or they might be conveyed to this compartment via an interaction with a yet unknown nuclear localized partner.

The large diversity among ERFs is well illustrated by the presence of distinctive motifs that are sufficient to discriminate between different subclasses. Recent studies already suggested a link between the structural classification of ERFs and their physiological function [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF], yet, this hypothesis has not been supported by strong experimental data at the level of whole ERF family. It was reported that within class E, the conserved specific N-terminal domain, initially described in tomato ERFs [START_REF] Tournier | New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element[END_REF], is dedicated to an oxygen dependent sequence of posttranslational modifications that leads to degradation of the protein under aerobic conditions. In hypoxia condition, the RAP2.12 ERF protein is relocated from plasma membrane to nucleus to activate gene expression associated with hypoxia acclimation [START_REF] Licausi | Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization[END_REF]. These results suggest that members of the subclass E are involved in hypoxia response.

Transient expression assays indicated that the activity of ERFs on synthetic GCC box promoter correlates well with their structural classification. That is, ERFs from subclass C are strong activators and those belonging to subclasses A, B and E are weak activators, whereas members of subclass F are clear repressors. By contrast, ERFs from subclasses E, G and H display no activity when challenged with the GCC box. Noteworthy, ERFs show weaker activity on the native complex osmotin promoter than on the synthetic promoter. This may be due to the high basal expression displayed by the native osmotin promoter in the absence of ERF effectors, which likely minimizes the induction effect observed in the presence of ERFs (Additional file 3). Moreover, the experimental data show that ERFs can regulate transcription from both types of ethylene-responsive promoters either containing (osmotin promoter) or lacking (E4 promoter) the GCC-box thus revealing their capacity to bind ciselements without the canonical GCC motif. While it cannot be ruled out that ERFs may induce indirectly the E4 promoter through the activation of primary target genes encoding transcriptional proteins active on the E4 promoter, our data suggest that ERFs can interact with other cis-acting elements beside the canonical GCC motif. This is in agreement with recent studies demonstrating that, in addition to the GCC box, ERFs can bind cis-acting elements such as VWRE and GCC-like [START_REF] Sasaki | Two novel AP2/ERF domain proteins interact with cis-element VWRE for wound-induced expression of the Tobacco tpoxN1 gene[END_REF][START_REF] Andriankaja | AP2-ERF transcription factors mediate Nod factor dependent Mt ENOD11 activation in root hairs via a novel cis-regulatory motif[END_REF][START_REF] Wu | ERF protein JERF1 that transcriptionally modulates the expression of abscisic acid biosynthesisrelated gene enhances the tolerance under salinity and cold in tobacco[END_REF][START_REF] Xu | Isolation and molecular characterization of the Triticum aestivum L. ethyleneresponsive factor 1 (TaERF1) that increases multiple stress tolerance[END_REF]. The clustering of the ERF proteins according to their activity on the GCC box, indicates that members of the same subclass tend to modulate the activity of the target promoters in the same way (Figure 3A). While the data show that the repressor and activator activities of ERFs correlate well with their structural classification, the recognition of the GCC-box by a particular ERF seems to depend on the promoter used and might be impacted by the nucleotide environment surrounding the cisacting element.

Indeed, gel shift assay experiments show that the environment of the GCC box greatly impacts the affinity of ERF proteins to the conserved cis-element. In particular, the data (Figure 4) clearly demonstrate that the nature of the nucleotides at position N 3 and N 12 can have different and sometime opposite effect on different ERFs, thus suggesting that these bases contribute to the binding specificity of various ERFs to target promoters. Taking together these results suggest that the two nucleotides directly flanking the GCC box at position N 4 and N 11 are essential for the binding of any ERF to the GCC-box and, therefore, their nature determines whether or not the cis-element is functional for the binding to an ERF. By contrast, the nature of nucleotides at position N 3 and N 12 can either enhance or reduce the binding affinity, which supports their contribution in determining the binding specificity of various ERFs to target promoters. Previous work carried out with native promoters already stressed the putative role of the GCC box flanking regions in impacting the binding activity of the ERFs [START_REF] Tournier | New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element[END_REF]. The same study also postulated that variation in amino acid composition within the binding domain may also impact the binding affinity of ERFs to their target promoters [START_REF] Tournier | New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element[END_REF]. It was also postulated that the conserved motifs lying outside the DNA binding domain may also contribute to the differential affinity displayed by ERF proteins towards the GCC box-containing promoters. Recent study demonstrated that the transactivating activity of some ERFs was localized in the acidic domain [START_REF] Jung | The barley ERFtype transcription factor HvRAF confers enhanced pathogen resistance and salt tolerance in Arabidopsis[END_REF]. In keeping with this observation, ERF.D.1 and ERF.D.2 are lacking the acidic activator domain and accordingly are unable to activate the synthetic GCC box promoter. On the other hand, subclasses A, B, C and E ERFs harbour one or more acidic domains, however, not all of them display transcriptional activation of the synthetic promoter. Members of class F have an EAR repressor domain in the C-terminal region of the protein and accordingly they are all repressors of the activity of the GCC box-containing promoters.

