Mohamed Zouine

Yongyao Fu

Anne-Laure Chateigner-Boutin

Isabelle Mila

Pierre Frasse

Hua Wang

Corinne Audran

Jean-Paul Roustan

Mondher Bouzayen

Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing

Background:

The phytohormone auxin is involved in a wide range of developmental processes and auxin signaling is known to modulate the expression of target genes via two types of transcriptional regulators, namely, Aux/IAA and Auxin Response Factors (ARF). ARFs play a major role in transcriptional activation or repression through direct binding to the promoter of auxin-responsive genes. The present study aims at gaining better insight on distinctive structural and functional features among ARF proteins.

Results: Building on the most updated tomato (Solanum lycopersicon) reference genome sequence, a comprehensive set of ARF genes was identified, extending the total number of family members to 22. Upon correction of structural annotation inconsistencies, renaming the tomato ARF family members provided a consensus nomenclature for all ARF genes across plant species. In silico search predicted the presence of putative target site for small interfering RNAs within twelve Sl-ARFs while sequence analysis of the 59-leader sequences revealed the presence of potential small uORF regulatory elements. Functional characterization carried out by transactivation assay partitioned tomato ARFs into repressors and activators of auxin-dependent gene transcription. Expression studies identified tomato ARFs potentially involved in the fruit set process. Genome-wide expression profiling using RNA-seq revealed that at least one third of the gene family members display alternative splicing mode of regulation during the flower to fruit transition. Moreover, the regulation of several tomato ARF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signaling pathways of these two hormones.

Conclusion:

All together, the data bring new insight on the complexity of the expression control of Sl-ARF genes at the transcriptional and post-transcriptional levels supporting the hypothesis that these transcriptional mediators might represent one of the main components that enable auxin to regulate a wide range of physiological processes in a highly specific and coordinated manner.

Introduction

The plant hormone auxin, indole-3-acetic acid (IAA), is a simple signaling molecule that plays a critical role in plant development and growth. This phytohormone regulates cell division and cell elongation and exerts pleiotropic effects on a wide range of developmental processes including organ differentiation, embryogenesis, lateral root initiation, apical dominance, gravitropism and phototropism, leaf elongation, shoot architecture and fruit development [START_REF] Quint | Auxin signaling[END_REF][START_REF] Teale | Auxin as a model for the integration of hormonal signal processing and transduction[END_REF][START_REF] Wang | Regulatory features underlying pollination-dependent and -independent tomato fruit set revealed by transcript and primary metabolite profiling[END_REF][START_REF] Sagar | SlARF4, an auxin response factor involved in the control of sugar metabolism during tomato fruit development[END_REF][START_REF] Sagar | Under-expression of the Auxin Response Factor Sl-ARF4 improves post-harvest behavior of tomato fruits[END_REF]. A critical move towards understanding the mechanisms underlying auxin action [START_REF] Theologis | Rapid induction of specific mRNAs by auxin in pea epicotyl tissue[END_REF] happened when it was shown that the hormone coordinates plant development essentially through transcriptional regulation of genes such as Aux/ IAA, Gretchen Hagen3 (GH3), Small Auxin Up RNA (SAUR) and Auxin Response Factor (ARF). It was subsequently found that these so-called early auxin-responsive genes contain in their promoter one or more copies of a conserved motif, TGTCTC or its variants, known as the auxin-responsive element (AuxRE) [START_REF] Guilfoyle | How does auxin turn on genes?[END_REF]. Experimental evidences were then provided showing that transcription factors from the ARF type specifically bind to this AuxRE to mediate the transcription of auxin responsive genes [START_REF] Tiwari | The roles of auxin response factor domains in auxin-responsive transcription[END_REF]. The components of the pathway linking auxin perception to gene expression are now well established indicating that ubiquitination of Aux/IAA proteins by the TIR1/AFB subunit of the SCF TIR1/AFB ubiquitin ligase leads to their degradation by the 26S proteasome thus releasing the Aux/IAA-mediated inhibition of ARFs which allows these transcription factors to modulate the expression of their target genes [START_REF] Hayashi | The interaction and integration of auxin signaling components[END_REF].

Three types of transcriptional regulators are required for the control of auxin-responsive genes, Auxin Response Factors (ARFs), Aux/IAAs and Topless (TPLs) [START_REF] Szemenyei | TOPLESS mediates auxindependent transcriptional repression during Arabidopsis embryogenesis[END_REF][START_REF] Causier | TOPLESS co-repressor interactions and their evolutionary conservation in plants[END_REF]. Members of the Aux/IAA and TPL families have been reported to function as repressors of auxin-induced gene expression [START_REF] Szemenyei | TOPLESS mediates auxindependent transcriptional repression during Arabidopsis embryogenesis[END_REF][START_REF] Ulmasov | Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements[END_REF][START_REF] Wang | Transcriptional corepressor TOPLESS complexes with pseudoresponse regulator proteins and histone deacetylases to regulate circadian transcription[END_REF][START_REF] Audran-Delalande | Genome-wide identification, functional analysis and expression profiling of the Aux/IAA gene family in tomato[END_REF]]. An increasing number of studies demonstrate the critical role of ARFs in a variety of developmental processes, such as embryo patterning [START_REF] Rademacher | Different auxin response machineries control distinct cell fates in the early plant embryo[END_REF][START_REF] Cole | DORNROSCHEN is a direct target of the auxin response factor MONO-PTEROS in the Arabidopsis embryo[END_REF], leaf expansion and senescence [START_REF] Lim | Auxin response factor 2 (ARF2) plays a major role in regulating auxin-mediated leaf longevity[END_REF][START_REF] Wilmoth | NPH4/ ARF7 and ARF19 promote leaf expansion and auxin-induced lateral root formation[END_REF][START_REF] Ellis | AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE FACTOR2 regulate senescence and floral organ abscission in Arabidopsis thaliana[END_REF], lateral root growth [START_REF] Wilmoth | NPH4/ ARF7 and ARF19 promote leaf expansion and auxin-induced lateral root formation[END_REF][START_REF] Marin | miR390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth[END_REF][START_REF] Yoon | Auxin and Abscisic Acid Responses of Auxin Response Factor 3 in Arabidopsis Lateral Root Development[END_REF], floral organ abscission and petal growth [START_REF] Ellis | AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE FACTOR2 regulate senescence and floral organ abscission in Arabidopsis thaliana[END_REF][START_REF] Varaud | AUXIN RESPONSE FACTOR8 regulates Arabidopsis petal growth by interacting with the bHLH transcription factor BIGPETALp[END_REF], fruit set and development [START_REF] Goetz | AUXIN RESPONSE FACTOR8 is a negative regulator of fruit initiation in Arabidopsis[END_REF][START_REF] Goetz | Expression of aberrant forms of AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and tomato[END_REF][START_REF] Guillon | Downregulation of an Auxin Response Factor in the tomato induces modification of fine pectin structure and tissue architecture[END_REF][START_REF] De Jong | The Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set and development[END_REF], apical hook formation [START_REF] Li | Convergence of signaling pathways in the control of differential cell growth in Arabidopsis[END_REF], and various responses to environmental stimuli [START_REF] Jain | Transcript profiling reveals diverse roles of auxinresponsive genes during reproductive development and abiotic stress in rice[END_REF]. In addition, ARF genes are involved in the cross-talk between auxin and other hormones like gibberellins [START_REF] De Jong | The Solanum lycopersicum AUXIN RESPONSE FACTOR 7 (SlARF7) mediates cross-talk between auxin and gibberellin signalling during tomato fruit set and development[END_REF], ethylene [START_REF] Li | A role for auxin response factor 19 in auxin and ethylene signaling in Arabidopsis[END_REF], ABA [START_REF] Liu | Auxin response factors and plant growth and development[END_REF] and brassinosteroid signaling [START_REF] Vert | Integration of auxin and brassinosteroid pathways by Auxin Response Factor 2[END_REF]. A typical ARF protein consists of a conserved N-terminal B3-type DNA Binding Domain (DBD) that regulates the expression of early auxin response genes, a variable middle region (MR) that function as a transcriptional activation or repression domain (AD or RD), and a conserved C-terminal dimerization domain (CTD) that contributes to the formation of either ARF/ARF homo-and heterodimers or ARF/Aux-IAA hetero-dimers [START_REF] Tiwari | The roles of auxin response factor domains in auxin-responsive transcription[END_REF][START_REF] Guilfoyle | Auxin response factors[END_REF][START_REF] Guilfoyle | Auxin response factors[END_REF]. The amino acid composition of MRs, located between the DBD and CTD, showed that AD types are rich in glutamine(Q), serine (S), and leucine (L) residues while RD types are rich in proline (P), serine (S), threonine (T), and glycine (G) residues [START_REF] Guilfoyle | Auxin response factors[END_REF][START_REF] Ulmasov | Activation and repression of transcription by auxin-response factors[END_REF].

Since the cloning of the first AtARF1 from Arabidopsis, 22 members of this family, distributed over all five chromosomes, have been identified [START_REF] Guilfoyle | Auxin response factors[END_REF]. The functional characterization of AtARF genes was revealed by mutant analysis approach. For instance, arf1 and arf2 T-DNA insertion mutations indicated that ARF2 regulates leaf senescence [START_REF] Lim | Auxin response factor 2 (ARF2) plays a major role in regulating auxin-mediated leaf longevity[END_REF] and floral organ abscission [START_REF] Ellis | AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE FACTOR2 regulate senescence and floral organ abscission in Arabidopsis thaliana[END_REF]. The arf7/arf19 double mutant had stronger auxin resistance than the single mutant and displayed phenotypes not seen in the single mutant [START_REF] Li | A role for auxin response factor 19 in auxin and ethylene signaling in Arabidopsis[END_REF]. ARF8 was reported to regulate fertilization and fruit development, and arf8-4 mutation results in the uncoupling of fruit development from pollination and fertilization giving rise to parthenocarpic fruit [START_REF] Goetz | AUXIN RESPONSE FACTOR8 is a negative regulator of fruit initiation in Arabidopsis[END_REF], while flowers in arf6/arf8 double mutant are arrested as infertile closed buds with short petals, short stamen filaments, undehiscent anthers and immature gynoecia [START_REF] Nagpal | Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation[END_REF]. In tomato, recent studies have shown the involvement of ARF genes in fruit set, development, ripening and fruit quality [START_REF] Wang | Regulatory features underlying pollination-dependent and -independent tomato fruit set revealed by transcript and primary metabolite profiling[END_REF][START_REF] Sagar | SlARF4, an auxin response factor involved in the control of sugar metabolism during tomato fruit development[END_REF][START_REF] Sagar | Under-expression of the Auxin Response Factor Sl-ARF4 improves post-harvest behavior of tomato fruits[END_REF][START_REF] Goetz | Expression of aberrant forms of AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and tomato[END_REF][START_REF] Guillon | Downregulation of an Auxin Response Factor in the tomato induces modification of fine pectin structure and tissue architecture[END_REF][START_REF] De Jong | The Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set and development[END_REF][START_REF] Jones | Down-regulation of DR12, an auxin-response-factor homolog, in the tomato results in a pleiotropic phenotype including dark green and blotchy ripening fruit[END_REF]. Because of these findings, members of this gene family are becoming one of the main targets towards improving fruit traits in tomato and more broadly in fleshy fruits.

Studies using different species have indicated a total of 25 ARF genes in rice (Oryza sativa), 39 ARF genes in Populus trichocarpa, 24 ARF genes in sorghum (Sorghum vulgare) and 31 ARF genes in maize [START_REF] Wang | Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa)[END_REF][START_REF] Kalluri | Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa[END_REF][START_REF] Paterson | The Sorghum bicolor genome and the diversification of grasses[END_REF][START_REF] Xing | Genome-wide identification and expression profiling of auxin response factor (ARF) gene family in maize[END_REF]. Though 21 ARF genes have been previously identified in the tomato (Solanum lycopersicum), yet, the list was incomplete and some the family members were either misannotated or suffered structural inconsistency due to the lack at that time of a high quality assembled tomato genome sequence [START_REF] Kumar | Genome-wide analysis of auxin response factor (ARF) gene family from tomato and analysis of their role in flower and fruit development[END_REF][START_REF] Wu | Identification, isolation and expression analysis of auxin response factor (ARF) genes in Solanum lycopersicum[END_REF]. The present study, while comprehensively revising the entire ARF gene family in tomato, brings new insight on the complexity of their expression control at the post-transcriptional level. The distinctive spatio-temporal pattern of expression of tomato ARF genes and their differential responsiveness to auxin and ethylene lay the foundation for a deeper functional characterization of these transcriptional mediators.

Results

Genome-wide search for tomato ARF genes

Comprehensive identification of the ARF gene family members in the tomato was achieved using all ARF proteins previously reported from Arabidopsis and other plant species in BLAST queries on the recently published tomato genome sequence (SL2.40 genome sequence and iTAG2.30 whole protein sequences). Twenty four significant hits corresponding to non-redundant putative Sl-ARF genes were identified. PCR amplification of full length coding sequences (CDS) revealed two structural annotation inconsistencies reducing the total number of ARFs in the tomato genome to 22 (Table 1). Indeed, four sequences previously annotated as distinct ARF genes in iTAG2.30 corresponded to C-terminal or N-terminal parts of two ARF proteins (So-lyc12g006340/Solyc00g196060; Solyc11g013480/So-lyc11g013470). The mapping of tomato RNA-Seq data allowed to further improve the annotation of tomato ARFs by identifying the 39 and/or 59 UTR regions for 13 Sl-ARF genes (Table 1). All Sl-ARF proteins were found to contain a typical DBD domain (Figure 1A) as revealed by the Pfam analysis tool (http://pfam. sanger.ac.uk/). The molecular weight of the deduced Sl-ARF proteins showed a large variation ranging from 68 to 126 kDa (Table 1). Of particular note, Sl-ARF6B contains a premature stop codon in the region corresponding to the DBD domain and is therefore likely to be a pseudo-gene whose expression at the protein level is not expected (data not shown). Using cNLS Mapper, nuclear localisation signals (NLS) were also identified in all of Sl-ARFs (data not shown).