Unravelling the expression pattern of ERF genes provides important clues towards the elucidation of their physiological function since physiological effects of transcriptional mediators not only depend on their activation or repression function, but also on their specific pattern of expression. While consistent with previous description concerning the expression of some ERF genes like Pti4 [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] and ERF1-4 [START_REF] Tournier | New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element[END_REF], the data distinguish different ERF groups depending on whether they are preferentially expressed in vegetative, or reproductive tissues or whether they show ubiquitous expression with no tissue preference. Noteworthy, structural subclasses did not display tissue or organ specialisation as exemplified by the fruit development process throughout which ERFs from structurally distinct subclasses are expressed from flower anthesis to fruit ripening. This could be in line with the hypothesis that different responses to ethylene taking place in different tissues and at different developmental stages are mediated by different ERF proteins. Tissue and abiotic stress expression pattern of tomato ERFs has been already reported [START_REF] Sharma | Identification, phylogeny, and transcript profiling of ERF family genes during development and abiotic stress treatments in tomato[END_REF], however, the hormone-dependent expression remained unknown. As expected, a high number of ERF genes are ethylene-responsive with 13 ERFs being up-regulated and 4 down-regulated upon ethylene treatment. Less expected, our study revealed that up to 12 tomato ERF genes were regulated by auxin among which 6 undergo double regulation by ethylene and auxin while the remaining 6 others are exclusively auxin-responsive. It was already reported that, beside ethylene, ERFs can be regulated by salicylic acid and jasmonic acid [START_REF] Gu | Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase[END_REF][START_REF] Lorenzo | ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense[END_REF][START_REF] Hirota | The auxin-regulated AP2/EREBP gene PUCHI is required for morphogenesis in the early lateral root primordium of Arabidopsis[END_REF], but the auxin regulation of such a high number of ERF genes has not been reported. The auxin regulation of the ERFs shown to be active on ethylene responsive promoters, suggests their potential contribution to the cross-talk between the two hormones. The phytohormones auxin and ethylene are essential regulators of plant development and it is well documented that ethylene and auxin regulate common physiological aspects such as hook formation [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], root hair differentiation [START_REF] Masucci | The rhd6 mutation of Arabidopsis thaliana alters roothair initiation through an auxin-and ethylene-associated process[END_REF], root elongation [START_REF] Pitts | Auxin and ethylene promote root hair elongation in Arabidopsis[END_REF], root growth [START_REF] Rahman | Auxin is a positive regulator for ethylene-mediated response in the growth of Arabidopsis roots[END_REF], and hypocotyl phototropism [START_REF] Harper | The NPH4 locus encodes the auxin response factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue[END_REF]. The mechanisms underlying the interactions between the two hormones are becoming better understood even though only few molecular players of this cross-talk have been identified so far. Besides acting independently on the same target genes, ethylene and auxin can mutually regulate each other's biosynthesis and response pathways. In support to this idea, we previously reported that the down-regulation of Sl-IAA3, encoding a tomato transcriptional regulator from the Aux/IAA type, results in phenotypic responses related to classical auxin and ethyleneregulated processes [START_REF] Chaabouni | Sl-IAA3, a tomato Aux/IAA at the crossroads of auxin and ethylene signalling involved in differential growth[END_REF][START_REF] Chaabouni | Tomato Aux/IAA3 and HOOKLESS are important actors of the interplay between auxin and ethylene during apical hook formation[END_REF]. Therefore, uncovering the auxin responsiveness of some members of the ERF gene family, may define new actors potentially involved in the crosstalk between the two hormones. Global transcriptomic analyses of tomato lines underexpressing Sl-IAA9, another tomato Aux/IAA gene [START_REF] Wang | Regulatory features underlying pollination-dependent andindependent tomato fruit Set revealed by transcript and primary metabolite profiling[END_REF][START_REF] Wang | The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis[END_REF], revealed the altered expression of a high number of ethylene-related genes, further supporting the idea of an active link between auxin and ethylene signalling during the flower to fruit transition. In keeping with the complex role of ERFs in mediating various hormone responses, ERF.A.1 and ERF.D.4 that are highly expressed in mature flowers, display strong up-regulation by auxin but not by ethylene. On the other hand, ERF.A.2 and ERF.C.1 are up-regulated by ethylene but not by auxin, and they are strongly expressed in the flower. Interestingly, many ERFs are expressed in flower and at early ripening stages (B and B+2), suggesting their putative auxin-dependent role in fruit set and fruit ripening process.

Conclusion

The present study provides some molecular clues on how ERFs can contribute to the specificity and selectivity of ethylene responses through (i) the differential expression of the gene family members, (ii) the ability to negatively or positively impact transcriptional activity and, (iii) the capacity to select with specificity their target genes based on the nucleotide environment of the GCC-box. Considering the diversity of their transcriptional activity and expression patterns, ERFs possess the necessary features for channelling ethylene signalling to a selected set of genes required for the appropriate developmental responses or the desired responses to environmental cues. The insight gained in this study opens new prospects towards assigning a specific role for individual ERFs in controlling developmental processes and for identifying the direct target genes for each member of the ERF family.

Methods

Plants growing

Tomato (Solanum lycopersicum cv MicroTom) plants were grown in climatic chamber. The conditions are the following: 14-h-day/10-h-night cycle, 25/20 °C day/night temperature, 80 % hygrometry, 250 μmol.m -2 .s -1 irradiance.

Cloning of over expressing and repressing ERF constructs

The full length CDS were obtained by RACE using BD SMART TM RACE cDNA Amplification kit (Clontech, www.clontech.com) and the complete ERF CDS were, cloned in over-expression vector, with or without SRDX fusion in C-terminal [START_REF] Hiratsu | Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis[END_REF] (Additional files 4, 5).

Ethylene and auxin treatments

Five day-old dark-grown seedlings were treated with air or ethylene gas (50 μL/L) for 5 hours and RNA was extracted from the corresponding tissues. Auxin treatments were performed on 7 day-old light-grown seedlings by soaking (3 hours) in auxin-containing (20 μM) or auxinfree MS solution. Three independent biological repeats were performed for each experiment.

Analysis of ERF gene expression

Phylogenetic analysis

In order to identify Sl-ERF genes in the tomato genome, the AP2/ERF domain of a tomato Ethylene Response Factor sequence (GenBank number NP_001234308) was used as BLAST query sequence against the tomato ITAG2.30(Sl2.40) protein database . The same database was also screened by an HMM analysis using a typical AP2/ERF domain PF00847 as query. One hundred forty six genes were identified as possibly encoding proteins containing the AP2/ERF domain (Table 1). The evolutionary history was inferred using the Neighbour-Joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]. The optimal tree with the sum of branch length = 13.53632771 is shown. The phylogenetic tree was linearized assuming equal evolutionary rates in all lineages [START_REF] Takezaki | The phylogenetic relationship of tetrapod, coelacanth, and lungfish revealed by the sequences of fortyfour nuclear genes[END_REF]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [START_REF] Zuckerkandl | Evolving Genes and Proteins: Evolutionary divergence and convergence in proteins[END_REF] and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 63 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 [START_REF] Tamura | MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0[END_REF]. Conserved motifs were determined using MEME version 3.5.5 [START_REF] Bailey | Fitting a mixture model by expectation maximization to discover motifs in biopolymers[END_REF].

Transcriptional activity tests by transient expression in a single cell system

A synthetic reporter construct (4XGCC-GFP) was generated by fusing a synthetic promoter containing 4 GCC-box repeats upstream of the minimal -42 to +8 TATA box from the 35S promoter of Cauliflower mosaic virus to the coding region of the Green Fluorescent Protein (GFP). Reporter constructs were also generated with native promoters, E4 (S44898) and Sl-Osmotine (C08HBa0235H18.1), fused to GFP. Effectors constructs were generated by fusing the 35S promoter to the CDS of the ERF genes. For transient assays, tobacco (Nicotiana tabacum) BY-2 protoplasts were co-transformed with reporter and effector constructs [START_REF] Chaabouni | Sl-IAA3, a tomato Aux/IAA at the crossroads of auxin and ethylene signalling involved in differential growth[END_REF].

Transformation assays were performed in three independent replicates. After 16 h, GFP expression was analysed and quantified by flow cytometry (FACS Calibur II instrument, BD Biosciences) on a flow cytometry platform (IRF31). Data were analysed using Cell Quest software. For each sample, 100-1000 protoplasts were gated on forward light scatter and the GFP fluorescence per population of cells corresponds to the average fluorescence intensity of the cell population after subtraction of autofluorescence determined with non-transformed BY-2 protoplasts. The data were normalized using an experiment with protoplasts transformed with the reporter vector in combination with the vector used as the effector plasmid but lacking the Sl-ERF coding sequence.

Subcellular localization

YFP N-terminal fusions were obtained with ERF.B1, D.1, H.1 and E.1 and used for tobacco protoplast BY-2 transfection according to Chaabouni et al. 2009 [START_REF] Chaabouni | Sl-IAA3, a tomato Aux/IAA at the crossroads of auxin and ethylene signalling involved in differential growth[END_REF]. The subcellular location of the fluorescence was determined after 20 hours using confocal microscopy.

Electro mobility shift assay

Proteins used for the gel shift assay were produced in vitro using a "TNT®T7 Quick kit for PCR DNA" (Promega, www.promega.com). Primers used for amplification of ERFs were design according to manufacture recommendation and include Kozak T7 sequences. Gel retardation experiments were performed with the GCC-box probe mutated in the flanking nucleotides according to the procedure described in Tournier et al. 2003 [17] (Additional file 7). Scanning and radioactivity quantification were achieved by a "Phosphoimaging Fujifilm Bas 5000" and the software "Image Gauge" (Fuji Film, www.fujifilm.com).

  ERF proteins display at least one canonical nuclear localization signal with the exception of ERF.B.1, D.1 and H.1 that lack any predictable nuclear targeting motif (Additional file 2). Since nuclear import of transcription factors is instrumental to their transcriptional activity, we investigated the subcellular localization of these three tomato ERFs by generating Nterminal fusions with the YFP expressed under the 35S promoter (35S:: YFP-ERF). Transient expression in tobacco BY-2 protoplasts coupled to confocal microscopy analysis clearly indicated that ERF B.1, D.1 and H.1 are exclusively targeted to the nuclear compartment similar to ERF.E.1 which contains a consensus nuclear localization signal (Figure2).

Figure 2

 2 Figure 2 Subcellular localization of Sl-ERFs. ERF.B.1, ERF.D.1, ERF.E.1 and ERF.H.1 proteins were fused to the YFP (Yellow Fluorescent Protein) in the N-terminal region and the chimerical proteins were transiently expressed in BY-2 tobacco protoplasts under the control of the 35S promoter. Subcellular localization was then analyzed by confocal laser scanning microscopy. The merged pictures of the yellow fluorescence channel (middle panels) and the corresponding bright field (left panels) are shown (right panels). Control cells expressing YFP alone are shown in the top panel. The scale bar indicates 10 μm

Figure 3 ERF

 3 Figure 3 ERF-mediated transcription from native and synthetic promoters. Transient expression in single cell system has been used to assess the transcriptional activity of ERF proteins from different subclasses. The fluorescence of the reporter gene was measured by flow cytometry upon co-transfection with a reporter construct (GCC:: GFP or Sl-Osmotin Promoter:: GFP or Sl-E4promoter:: GFP) and an effector construct (35S::Sl-ERF or 35S::Sl-ERF-SRDX). The basal fluorescence obtained in the assay transfected with the reporter construct and an empty effector construct was taken as reference (100 % relative fluorescence). (A) ERF activity on synthetic promoter containing 4 direct repeats of the GCC-box. (B) ERF activity on osmotin native promoter containing the canonical GCC motif.

Figure 4

 4 Figure 4 Binding affinity of ERFs to the GCC-box is impacted by the nucleotide composition of the flanking regions. (A)To assess the role of the nucleotide composition of the close environment of the GCC box, nucleotides flanking the chitinase GCC box were mutated. Different mutated GCC boxes were used as probe in gel shift assay to test the binding affinity of ERFs. (B) Binding affinity of ERFs to the mutated probes. Relative affinity is calculated with non mutated Sl-Chitinase (Solyc10g055810.1) as reference. The data are mean of 3 independent replicates. Analysis of variance with the R package reveals that the flanking region of the GCC box is significantly involved in the affinity of the binding (p<0.05)

  Figure 4

Figure 6

 6 Figure 6

Table 1 Summary of the tomato AP2/ERF superfamily Classification Number

	AP2 family	18
	DREB family	48
	ERF family	77
	RAV family	3
	Total	146

Figure 1 Phylogenetic tree of Arabidopsis and Tomato ERFs. Different

	subclasses are
	named by 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 (Additional file 1) 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 neighbour joining method

Table 2 The distribution of members of the ERF gene family among subclasses constructed in the present study and in previous classifications

 2

		Tomato		Arabidopsis
	(This study)	[17]	[39]	[13]	[10]

Table 4 Presence of intron on tomato ERFs ERF gene Class Intron numbers Solyc01g008890

 4

Table 5 Distribution of ERFs on tomato chromosomes Chromosomes genes per cluster Number of ERFs genes Clusters of ERFs/ERFs

 5

	Ch01	13	1/5
	Ch02	5	2/2
	Ch03	11	1/3
	Ch04	6
	Ch05	9	1/3
	Ch06	8
	Ch07	4
	Ch08	5	1/3
	Ch09	4	1/2
	Ch10	3
	Ch11	3	1/2
	Ch12	6
	Total	77	8 clusters

  1, Sl-ERF.E.1, Sl-ERF.F.4, and Sl-ERF.H.1) are up-regulated by both hormones whereas ERF.F.5 undergo opposite regulation by auxin and ethylene. More surprisingly, 6 ERF genes (ERF.A.1, ERF.A.3, ERF.C.4, ERF.C.6, ERF.D.4 and ERF.F.1) are significantly up-regulated by auxin while they are not responsive to ethylene.