Building on the available tomato genome assembly sequence, the mapping of Sl-ARF genes revealed that Sl-ARF family members are distributed among the 12 tomato chromosomes. Chromosome 7 and 11 are found to harbor three ARFgenes each; chromosome 1, 2, 3, 5, 8 and 12 bear two ARFs, while each of chromosome 4, 6, 9 and 10 contains only a single ARF gene (Figure S1 in File S1). Unlike the situation prevailing in Arabidopsis, there is no evidence for tandem or segmental duplication events involving members of the tomato ARF family.

Phylogenetic relationship and consensus nomenclature for Sl-ARFs

To explore phylogenetic relationship among ARF proteins in largely distributed land plant species, a phylogenetic tree (Figure 1B) was constructed that included ARF family members from tomato, Arabidopsis, potato, grape and rice. The phylogenetic distribution revealed that ARF genes group into four major classes named Class I, II, III and IV. ARFs predicted to function as transcriptional activators, based on the presence the Q-rich activation domain in their middle region, belong to sub-class IIa (Sl-ARF5, Sl-ARF6A, Sl-ARF7A, Sl-ARF7B, Sl-ARF8A, Sl-ARF8B and Sl-ARF19) while ARFs from the remaining classes (Ia, IIb, III and IV) all harbor a repression domain in the middle region and are consequently predicted to function as transcriptional repressors.

Compared to Arabidopsis which contains 23 members, the size of the tomato Sl-ARF gene family is slightly contracted to 22 members. In order to reach a consensual nomenclature for ARF genes across species, the tomato members of this gene family were renamed, based on phylogenetic relationship and according to the numbering of the closest Arabidopsis homolog. While complying with the most complete classification available in Arabidopsis [START_REF] Guilfoyle | Auxin response factors[END_REF], the proposed nomenclature better clarifies the correspondence between ARF subclasses in plant species. Noteworthy, sub-class Ib which has no representative in the tomato, contains 7 members in Arabidopsis that are likely to derive from multiple duplications of At-ARF13 which has no ortholog in any of the plant species tested in the present study. A distinctive feature of the tomato ARF family is the expanded size of the activators' sub-class (IIa) which represents 36.5% of the ARF genes whereas the activators only account for 21.7% of Arabidopsis ARFs. Another specific feature of the tomato ARF family is the presence of Sl-ARF24 (sub-class IV) that is not found out of the Solanaceae family. Interestingly, this presumably Solanaceae-specific gene encodes an ARF protein that lacks domain III and IV involved in protein/protein interactions and required for the binding to Aux/IAA proteins. Likewise, Sl-ARF3, Sl-ARF16B and Sl-ARF17 are also deprived of domain III and IV necessary for interaction with Aux/IAAs (Figure 1A and Figure S2 in File S1). It is therefore likely that these Sl-ARFs escape the classical mechanism underlying auxin signaling which implies the sequestration of ARF proteins through interaction with Aux/IAAs.

Predicted siRNA-mediated degradation and multiple upstream ORFs in the 59 leader sequences of tomato ARF transcripts ARF genes have been already reported to undergo posttranscriptional regulation involving small interfering RNAs. In silico analysis at the RNA level predicted that 12 out of the 22 tomato Sl-ARFs have a putative target site for small interfering RNAs (Figure 1A). That is, Sl-ARF2A, Sl-ARF2B, Sl-ARF3 and Sl-ARF4 are predicted to be potentially targeted by TAS3; Sl-ARF6A, Sl-ARF8A and Sl-ARF8B by miR167; and Sl-ARF10A, Sl-ARF10B, Sl-ARF16A, Sl-ARF16B and Sl-ARF17 by miR160.

The uORFs are elements found in the 59-leader sequences of specific mRNAs that modulate the translation of downstream ORFs by ribosomal stalling and inefficient re-initiation or by affecting transcript accumulation through nonsense-mediated mRNA decay pathway. Among the 19 Sl-ARFs for which the 59 leader sequences are available in iTAG2.30 (8 members) or identified in this study (11 members), uORFs were predicted for 17 genes, ranging from 1 to 52 amino acids in size with four genes (Sl-ARF2A, Sl-ARF5, Sl-ARF10A and Sl-ARF16A) having five or more uORFs (Table S1 in File S1).The average number of uORF per Sl-ARF gene is similar in tomato (2.8/leader) and Arabidopsis (3.3/leader), indicating that tomato ARFs are suitable candidates to be regulated through translational uORFs depending mechanism

Transcriptional activation and repression activities of tomato ARFs

To characterize the capacity of tomato ARF proteins to in vivo activate or repress gene transcription, tobacco cells were cotransfected with an effector construct expressing the full-length coding sequence of Sl-ARFs and a reporter construct carrying the auxin-responsive DR5 promoter fused to GFP coding sequence [START_REF] Ottenschlager | Gravity-regulated differential auxin transport from columella to lateral root cap cells[END_REF]. DR5 is a synthetic auxin-responsive promoter made of 9 inverted repeats of the conserved Auxin-Responsive Element, the so-called TGTCTC box, fused to a CaMV35S minimal promoter. The DR5-driven GFP chimeric gene showed low basal activity which was induced up to 5-fold by exogenous auxin treatment (Figure 2). Co-transfection of tobacco protoplasts with the DR5::GFP reporter construct and effector plasmids expressing either Sl-ARF1, Sl-ARF2A, Sl-ARF2B, Sl-ARF3, Sl-ARF4, Sl-ARF9A, Sl-ARF10A orSl-ARF17 coding sequences, resulted in repression of the auxin-induced expression of the reporter gene (Figure 2). By contrast, co-transfection with effector constructs expressing Sl-ARF5, Sl-ARF6A, Sl-ARF7, Sl-ARF8B or Sl-ARF19 enhanced slightly the auxin-induced expression of the reporter gene. Noteworthy, with the exception of Sl-ARF6A and Sl-ARF7, these activator ARFs were unable to enhance the basal activity of the DR5 promoter in the absence of auxin treatment (Figure 2) suggesting that most ARFs require the input of an active auxin signalling for transcriptional activation of target genes.