	Figure 6 Expression of ERFs in response to ethylene and auxin treatments. Quantitative
	RT-PCR of ERF transcripts in total RNA samples extracted from 5-days dark growing
	seedlings treated for 5 hours with ethylene or with IAA for 3 hours. ΔΔCt refers to the fold of
	difference in ERF expression relative to the untreated seedlings. Stars indicate a statistical
	significance (p<0.05) using Mann-Withney test

Acknowledgement

This work forms part of the requirement for the degree of Ph.D. for JP. This research was supported by the European Integrated Project EU-SOL (FOOD-CT-2006-016214), by IFCPAR-CEFIPRA Indo-French program (Grant 3303-02) and by funds from the "Laboratoire d'Excellence" (LABEX) entitled TULIP (ANR -10-LABX-41). BCNP was recipient of postdoctoral fellowship from the IFCPAR-CEFIPRA. The work benefited from the networking activities within the European COST Action FA1106. Microscopy and cytometry analyses were performed at RIO Imaging Platform under the technical assistance of A. Jauneau and J.C Lepert.

Sequences

Coding DNA sequences of the 28 studied ERFs are provided in additional file 8.

Authors' contribution

JP and BCNP participated in the design of the study, performed the experiments, and contributed to the writing of the manuscript. WZ contributed to the cloning of ERF genes and to qRT-PCR experiments. KC participated in the design of the project. IM carried out the transcriptional activity experiments in the single cell system. MZ contributed to the in silico analysis of the ERF gene families. AL and JCP participated in the ethylene and auxin treatment experiments. MOT contributed to the design of the SRDX-repressor motif experiments. FR contributed to the design of the study and of the experiments, to bioinformatics analysis and to drafting the manuscript. MB conceived the study, and participated in its design and coordination and contributed to the writing of the final manuscript. All authors read and approved the final manuscript.

Additional files

Additional_file_1 as XLS

Additional file 1 Correspondance between Sl-ERF nomenclature and ITAG 2.3 nomenclature.

Additional_file_2 as PDF

Additional file 2 Schematic diagram of protein structure of the isolated 28 tomato ERFs defining the 8 subclass. Each colored box represents the AP2/ERF domain and conserved motifs, as indicated below the diagram. The position of the motif is indicating by the number on the top of the diagram. The name of motif by Nakano is given inside the box [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF] Additional_file_3 as PDF Additional file 3 Comparative basal activity of the synthetic promoter (4 X GCC) and the native promoters, fused to the GFP in the absence of added ERF effectors.

Additional_file_4 as XLS

Additional file 4 Primers used for RACE amplification.

Additional_file_5 as XLS

Additional file 5 Primers used for CDS amplification. Each pair of primers encompasses the complete CDS from the ATG to the STOP codon. Adaptor attB1 and attB2, have been added at the 5′ end of sense and antisense primers, respectively.

Additional_file_6 as XLS

Additional file 6 Primers used for qRT-PCR. Primers designed with PRIMER EXPRESS software in sequence specific regions for each ERF. Specificity was further checked using BLAST against all tomato unigenes (Tomato unigene database).

Additional_file_7 as XLS

Additional file 7 Primers used for the synthesis of radioactive GCC box probes utilized in EMSA experiments.

Additional_file_8 as DOCX

Additional file 8 (DOCX 22 kb) Coding sequences of the 28 studied ERF.


Saoussen Cheikhrouhou
email: saoussen.cheikhrouhou@redcad.org

Slim Kallel
email: slim.kallel@fsegs.rnu.tn

Nawal Guermouche
email: nawal.guermouche@laas.fr

Mohamed Jmaiel
email: mohamed.jmaiel@enis.rnu.tn

A Survey on Time-aware Business Process Modeling

Keywords: Temporal constraints and dependencies, Business Process Modeling (BPM), Workflow, Web service composition, Inter-Organisational Business Process (IOBP)

One key perspective when dealing with Business Process Management (BPM) is time. All business experts agree upon the fact that time is a key resource for each business process within an organisation. Indeed, time managing is an effective cost and error reduction strategy and thus ensures profit maximization for the organisation. As a result, business managers, researchers, and academicians in management are striving to have full-support of temporal aspects in business process management suites. Consequently, modeling and managing temporal requirements in the business process field is becoming a topic of intensive research. This paper presents a survey of the existing approaches to specifying and verifying the temporal perspective in business processes.

INTRODUCTION

Nowadays, business is migrating from Business-to-Comsumer (B2C) applications to Business-to-Business (B2B) ones in order to deal with the ever increasing economic pressure and to enhance the overall competitiveness.

When addressing the issue of B2B, one organisation may collaborate with many others with complementary skills to form an Inter-Organisational Business Process (IOBP). The emergence of the IOBP field gave already a major contribution to different sectors such as aeronautic domain, in which more than 50% of the supply chain is subcontracted. Furthermore, the aeronautic sector's strategy is migrating from one-tier subcontractor to an important number of subcontractors with a ditributed control over the different sub-contracting processes. Satisfying time constaints such as time deadlines is vital for the processes of the aviation in-dustry, since the violation of such constraints may lead to critical situations and could even threaten the aviation safety. Obviously, it is not straightforward to adress the temporal requirements in IOBPs.

Different specification methods and verification techniques and tools have been developed to deal with such setting [START_REF] Bettini | Temporal Reasoning in Workflow Systems[END_REF]Kazhamiakin et al., 2006a;[START_REF] Guermouche | Etude des Interactions Temporisées dans la Composition de Services Web[END_REF][START_REF] Watahiki | Formal Verification of Business Processes with Temporal and Resource constraints[END_REF]. Nevertheless, the temporal resource management in business processes, especially in huge and collaborative processes as used in the aviation industry, is still a challenging research task.

This paper surveys the current state of the art in specifying and verifying the temporal perspective in business processes. But the main focus of this paper is the specification step. Few research attempts however have been made to carry overviews on the business process field (see, for instance [START_REF] Wang | Formal verification of timed systems: a survey and perspective[END_REF][START_REF] Dustdar | A survey on web services composition[END_REF]). Nevertheless, these overviews are not as focused as the one presented here since they do not elect time as a first time citizen in the business process modeling (BPM) phase.

This paper is structured as follows. Section 1 gives an overview on the existing temporal constraints specification and verification methods in the business process field, followed by a rich evaluation and discussion in section 2. The last section concludes and highlights the emerging research challenges to address in the field of business process time management.

OVERVIEW ON THE EXISTING TEMPORAL CONSTRAINTS SPECIFICATION METHODS

As preliminary step of this work, we give a classification of the existing temporal constraints models. Mainly, the studied approaches are collected from three research areas: workflows, Web service composition, and inter-organisational domain. These research areas can be generalized and seen from a business process field perspective.

Temporal constraints in the workflow research area

The major contribution of Time-BPMN [START_REF] Gagné | Time-BPMN[END_REF], is the extension of Business Process Modeling Notation BPMN (OMG, 2008) with a large set of required temporalities. This extension deals with various temporal constraints and dependencies between business process activities. However, this extension does not permit to model temporal constraints relating to the duration of business process activities (eg. A given activity lasts x time units and x may be defined as a time interval). In addition, Time-BPMN is limited to the specification phase since no verification mechanism of temporal constraints conflicts is provided.

The work presented in [START_REF] Watahiki | Formal Verification of Business Processes with Temporal and Resource constraints[END_REF] proposes a formal specification of BPMN (OMG, 2008) with timed automata. First, the authors extend BPMN to handle temporal constraints (i.e., the minimum and maximum execution time of a task), resource constraints, and concurrency constraints (i.e., the number of instances executable in parallel). Second, they provide an automatic mapping of the extended BPMN onto timed automata. Computation tree logic (CTL) formulas are used to specify the different properties to be verified by the UPAAL model checker. This approach aims at verifying some features, such as deadlocks and bottlenecks. The scope of this paper is limited to a small subset of BPMN elements. Additionnally, this BPMN extension permits to specify temporal constraints related to only one activity within the business process model. [START_REF] Huai | Towards Trustworthy Composite Service Through Business Process Model Verification[END_REF] present a method for verifying BPMN (OMG, 2008) models based on time Petri nets. The proposed method supports the analysis of model structure (dead task, deadlock and infinite loops) and tests the time conflicts of the model.

First, the authors translate the BPMN model to time Petri nets. Second, they construct the reachability graph of the Petri nets in order to verify the model structure. Furthermore, they exhibit the time choreography verification algorithm to verify time conflicts. The proposed algorithm proceeds by the accumulation of clock constraints of terminated activities and assignes them to the corresponding activated activities within the tested path. This work presents the advantage that clock constraints are propagated to tasks and message flows, which makes explicit the implicit timed conflicts due to service interaction. The first limitation is related to the lack of temporal dependencies between multiple activities of the business process, which makes the proposed time choreography verification algorithm very limited.

The major contribution of the approach cited in [START_REF] Du | Dynamic Checking and Solution to Temporal Violations in Concurrent Workflow Processes[END_REF] is that it can dynamically check the temporal violations of multiple concurrent workflow processes with resource constraints. First, the authors construct the sprouting graph models of the time workflow nets (TWF-nets) [START_REF] Ling | Time Petri nets for workflow modelling and analysis[END_REF] for multiple workflow processes. Second, they update the sprouting graph at different checking points and check the temporal constraints. Finally, and most importantly, the violation paths and solutions (by modifying the duration of some activities) are given. Moreover, they use the UPAAL model checker to verify the correctness of their approach. This work verifies only temporal constraints of the form: an activity a j should end its execution no later than x time units after the activity a i starts. The complexity of the construction of the sprouting graph becomes high when the number of resource constraints increases. This is identified as the major weakness of this approach.

The particularity of the approach of Bettini et al. [START_REF] Bettini | Temporal Reasoning in Workflow Systems[END_REF] is that it merges several research directions on temporal workflow models and on temporal constraint networks.

Regarding the modeling of temporal constraints, Temporal Constraint with Granularity TCG graph is used for this purpose. Additionally, this paper provides temporal constraints reasoning and management tool offering the following services: First, it checks the consistency of complex temporal requirements. Second, it monitors workflow activities and predicts their starting and ending time. Finally it provides the enactment service with useful temporal information for activity scheduling. Hence, the concept of Free schedules is a relevant contribution of this paper. A schedule is free when it is possible to statically fix the start times of all tasks of the workflow without constraining their durations while satisfying all the other temporal constraints.

Time modeling and management in the clinical workflow domain has been widely investigated by [START_REF] Combi | Conceptual Modeling of Temporal Clinical Workflows[END_REF][START_REF] Combi | Controllability in Temporal Conceptual Workflow Schemata[END_REF]. Among the proposed temporal constructs, we can notice: the duration (the activity duration) and delays (the edge duration), the relative constraints, the absolute constraints, and the periodic constraints. To this regard, the authors developed a tool named Temporal Workflow Analyzer (TWA) to support workflow modeling at workflow design time. Furthermore, in [START_REF] Combi | Controllability in Temporal Conceptual Workflow Schemata[END_REF], the authors introduced and discussed the concept of controllability checking which refers to the capability of executing a workflow for any possible duration of tasks.

Wong et al. have described in [START_REF] Wong | A Relative Timed Semantics for BPMN[END_REF] a relative-timed semantic model for BPMN in the language of Communicating Sequential Processes (CSP). This model helps to introduce the notion of relative time in the form of delays and durations. The model checker FDR is accordingly used to verify the time compatibility issue in business collaborations.