Expression of Sl-ARF genes in different tomato tissues

To gain clues on the physiological function of tomato ARFs, the spatio-temporal expression of individual members of the gene family was examined at the transcriptional level using qRT-PCR. Transcript accumulation could be assessed for 15 ARF genes in different tissues including root, stem, leaves, flower and fruit at various developmental stages. For the remaining 7 tomato ARF genes, transcript detection was unsuccessful in any of the samples tested suggesting their extremely low expression in these tissues. The data indicate that the expression of ARF genes is ubiquitous in all tissues with most genes being expressed in reproductive tissues suggesting their putative role in flower and fruit development (Figure 3). Heatmap representation (Figure 4) allowed the clustering of tomato ARFs into two main groups based on their expression pattern: group I (Sl-ARF1, Sl-ARF2A, Sl-ARF2B, Sl-ARF4, Sl-ARF7A, Sl-ARF6B and Sl-ARF18) are genes preferentially expressed in roots and group II Sl-ARFs in the areal part of the plant.

Sl-ARF6B displays a very low expression in all tomato tissues analyzed and the corresponding CT values showed high variability among repeats making these Sl-ARF6B expression data not meaningful. Therefore, they were not included in Figure 3 but retained for the heat map (Figure 4) in despite of the variability between the repeats in order to give a general idea about its expression in different tissues.

Auxin and ethylene regulation of Sl-ARF genes

Screening for cis-acting elements corresponding to Auxin Response Elements (AuxRE) within the promoter regions using the Place database (http://www.dna.affrc.go.jp/PLACE/ signalscan.html) identified conserved (TGTCTC) and degenerate (TGTCCC) motifs in most tomato ARF promoters. In addition to these AuxRE, Sl-ARF promoters contain conserved Ethylene-Response motifs, the so-called ERELEE4 motif found in the promoter of tomato E4 gene (AWTTCAAA) (Table S2 in File S1). The presence of these cis-regulatory elements suggests a potential regulation of ARF genes by both auxin and ethylene. To test the responsiveness of tomato ARF genes to both hormones, transcript accumulation was assessed by qRT-PCR in seedlings treated with auxin or ethylene. All Sl-ARFs were found to be auxin-responsive after 2 hour treatment (Figure 5A), with Sl-ARF4, Sl-ARF5 and Sl-ARF2A showing the highest up-regulation whereas Sl-ARF1, Sl-ARF7 Sl-ARF10 displayed the most significant down-regulation. On the other hand, the expression of Sl-ARF2B, Sl-ARF5 and Sl-ARF9A showed strong up-regulation (more than four folds increase) when treated 5 hours with ethylene (Figure 5B). Of particular interest, Sl-ARF5 is strongly up-regulated by both hormones and may therefore be involved in mediating responses to both hormones.

Expression of Sl-ARF genes during tomato fruit set

The expression of a high number of Sl-ARFs in reproductive tissues (Figure 3 and4) along with the previously reported role of auxin in controlling the fruit set process, prompted us to investigate the expression of Sl-ARF genes during the flower-tofruit transition. To determine the expression dynamics throughout the fruit set process, transcript accumulation of tomato ARFs was monitored by RNA-seq approach at flower buds, anthesis and posanthesis stages (young fruit at 4 DPA). For each stage, RNA libraries were generated from three independent biological replicates and subjected to Illumina mRNA-Seq technology sequencing (Data desposited at NCBI SRA database under accession number SRP029978). Reads were then mapped on the tomato genome sequence and read counts were determined as described in Maza et al. 2013 [START_REF] Maza | Comparison of normalization methods for differential gene expression analysis in RNA-Seq experiments: A matter of relative size of studied transcriptomes[END_REF]. The data indicate that most Sl-ARFs undergo a strong change in their expression associated with the flower-to-fruit transition (Figure 6). Three groups could be discriminated based on RNA counts distribution during the fruit set process. Group 1 corresponds to Sl-ARFs whose expression increased following pollination, Group 2 to ARFs with unchanged expression and Group 3 to Sl-ARFs displaying decreased expression following pollination (Figure 6).

Sl-ARF transcripts undergo intense alternative splicing during tomato fruit set

Closer analysis of the mapping of RNA-seq data on the gene models revealed possible alternative splicing regulation during fruit set for 30% of Sl-ARF genes. Sl-ARF2B and ARF19 shows one possible alternative splicing occurring at intron 11 and intron 1, respectively (Figure 6A and Figures S3.1 in File S1). Sl-ARF3 and Sl-ARF4 could putatively give rise to two alternative splicing events at introns 7 and 9, and at introns 6 and 10, respectively. Three possible alternative splicings were found at introns 3, 6 and 10 in Sl-ARF8A and at introns 9, 11 and 13 in Sl-ARF8B. Finally, Sl-ARF24 offers up to four alternative splicing possibilities at introns ). In all cases, the detected Sl-ARF splice variants resulted in a frame shift within the coding region that generates a premature stop codon. To further validate the occurrence of the alternative splicing forms and assess the relative levels of the various splice variants, a semi quantitative PCR approach was conducted. To this purpose, two pairs of primers were designed, one aiming to specifically amplify the retained intron fragment while the second pair was designed in the margins of the two exons framing the retained intron. A PCR product with the expect size was detected for all genes confirming the presence of the splice variant in each RNA extraction (Figure 7B). Interestingly, the data indicate that the abundance of the Sl-ARF8B_int11 transcript variant decreases dramatically in young fruits whereas the global expression of the corresponding Sl-ARF8B gene increases significantly. This finding suggests that the down-regulation of the Sl-ARF8B_int11 transcript variant may potentially play a role in the regulation of the flower to fruit transition. By contrast, increased accumulation of the Sl-ARF19_int1 was observed concomitant to the transition from flower to fruit. Taking together, these data uncover a potential role for alternative splicing in regulating the expression of tomato ARFs during the fruit set process.

Discussion

Being down-stream components of auxin signalling pathway, ARFs likely contribute to the specificity of the hormone responses. Hence, the functional characterization of these transcriptional mediators is essential towards understanding the mechanisms by which auxin triggers appropriate growth and developmental responses in a timely and tissue-specific manner. To better define the role of ARFs in mediating specific auxin responses, the present study brings a complete picture on the main structural features of the tomato ARF gene family. Identification of tomato ARFs has been already described but this attempt built on a draft tomato genome sequence and ESTs and could therefore not be comprehensive [START_REF] Kumar | Genome-wide analysis of auxin response factor (ARF) gene family from tomato and analysis of their role in flower and fruit development[END_REF][START_REF] Wu | Identification, isolation and expression analysis of auxin response factor (ARF) genes in Solanum lycopersicum[END_REF]. The present work takes advantage of the most updated tomato reference genome sequence [START_REF]The tomato genome sequence provides insights into fleshy fruit evolution[END_REF] to isolate the complete ARF family members and perform functional analysis and expression profiling of these transcriptional regulators. Using these extended resources, the list of tomato ARFs has been enlarged to 22 members and manual annotation based on deep RNA-Seq data, allowed the curation of some structural annotation inconsistencies as well as the identification of the 39 and 59 UTR regions for more than 50% of the Sl-ARF gene family. The tomato members of the ARF family were renamed according to the numbering of the closest Arabidopsis homolog, which provides a consensus nomenclature for ARF genes across plant species. In this way, the proposed nomenclature better clarifies the correspondence between ARF subclasses in various plant species. The phyllogenetic approach applied on a well distributed set of plant ARFs allowed to identify a specific sub-class (sub-class IV) that is absent out of the Solanaceae family. Interestingly, this subclass contains a specific gene, Sl-ARF24, encoding a putative ARF protein that lacks the two protein/protein interaction domains, known as domain III and IV and required for the binding to Aux/ IAA proteins. It is therefore likely that Sl-ARF24 escapes the classical mechanism underlying auxin signaling which implies the sequestration of ARF proteins through interaction with Aux/IAAs.

As a preliminary approach towards functional characterization of members of the tomato ARF family, the present study describes their expression pattern, their post-transcriptional regulation and their ability to activate or repress transcriptional activity on synthetic or native auxin-responsive promoters. Transactivation assays revealed that 36% of tomato ARFs are strong repressors of transcriptional activity while only 22% are transcriptional activators. The repressor/activator ratio among ARFs is more than twice higher in tomato (3.6) compared to Arabidopsis (1.7), yet, it remains to be elucidated whether this feature may account for differences in developmental and growth behaviour between the two species. In contrast to repressor ARFs, most activator Sl-ARFs promote transcription of target genes only upon exogenous auxin treatment thus suggesting that activator ARFs require some input from a highly activated auxin signalling pathway in order to potentiate transcriptional activity. It is conceivable that when the auxin level is low, the amount of Aux/IAA proteins available is sufficient to block ARFs at the protein level thus preventing these latter from activating the transcription of the target genes. In this perspective, it has to be postulated that Aux/IAAs are present in excess in the cell when the tissue is not subjected to auxin treatment.

The spatio-temporal pattern of expression indicated that all Sl-ARF genes are expressed in flower and fruit suggesting a putative important role in reproductive tissue development. The shift from the static flower ovary to fast-growing young fruit is a phenomenon known as fruit set and auxin has been shown to play a crucial role in controlling this developmental process [START_REF] Serrani | Auxin-induced fruit-set in tomato is mediated in part by gibberellins[END_REF][START_REF] De Jong | The role of auxin and gibberellin in tomato fruit set[END_REF] representing an important step in the development of all sexually reproducing higher plants. Adding to the primary role of Aux/IAAs in triggering the fruit set process previously reported [START_REF] Wang | Regulatory features underlying pollination-dependent and -independent 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], the present study reveals the potential active role of a number of Sl-ARFs during this process based on genome-wide transcriptomic profiling of the flower to fruit transition. The expression of 12 members of the gene family sharply increases upon pollination/ fertilization, while the expression of a fewer number of Sl-ARF genes peaks at anthesis and then dramatically declines at postpollination stage. Given the role of auxin signaling in the fruit set process [START_REF] De Jong | The role of auxin and gibberellin in tomato fruit set[END_REF][START_REF] Devoghalaere | A genomics approach to understanding the role of auxin in apple (Malus x domestica) fruit size control[END_REF], the dynamics of the expression pattern of these Sl-ARFs is indicative of their putative involvement in mediating auxin responses during the flower-to-fruit transition. This is consistent with the prominent role reported for Sl-ARF8A and Sl-ARF7 (referred as Sl-ARF19 in the present study) during fruit set and parthenocarpy in Arabidopsis and tomato, respectively [START_REF] Goetz | Expression of aberrant forms of AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and tomato[END_REF][START_REF] De Jong | The Solanum lycopersicum auxin response factor 7 (SlARF7) regulates auxin signaling during tomato fruit set and development[END_REF]. Of particular interest, Sl-ARF8A shows the most dramatic rise in expression at post-anthesis stage which may designate this ARF among all family members as the main actor of the fruit set process.

The data indicate that tomato ARFs are subject to multi-levels post-transcriptional regulation of their expression. In line with Arabidopsis ARFs [START_REF] Wang | Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis[END_REF][START_REF] Williams | A database analysis method identifies an endogenous trans-acting short-interfering RNA that targets the Arabidopsis ARF2, ARF3, and ARF4 genes[END_REF][START_REF] Wu | Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction[END_REF], it is shown here that 11 out of the 22 tomato ARF genes are potentially regulated by siRNAs. Moreover, the direct evidence for active alternative splicing described here uncover a new layer of complexity in the post-transcriptional regulation of ARF genes in the tomato. This mode of regulation may account for a significant part of the control of ARF expression in processes such as fruit set in the tomato as indicated by the abundance of some transcript splice variants concomitant to the flower to fruit transition. An additional mean towards controlling ARF expression in the tomato may also take place at the translational level via upstream ORFs (uORFs) that have been predicted in most members of ARF genes. This mode of regulation has been first suggested in Arabidopsis where in silico search revealed an enrichment of uORFs in the ARF 59-leader sequences that is not seen in other auxin-related genes such Aux/ IAA, YUCCA, TIR1 auxin receptors homologs and PIN family of auxin transporters [START_REF] Zhou | Translation reinitiation and development are compromised in similar ways by mutations in translation initiation factor eIF3h and the ribosomal protein RPL24[END_REF]. Subsequently, translational control of AtARFs by upstream ORF (uORFs) has been proposed as a regulatory mechanism required in modulating auxin responses during plant development [START_REF] Rosado | Arabidopsis ribosomal proteins control developmental programs through translational regulation of auxin response factors[END_REF]. Though direct experimental evidence is still lacking, tomato ARFs may also undergo the same mode of regulation.

In addition of being auxin-responsive, the expression of some Sl-ARFs was found to be regulated by ethylene. The presence of auxin and ethylene cis-regulatory elements in the promoter region of a number of Sl-ARFs, supports the potential regulation of ARF genes by both auxin and ethylene and suggests that these transcription factors have the ability to mediate both auxin and ethylene responses. In support to this hypothesis, Arabidopsis ARF19 has been shown to be inducible by ethylene and has been reported to contribute to ethylene sensitivity through a cross-talk between auxin and ethylene signalling [START_REF] Li | Convergence of signaling pathways in the control of differential cell growth in Arabidopsis[END_REF][START_REF] Li | A role for auxin response factor 19 in auxin and ethylene signaling in Arabidopsis[END_REF]. Also, ARF2 has been shown to regulate the hook curvature of etiolated Arabidopsis seedlings, a typical ethylene response [START_REF] Li | Convergence of signaling pathways in the control of differential cell growth in Arabidopsis[END_REF]. Taking together, these data suggest that ARFs may act at the crossroads of auxin and ethylene signaling.