Temporal constraints in web service composition research field

This subsection covers the basics of temporal constraints specification and verification approaches in the web service composition research field. The authors in (Kazhamiakin et al., 2006a) address the problem of qualitative and quantitative analysis of timing aspects of web service compositions. To capture the timing aspects of BPEL4WS processes, the Web Service Timed State Transition Systems (WSTTS) formalism is previously introduced in [START_REF] Kazhamiakin | Timed Modelling and Analysis in Web Service Compositions[END_REF]. For the verification, they use the NuSMV model checker. The authors verify the composition against a large set of temporal properties such as deadlock and the termination of the procedure within a given delay. Furthermore, the approach aims at calculating the maximal and minimal duration time of the process. This work has the advantage not only to check whether a certain time-related requirement is satisfied, but also to compute extreme time bounds that satisfy such requirement. Nevertheless, considering only timing aspects of BPEL4WS processes limits the mentioned approach to the service oriented research field. Verifying the timing requirements on the model (exp. BPMN) results in a generalized approach applicable to a service oriented implementation as well as to other possible implementations.

The approach proposed in [START_REF] Kallel | Specifying and Monitoring Temporal Properties in Web Services Compositions[END_REF] covers the specification of temporal con-straints for the web service domain using a new proposed language, XTUS-Automata. In the specification phase, this work presents temporal specification patterns ( i.e. patterns for duration properties, pattern for temporal properties over cardinalities, and pattern for absolute time properties). This work combines timed automata (TA) and extended time unit system (XTUS) to allow specifying temporal properties involving relative time as well as absolute time. Furthermore, this work conducts a formal verification of deadlock using the model checker UPAAL. Finally, it presents an aspect-based monitoring mechanism, in which formally specified temporal constraints are translated automatically to modular aspect code in the aspect-oriented workflow language AO4BPEL. It is worth noting that this paper offers interesting specification patterns by which we can cover a large set of real world workflow temporal constraints. Nevertheless, this work is unable to verify the existence of temporal constraint conflicts.

In [START_REF] Guermouche | Etude des Interactions Temporisées dans la Composition de Services Web[END_REF], the author uses temporal properties in order to analyze the compatibility in web service composition. A formal model abstracting messages, data, data constraints as well as temporal constraints, based on timed automata is proposed. A compatibility analysis of the web service choreography is conducted. The UP-AAL model checker was used to detect some structural problems due to temporal conflicts. So far in this approach, the focus has been on the construction of a correct web service composition. For this end, a mediator is generated, whenever it is possible, to overcome the web service collaboration incompatibility issues. The clock ordering process is used to verify deadlock freeness due to time constraints conflicts. Nevertheless, the scope of this paper is limited to the verification of time constraints only caused by message interaction between services of the process.

In [START_REF] Guermouche | Towards Timed Requirement Verification for Service Choreographies[END_REF], the authors propose a framework to check temporal requirements on choreographies. This is achieved by the verification of the composed annotated BPEL processes. This work enables efficiently to specify time constraints such as the estimated execution time of ac-tivities and the temporal delay between two activities or messages. Furthermore, complex temporal requirements could be expressed.

For instance, the absence pattern with delay and the response pattern with delay. The timed business processes are automatically translated into the formal modeling language, Fiacre [START_REF] Berthomieu | Fiacre: an Intermediate Language for Model Verification in the Topcased Environment[END_REF]. The TIme petri Net Analyzer (TINA) [START_REF] Berthomieu | The tool tina âĂŞ construction of abstract state spaces for petri nets and time petri nets[END_REF]) model checker tool is used for complex real-time requirements automatic checking. This work has the advantage of supporting synchronous as well as asynchronous services. This work has attempted to provide a modeling environment inspired from BPMN to visually specify the temporal requirements whereas no translation mechanism from the graphical specification to the used formal language is provided.

Benatallah et al. have widely invested in checking compatibility and replaceability analysis in timed ptotocols of web services [START_REF] Benatallah | Analysis and Management of Web Service Protocols[END_REF][START_REF] Benatallah | On Temporal Abstractions of Web Service Protocols[END_REF]. The approach detailed in [START_REF] Benatallah | On Temporal Abstractions of Web Service Protocols[END_REF] models business protocols as deterministic finite state machines. The scope of this work is limited to synchronous services and temporal requirements can only be associted to messages inside the same service.

Temporal constraints in the inter-organisational research field

This subsection is dedicated to explaining research attempts to specify and verify temporal constraints in inter-organisational business processes, crossing the organizational boundaries.

Eder et al. (Eder and Tahamtan, 2008b) focuses on checking temporal consistency in interorganizational workflows. In this context each organization contributes to the interorganizational workflow through its process view. Process views are a prevalent modeling approach for interorganizational workflows. They include a subset of the activities of the organisational private workflow needed for communication. Indeed, it allows to or-ganisations to well interact with others while preserving their organisational privacy. The proposed approach checks if the interorganizational workflow is temporally consistent by checking if its participating views are temporally consistent. For that, the authors use the concept of temporal plans and use timed activity graphs as the basic modeling language. It is clear that assuming that the different activities of the workflow have a deterministic duration is quite restrictive. Additionnally, this approach enables to specify only deadline constraints between the corresponding activities of the process(i.e. activities that communicate with each other). By checking only the corresponding activities of the different views, the authors implicitly suppose that the temporal constraints are synchronized based on message synchronization which is not always true because the views may not start executing at the same time and thus it is insufficient to compare only the intervals of the corresponding activities. In addition, the authors do not mention any other issue for full consistency like messaging conformance, data flow conformance or structural conformance. Time conformance has been studied by Eder and Tahamtan in (Eder and Tahamtan, 2008a). It consists in checking whether a timed orchestration satisfies a timed choreography by generating temporal execution plans. The algorithm calculating the timed graphs and checking temporal conformance is detailed in (Eder and Tahamtan, 2008a). The conformance condition verifies that for each activity the sum of its earliest possible start and its duration must be less or equal to its latest allowed end for both best and worst cases. The durations of activities are presented by deterministic values which limits the scope of this work. The authors do not consider temporal constraints crossing the boundary of an activity or event-related temporal constraints.

In the context of Inter-Organizational Workflows, the approach detailed in [START_REF] Makni | Satisfaction and Coherence of Deadline Constraints in Inter-Organizational Workflows[END_REF] deals with the deadline constraints conformance verification, and thus without exposing the private processes of the involved partners. The authors demonstrate how missing deadlines while delivering the re-quired services may cause a global failure execution, even if the business behavior complementarity of the involved services is ensured. Based on the CoopFlow approach and using Time Petri nets theory, the authors propose a method for modeling and advertising temporal requirements for cooperative activities on the abstracted version of business processes by using observers. In fact, they prove that a deadline local verification process executed by a partner can lead to a deadline conformance in the resulting interconnected workflow. However, no method is proposed for the deadline local verification process. Additionally, this work permits only to add deadline constraints between cooperative activities (wich are visible by the cooperation candidates).