Altogether, the data provide molecular clues on how ARFs can contribute to the specificity and selectivity of auxin responses through (i) structural features, (ii) differential expression of family members at the tissue and organ levels and, (iii) ability to negatively or positively impact transcriptional activity of target genes. The auxin and ethylene regulation of some ARF members suggest their specific role in the multi-hormonal cross-talks. The regulation of the expression of ARFs by alternative splicing during fruit set provides new insight into the complexity of regulation of these genes at the post-transcriptional level.

Materials and Methods

Plant material and growth conditions

Tomato seeds (Solanumlycopersicum cv MicroTom or Ailsa Craig) were sterilized, rinsed in sterile water and sown in recipient Magenta vessels containing 50 mL of 50% Murashige and Skoog (MS) culture medium added with R3 vitamin (0.5 mg L 21 thiamine, 0.25 mg L 21 nicotinic acid and 0.5 mg L 21 pyridoxine), 1.5% (w/v) sucrose and 0.8% (w/v) agar, pH 5.9. Plants were grown under standard greenhouse conditions. The culture chamber rooms are set as follows: 14-h-day/10-h-night cycle, 25/20uC day/night temperature, 80% hygrometry, 250 mmol m 22 s 21 intense luminosity.

In silico Identification of the tomato ARFs

All the ARF gene sequences (ITAG2.3_gene_models.gff3) are download from the Sol Genomics Network (http://solgenomics. net/), and analyzed in Notepad++ software. The NLS location was searched using cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/ cgi-bin/NLS_Mapper_form.cgi). All the obtained sequences were sorted for the unique sequences and these were further used for B3, AUX_RESP, and Aux/IAA domain search using InterProScan Sequence Search (http://www.ebi.ac.uk/Tools/pfa/iprscan/ ). The UTR of Sl-ARFs were found by two steps, first, the whole tomato genome and Sl-ARF gene structures (ITAG2.3_gene_models.gff3) were loaded into the Java, and then, the complete cDNA sequences from RNA-Seq data including three stages (flower bud, anthesis and post-anthesis) were blast with Sl-ARF gene structures to identify the final 59 or 39 UTRs in Sl-ARFs. The miRNA location on the Sl-ARFs were searched depend on the GBF data (http://tata.toulouse.inra.fr/gbf/blast/blast.html) and SGN Blast tools. Taken together, all of the Sl-ARF family structures were

Transient Expression Using a Single Cell System

Protoplasts were obtained from suspension-cultured tobacco (Nicotianatabacum) BY-2 cells and transfected by a modified polyethylene glycol method as described by Abel and Theologis [START_REF] Abel | Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression[END_REF]. For nuclear localization of the selected ARF fusion proteins, the coding sequence of genes were cloned as a C-terminal fusion in frame with GFP under the control of the 35S CaMV, a cauliflower mosaic virus promoter. Transfected protoplasts were incubated for 16 h at 25uC and analysed for GFP fluorescence by confocal microscopy. For co-transfection assays, aliquots of protoplasts (0.5610 6 ) were transformed either with 10 mg of the reporter vector alone containing the promoter fused to the GFP reporter gene or in combination with 10 mg of ARF contructs as the effector plasmid. Transformation assays were performed in three independent replicates. After 16 h, GFP expression was analyzed and quantified by flow cytometry (FACS Calibur II instrument, BD Biosciences, San Jose, CA) on the flow cytometry platform, IRF31, Inserm, Toulouse and and cell sorting platform, INSERM UPS UMR 1048, Toulouse RIO imaging platform. Data were analyzed using Cell Quest software. For each sample, 100 to 1000 protoplasts were gated on forward light scatter and the GFP fluorescence per population of cells corresponds to the average fluorescence intensity of the population of cells after subtraction of autofluorescence determined with non transformed BY-2 protoplasts. The data are normalised using an experiment, in presence of 50 mM 2.4 D, with protoplasts transformed with the reporter vector in combination with the vector used as the effector plasmid but lacking Sl-ARF coding region.

RNA isolation and Quantitative RT-PCR

Total RNA from fruit was extracted according to the method of Hamilton et al. [START_REF] Hamilton | Antisense Gene That Inhibits Synthesis of the Hormone Ethylene in Transgenic Plants[END_REF]. Total RNA from leaves and seedlings was extracted using a Plant RNeasy Mini kit (Qiagen) according to the manufacturer's instruction. Total RNA was treated by DNase I to remove any genomic DNA contamination. First strand cDNA was reverse transcribed from 2 mg of total RNA using Omniscript kit (Qiagen) according to the manufacturer's instruction.The qRT-PCR analysis was performed as previously described [START_REF] Wang | Regulatory features underlying pollination-dependent and -independent tomato fruit set revealed by transcript and primary metabolite profiling[END_REF]. The sequences of primers are listed in Table S3 in File S1. Relative fold differences were calculated based on the comparative Ct method using the Sl-Actin as an internal standard. To determine relative fold differences for each sample in each experiment, the Ct value of genes was normalized to the Ct value for Sl-Actin-51 (accession number Q96483/Solyc11g005330) and was calculated relative to a calibrator using the formula 2 2DDCt . At least two to three independent RNA isolations were used for cDNA synthesis and each cDNA sample was subjected to real-time PCR analysis in triplicate. Heat map representation was performed using centring and normalized DCt value, with Cluster 3.0 software and Java Tree view to visualize dendogram.

Hormone treatment

For auxin treatment on light grown seedlings, 12-day-old tomato seedlings (30 seedlings) were soaked in liquid MS medium with or without (mock treatment) 10 mM IAA for 2 hours. The efficiency of the treatment was checked by measuring the induction of the tomato early auxin-responsive SAUR gene. For ethylene treatment on dark grown seedlings, 5-days-old Micro-Tom seedlings (100 seedlings) were treated with air or ethylene gas (50 mL/L) for 5 hours. The efficiency of the treatment was checked by measuring the induction of the tomato ethyleneresponsive E4 gene. Experiment was repeated for 3 biological times.

RNA-Sequencing and RT-PCR

Total RNA was extracted from bud, flower and post-flower (4DPA) for three biological repeats using a TRIZOL Reagent (invitrogen) according to the manufacturer's instruction. Total RNA was treated by DNase I to remove any genomic DNA contamination and checked by RNA gel and Agilent RNA 6000 Nano Assay, which the RIN value above 7 was determined to be qualified. After that, the best RNA were sent out for deep RNA sequencing using Illumina Hiseq2000 and the reads generated were mapped to the tomato genome sequence SL2.40. The data are desposited at NCBI SRA database under the accession number SRP029978 The gene expression was calculated for each annotated tomato gene (iTAG2.30). For continuous validation, first strand cDNA was synthesized as previously described and PCR was performed using primers designed from the intron and exon of 7 Sl-ARF genes. The primer sequences are listed in Table S4 in File S1. An aliquot of 1 ul of the product was used as a template. The PCR amplification cycle was as follows: 95uC for 30 s, 56-60uC for 40 s, 72uC for 30 s-2.5 min. Samples were taken after 25, 30 or 35 cycles and 10 ul of the PCR product was visualized on a 2-2.5% agarose gel. All PCRs were carried out in a Mastercycler (Eppendorf, Hamburg, Germany). DNA was stained with ethidium bromide in the gel. Sl-Ubi3 expression was used as an internal control.