The authors in [START_REF] Makni | Negotiating Deadline Constraints in Interorganizational Logistic Systems: A Healthcare Case Study[END_REF] discuss the application of Inter-Organizational Workflows (IOW) for automating processes in the collaborative context. A case study of emergency healthcare is presented in order to show the feasibility of the proposed temporal extension of the CoopFlow approach detailed in [START_REF] Makni | Satisfaction and Coherence of Deadline Constraints in Inter-Organizational Workflows[END_REF]. This paper presents a proof of concept for automating the temporal conformance process in CoopFlow. The author noticed the use of TINA and Little Parametric Tool (LPT) tools [START_REF] Godary | LPT : Little Parametric Tool, outil pour la validation d'une borne temporelle paramétrée[END_REF] for verification purposes. Nevertheless, no more details are provided. Throughout this paper, the authors focused on mentioning negotiation aspects of temporal constraints in the presented case study but no negotiation strategy is detailed.

EVALUATION AND DISCUSSION

Throughout this survey paper, we provided a representative overview of the major efforts of time management in the business process field. The evaluation results are presented in table 1. Space limitations prevent presenting all the approaches discussed above, so we have omitted some of them being only focusing on a very limited scope of time management.

Table 1 highlights the different character-istics of each approach according to the following criteria:

1. How temporal requirements are modeled within the approach? Eg. the standard BPMN, the TWF-nets, etc.

What properties against which the business process is verified?

Eg. the structural properties (i.e. the analysis of dead tasks, bottlenecks, deadlocks and loops), the time conflicts of the model, the user-defined temporal constraints (exp. the deadline constraints and the absence constraint), etc.

How this verification is proceeded?

Eg. model checker tools, algorithms, etc.

Based on the above observations, we identified that most of the already studied constraints include the temporal perspective.

The temporal constraints are usually correlated with other constraints such as data [START_REF] Guermouche | Etude des Interactions Temporisées dans la Composition de Services Web[END_REF] and resource constraints [START_REF] Watahiki | Formal Verification of Business Processes with Temporal and Resource constraints[END_REF][START_REF] Du | Dynamic Checking and Solution to Temporal Violations in Concurrent Workflow Processes[END_REF].

Consequently, there are been several attempts to model the different constraints in the business process diagram itself using the defacto industrial standard for business process modeling, BPMN [START_REF] Gagné | Time-BPMN[END_REF][START_REF] Guermouche | Towards Timed Requirement Verification for Service Choreographies[END_REF]. The use of a graph-based modeling approach of business processes as BPMN, is a competitive advantage. Indedeed, the visual appeal of the graph-based modeling approaches makes them useful for all kinds of workflow designers since no technical background is required. Similarly, other research efforts [START_REF] Makni | Satisfaction and Coherence of Deadline Constraints in Inter-Organizational Workflows[END_REF][START_REF] Makni | Negotiating Deadline Constraints in Interorganizational Logistic Systems: A Healthcare Case Study[END_REF][START_REF] Kallel | Specifying and Monitoring Temporal Properties in Web Services Compositions[END_REF][START_REF] Guermouche | Etude des Interactions Temporisées dans la Composition de Services Web[END_REF][START_REF] Du | Dynamic Checking and Solution to Temporal Violations in Concurrent Workflow Processes[END_REF][START_REF] Huai | Towards Trustworthy Composite Service Through Business Process Model Verification[END_REF] opted for formal specification languages with modeling capabilities such as Petri nets and Timed Automata. Whereas, other approaches like [START_REF] Wong | A Relative Timed Semantics for BPMN[END_REF] opted for CSP as a process algebra language, which lacks for graphical support. The approach followed by [START_REF] Pesic | Constraint-Based Workflow Models: Change Made Easy[END_REF] is somewhat different from the others since it models the constraints apart from the business process model (eg. by LTL formulas).

When considering the business process model itself, there are works specifying different constraints for one business process. Others take into account the cooperation between more than one business process both in the web service composition field and in the interorganizational business process field. When addressing the issue of IOBP, it is inevitable to reason about the migration of the different constraints between the private and the public workflows. There are several ideas for further research especially in the IOBP field. We can, first focus on elaborating a generic modeling approach which supports different constraint modeling such as temporal constraints and other associated constraints, namely, resource and data constraints. When dealing with temporal constraints, we remarked the lack of absolute time constraints in the majority of the works. In addition to that, just one work has modeled time points obtained from the execution phase [START_REF] Kallel | Specifying and Monitoring Temporal Properties in Web Services Compositions[END_REF]. We propose to deal with sequence, choice and concurrency structures and thus adopting best and worst cases of execution.

We now turn our attention to the temporal requirements verification problem. Temporal verification mechanisms are of paramount importance since they enable to detect, early on, possible temporal conflicts and to react to them effectively. In this context, although many efforts confound the time conflict verification of the model (i.e. the violation of some temporal requirements) with the structure verification (i.e. the analysis of dead tasks, bottlenecks, deadlocks and loops) [START_REF] Kallel | Specifying and Monitoring Temporal Properties in Web Services Compositions[END_REF][START_REF] Watahiki | Formal Verification of Business Processes with Temporal and Resource constraints[END_REF], there are some works which have tried to differentiate the two verification processes [START_REF] Guermouche | Etude des Interactions Temporisées dans la Composition de Services Web[END_REF][START_REF] Huai | Towards Trustworthy Composite Service Through Business Process Model Verification[END_REF][START_REF] Du | Dynamic Checking and Solution to Temporal Violations in Concurrent Workflow Processes[END_REF].