Figure 1 .

 1 Figure 1. The ARFfamily structures in tomato and phylogenetic relationship between rice, potato, tomato, grape and Arabidopsis. (A) The generic structures of Sl-ARF family except Sl-ARF6A. The gene size (kb) is indicated in the upper panel. The domain of Sl-ARF gene is indicated by different colours. The marker in Sl-ARF family showsSl-ARF2A, 2B, 3 and 4genes are spliced by TAS 3, Sl-ARF8A and 8B spliced by miRl67, and Sl-ARF10A, 10B, 16A, 16B and 17 spliced by miR160.(B) The unrooted tree was generated using MEGA4 program by neighbor-joining method. Bootstrap values (above 50%) from 1000 replicates are indicated at each branch. All Sl-ARFs contain a DBD (brown). Most of the Sl-ARF proteins except Sl-ARF3, 10, 24, 16 and 17 contain a carboxy-terminal domain related to the domains III and IV found in the Aux/IAA proteins (blue).Sl-ARF5, 6A, 7, 8A, 8B, 19 contains a middle region that corresponds to the predicted activation domain (green) found in some AtARFs. The remaining Sl-ARFs contains a predicted repression domain (red). Sl-ARF-6B and AtARF23 contain only a truncated DBD (B3 domain). doi:10.1371/journal.pone.0084203.g001

Figure 2 .

 2 Figure 2. Sl-ARF factors differentially regulate the expression of reporter genes driven by synthetic and native auxin-responsive promoters. Sl-ARF factors were challenged with a synthetic auxin-responsive promoter called DR5, consisting of seven tandem copies of the AuxREtgtctc element. A transient expression using a single cell system was performed to measure the reporter gene activity. The fluorescence was measured by flux cytometry. Because of the very low basal activity of the DR5 promoter without auxin treatment, the auxin inducible fluorescence obtained by co-transformation with the promoter fused to the reporter gene and with the empty vector was standardized to 100 and taken as reference. Biological triplicates were averaged and analysed statistically using Student's t-test at (P,0.05). (*) indicates significant changes corresponding to co-transformation with effector Sl-ARF and reporter DR5-GFP constructs compared to basal activity of DR5 promoter in the absence of auxin treatment. (**) indicates significant changes for the same experiment carried out in the presence of auxin Bars indicate the SEM. doi:10.1371/journal.pone.0084203.g002

Figure 3 .

 3 Figure 3. Real-time PCR expression profiles of individual Sl-ARF genes. Total of 15 Sl-ARFgenes were performed in different tomato organs (root, stem, leaf, flower, 8DPA, Mature Green, Breaker and Red). X-axis represents different Sl-ARF genes, while Y-axis represents three relative expressions of those genes. 8DPA: 8 days after pollination, Mature Green, Breaker and Red represent different stage of the fruit development. doi:10.1371/journal.pone.0084203.g003

Figure 4 .

 4 Figure 4. Heatmap showing Sl-ARF gene expression in different tomato tissues. Changes in RNA accumulation in different tomato tissues (Roots, Leaves, Stems, Flowers, Early Immature Green (8 DPA), Mature Green, Breaker, Red (Breaker + 7 days) as schematically depicted above the displayed array data, are shown relative to the RNA accumulation levels in roots. Levels of down expression (green) or up expression (red) are shown on a log2 scale from the high to the low expression of each Sl-ARF gene. doi:10.1371/journal.pone.0084203.g004

Figure 5 .

 5 Figure 5. The expression of Sl-ARF family genes in response to auxin and ethylene. (A) Auxin induction of Sl-ARF genes on light grown seedlings. Quantitative RT-PCR of Sl-ARF transcripts in RNA samples extracted from 12-day-old tomato seedlings soaked in liquid MS medium with 10 mM IAA for 2 hours. DDCT refers to the fold of difference in Sl-ARF expression to the untreated seedlings. The SAUR gene was used as control to validate the auxin treatment.(B) Ethylene regulation of Sl-ARF genes on dark grown seedlings. Quantitative RT-PCR of Sl-ARF transcripts in RNA samples extracted from5-days dark-grown tomato seedlings treated 5 hours with ethylene (50 mL/L). DDCT refers to fold differences in Sl-ARF expression relative to untreated seedlings. The E4 gene was used as control for efficient ethylene treatment. doi:10.1371/journal.pone.0084203.g005

Figure 6 .

 6 Figure 6. The expression profile of Sl-ARF family genes in tomato fruit set. (A)12 Sl-ARF genes are over-expressed after pollination and fertilization (4DPA), which are Sl-ARF9A, 4, 18, 8A, 1, 7B, 5, 8B, 2A, 3, 7A and 2B genes in turn according to the log change of P/A (Post-anthesis/ Anthesis). (B) 5 Sl-ARF genes keep stable expression from flower bud to post-anthesis, includingSl-ARF10A, 10B, 6B, 9B, 17 genes.(C) 3 Sl-ARF genes are up-regulated from flower bud to anthesis and down-regulated after pollination and fertilization (4DPA), including Sl-ARF24, 19, and 16A genes. The expression values are taken from RNA-sequencing data and the colors represent different Sl-ARF genes. doi:10.1371/journal.pone.0084203.g006

Figure 7 .

 7 Figure 7. The ARF family genes showed alternative spilcing mode of regulation in tomato fruit set. (A) RNA-seq reads generated during the fruit-set and mapped on Sl-ARF19 gene structure showing one alternative spicing that can be generated in the Intron 1. Reads are represented by red and blue rod arrows (B) The RT-PCR was carried out using pairs of primers designed within the introns of 7 Sl-ARF genes highlighted in Figures S3.1 to S3.6 in File S1, such as Sl-ARF8A_Intron 6, Sl-ARF8B_Intron 11, Sl-ARF3_Intron 9, Sl-ARF24_Intron 3, Sl-ARF19_Intron 1, Sl-ARF4_Intron 6 and Sl-ARF2B_Intron 11. The ubiquitin gene was used as the reference. (C) The RT-PCR was performed using pairs of primers nested in the two exons encompassing the intron of target Sl-ARF genes, such as Exon1-Exon2 in Sl-ARF19 and Exon6-Exon7 in Sl-ARF8A. The cDNAs generated from flower bud (B), flower at anthesis (F) and young fruit 4 days post-pollination (P) tissues were used as the template. The ubiquitin was used as the reference. doi:10.1371/journal.pone.0084203.g007

Table 1 .