Approaches

How TC What properties How this verification [REF] are modeled? are verified? is proceeded? [START_REF] Gagné | Time-BPMN[END_REF] BPMN [START_REF] Watahiki | Formal Verification of Business Processes with Temporal and Resource constraints[END_REF] BPMN bottlenecks UPAAL model checker + + Timed Automata deadlocks [START_REF] Huai | Towards Trustworthy Composite Service Through Business Process Model Verification[END_REF] Time Petri nets dead task + deadlock +infinite loops the reachability graph of the Petri nets the time conflicts of the model the time choreography verification algorithm [START_REF] Du | Dynamic Checking and Solution to Temporal Violations in Concurrent Workflow Processes[END_REF] Time workflow nets duration between two activitie Algorithms (TWF-net) less than s time units + UPAAL model checker (Kazhamiakin et al., 2006a) Web Service Timed deadlock model checker NuSMV State Transition Systems (WSTTS) + maximal and minimal duration of the process [START_REF] Kallel | Specifying and Monitoring Temporal Properties in Web Services Compositions[END_REF] XTUS-Automata deadlock UPAALmodel checker [START_REF] Guermouche | Etude des Interactions Temporisées dans la Composition de Services Web[END_REF] Timed Automata compatibility analysis of the web Algorithms service choreography deadlock UPAAL model checker (Eder and Tahamtan, 2008b) Timed activity graphs Temporal consistency Algorithms (Eder and Tahamtan, 2008a) + Time conformance [START_REF] Makni | Satisfaction and Coherence of Deadline Constraints in Inter-Organizational Workflows[END_REF] Time Petri nets Time conformance TINA model checker and LPT [START_REF] Makni | Negotiating Deadline Constraints in Interorganizational Logistic Systems: A Healthcare Case Study[END_REF]) [START_REF] Guermouche | Towards Timed Requirement Verification for Service Choreographies[END_REF] A modeling environment the absence constraint with delay inspired from BPMN + the response constraint with delay TINA model checker The Fiacre specification [START_REF] Bettini | Temporal Reasoning in Workflow Systems[END_REF] Temporal Constraint with Granularity the consistency Algorithms (TCG) graph [START_REF] Combi | Conceptual Modeling of Temporal Clinical Workflows[END_REF] A proposed conceptual The controllability checking Algorithms [START_REF] Combi | Controllability in Temporal Conceptual Workflow Schemata[END_REF] workflow model [START_REF] Wong | A Relative Timed Semantics for BPMN[END_REF] Communicating Sequential Time compatibility FDR model checker Processes (CSP)

To cope with the time conflict verification, there are some works which have neglected the intra-activity temporal requirements (eg. the duration of the modeled activities) [START_REF] Guermouche | Etude des Interactions Temporisées dans la Composition de Services Web[END_REF]. On the other hand, there are some approaches which have neglected the inter-activity temporal dependencies [START_REF] Watahiki | Formal Verification of Business Processes with Temporal and Resource constraints[END_REF][START_REF] Huai | Towards Trustworthy Composite Service Through Business Process Model Verification[END_REF]. Besides, the approach detailed in [START_REF] Du | Dynamic Checking and Solution to Temporal Violations in Concurrent Workflow Processes[END_REF] has tried to include the two different temporal requirements.

Additionally, there are some efforts concentrating on verifying other issues such as time conformance (Eder and Tahamtan, 2008a;[START_REF] Makni | Satisfaction and Coherence of Deadline Constraints in Inter-Organizational Workflows[END_REF], the absence constraint [START_REF] Guermouche | Towards Timed Requirement Verification for Service Choreographies[END_REF], and controllability [START_REF] Combi | Controllability in Temporal Conceptual Workflow Schemata[END_REF].

Once the verification process is conducted and a possible violation is detected, only few approaches (Du et al., 2010) tried to detect erroneous paths and to propose solutions. So far, the approach detailed in [START_REF] Du | Dynamic Checking and Solution to Temporal Violations in Concurrent Workflow Processes[END_REF] has proposed the modification of the duration of some activities as solution to the temporal violation in concurrent workflow processes with resource constraints. Additionnally, the author in (Guermouche, 2010) has considered the use of mediators when dealing with the compatibility analysis of the web service choreography. The idea of mediators proposed in [START_REF] Guermouche | Etude des Interactions Temporisées dans la Composition de Services Web[END_REF] sounds very promising since it has succeeded in resolving a large set of temporal violations in an efficient manner.

Typically, researchers in the field of time management in the business process field are invited to widen the set of possible solutions to temporal constraints violations. From the research directions that have to be considered, we can notice the modification of the allocation policy of the shared resources and the change of the overall business process structure arriving at the substitution of some activities.

Finally, we can proceed by monitoring or enforcing of the different constraints in the execution phase. Another line of research is to study constraint-based business process models which offer design decisions at the execution time and enable different process variants.

RESEARCH CHALLENGES AND CONCLUSION

Business managers, researchers, and academicians in management are striving to have full-support of temporal aspects in current business process management suites. Obviously, modeling and managing temporal requirements has long been a topic of intensive researches. Hence, with the help of the critical and comprehensive analysis presented within this survey paper, we pointed out that this emerging research field still face a multitude of challenges. The succeeding listing illustrates the major challenges to be addressed to substantially enhance the time management in the business process management field.

• Proposing a business process model supporting different temporal requirements : to enable the specification of temporal constraints related to one activity as well to Ad-Hoc sub processes and concurrent business processes sharing resources and exchanging messages. • Improving the existing process view generation methods in order to define the mapping of a large set of temporal requirements from private to public process models. • Defining a mapping mechanism from the business process model to a suitable formal language for future verification purposes. And if necessary, proposing a new formal language to well support the specification of all the temporal requirements. • Suggesting efficient verification approaches to diagnose potential temporal violations of the process model early enough. In this context, it is beneficial to verify the business process against several issues such as structural properties (i.e. the analysis of dead tasks, bottlenecks, deadlocks and loops), time conflicts of the model, user-defined temporal constraints (exp. the deadline constraints and the absence constraint), time conformance of the IOBP (similarly the compatibility analysis in the web service field) as well as the controllability checking.

• Defining violation identification mechanisms and proposing relevant primitives to resolve violations.

To summarize, in this paper we have analyzed and compared existing approaches for modeling and verifying time-related properties on business processes. Based on this analysis and evaluation, we have pointed out the challenges which sets foundations for full temporal support in business process modeling area. We are convinced that finding solutions to these challenges will significantly improve the interorganisational business process temporal support, helps to achieve process automatisation and thus helps the organisation to get advantage over competitors and to maximize its revenue.

Table 1 :

 1 Evaluation-How existing research approaches model and verify the temporal constraints (TC)