 1 Sl-ARF gene family in tomato. Gene Model modification type: UTR means that the UTR sequence have been identified and annotated, CDS means that the the coding sequence have been corrected. g New locations in the tomato genome version Sl2.40 taking into account the manual curation of the previous gene annotation in iTAG2.30 doi:10.1371/journal.pone.0084203.t001

	Generic name a	Alias b	CDS length c	Str	Chr	Domains d	Name in Wu et al. e	improvement f	New location g
	Sl-ARF1	Solyc01g103050	1965	+	1	B3,ARF,AUX/IAA,SPL-Rich RD	SlARF1(HM061154.1)	-
	Sl-ARF2A	Solyc03g118290	2541	+	3	B3,ARF,AUX/IAA,SPL-Rich RD	SlARF2(DQ340255.1)	-
	Sl-ARF2B	Solyc12g042070	2490	-	12	B3,ARF,AUX/IAA,SPL-Rich RD	SlARF11(HM143940.1)	59, 39 UTR	42538600..42544937
	Sl-ARF3	Solyc02g077560	2244	+	2	B3,ARF,SL/G-Rich RD	SlARF3(DQ340254.1)	-
	Sl-ARF4	Solyc11g069190	2436	-	11	B3,ARF,AUX/IAA,SPL-Rich RD	SlARF4(DQ340259.1)	59, 39 UTR	50900912..50910023
	Sl-ARF5	Solyc04g081240	2793	-	4	B3,ARF,AUX/IAA,QSL-Rich AD	SlARF5(HM195248.1)	-
	Sl-ARF6A	Solyc12g006340(Nter);	2643	2/+	12/0	B3,ARF,AUX/IAA,	SlARF6(HM594684.1)	59 UTR, CDS	857256..859656(Nter)
		Solyc00g196060(Cter)				QSL-Rich AD
	Sl-ARF6B	Solyc07g043620	2673	-	7	B3,ARF,QSL-Rich AD	SlARF6-1(NM_001247611.1)	59UTR	54884781..54890560
	Sl-ARF7A	Solyc07g016180	3339	-	7	B3,ARF,AUX/IAA,QSL-Rich AD	SlARF19(HM130544.1)	-
	Sl-ARF7B	Solyc05g047460	3294	-	5	B3,ARF,AUX/IAA,QSL-Rich AD	SlARF19-1(HM565130.1)	59UTR	58050744..58057040
	Sl-ARF8A	Solyc03g031970	2535	+	3	B3,ARF,AUX/IAA,QSL-Rich AD	SlARF8-1(HM560979.1)	59UTR	8739535..8747501
	Sl-ARF8B	Solyc02g037530	2529	+	2	B3,ARF,AUX/IAA,QSL-Rich AD	SlARF8(EF66734F2.1)	59UTR	21756022..21766699
	Sl-ARF9A	Solyc08g082630	1977	+	8	B3,ARF,AUX/IAA,SPL-Rich RD	SlARF9(HM037250.1)	59UTR	62527409..62531812
	Sl-ARF9B	Solyc08g008380	2052	+	8	B3,ARF,AUX/IAA,SPL-Rich RD	SlARF12(HM565127.1)	59UTR	2807931..2812983
	Sl-ARF10A	Solyc11g069500	2100	-	11	B3,ARF,AUX/IAA,SL/G-Rich RD	SlARF10(HM143941.1)	59, 39 UTR	51188434..51192539
	Sl-ARF10B	Solyc06g075150	2016	+	6	B3,ARF,AUX/IAA,SL/G-Rich RD	SlARF16(HM195247.1)	39 UTR	43020594..43023604
	Sl-ARF24	Solyc05g056040	1953	-	5	B3,ARF,SPL-Rich RD	SlARF13(HM565128.1); SlARF13-	-
							1(HM565129.1)
	Sl-ARF16A	Solyc09g007810	2085	-	9	B3,ARF,AUX/IAA,SL/G-Rich RD	No	39 UTR	1332230..1335760
	Sl-ARF16B	Solyc10g086130	1896	-	10	B3,ARF,SL/G-Rich RD	SlARF16(NM_001247861.1)	-
	Sl-ARF17	Solyc11g013480(Nter);	1869	-	11	B3,ARF,SL/G-Rich RD	SlARF17(HQ456923)	39 UTR, CDS	6495469..6511349
		Solyc11g013470(Cter)
	Sl-ARF18	Solyc01g096070	2058	+	1	ARF,AUX/IAA,SPL-Rich RD	No	59UTR	78941268..78946012
	Sl-ARF19	Solyc07g042260	3357	-	7	B3,ARF,AUX/IAA,QSL-Rich AD	SlARF7(EF121545.1)	-

a Sl-ARF gene names b the alias of each ARF gene in iTAG2.30 genome annotation c Length of the corresponding Coding Sequence (CDS) in base pairs. d Conserved Domains found in PFAM database: B3 means DNA binding domain, ARF means Auxin response Factor conserved domain, AUX/IAA means AUX/IAA dimerization domain, AD means transcriptional activation domain, RD means transcriptional repression domain. e Corresponding names in Wu et al.; accession numbers are in the parentheses. f

Acknowledgments

This work benefited from the networking activities within the European funded COST ACTION FA1106 ''Qualityfruit''. The authors thank C. Pecher and A. Zakaroff-Girard for their technical assistance and expertise in flow cytometry (Cytometry and cell sorting platform, INSERM UPS UMR 1048, Toulouse RIO imaging platform). The authors also thank O. Boucher and N. Marsaud for their technical assistance and expertise in RNA-seq data production (plateforme genotoul, GeT).

Supporting Information

File S1 Supporting tables and figures. Table S1. uORF prediction in the 59UTR leader sequences of Sl-ARFs. Table S2.

In silico analysis of Sl-ARF gene promoters. Table S3. Quantitative RT-PCR primers of Sl-ARF genes. Table S4. PCR primers for identifying the alternative splicing expressed forms in Sl-ARF genes.


Antoine Beyer
email: antoine.beyer@ifsttar.fr

Laetitia Verhaeghe

Cooperation between Waterways and Railways, an Unnatural Alliance: Rail Strategic Development of River Ports in the Greater Paris Region

Keywords: Rail, River port, Combined transport, Ile-de-France, urban logistics Résumé Rail, Ports fluviaux, Transport combinés, Ile-de-France, logistique urbaine ___________

whether they are published or not. The documents may come

Introduction.

Rail and waterways are mainly regarded as competing transport modes, especially in the massed cargo connections from the seaports. Combining two bulk transports is complicated and seems to make less sense from an economical point of view. However, rare exceptions of such offers exist on the Rhine in Duisburg and Basel, as main continental hubs where goods and especially containers are partly shifted from the barge to the train. A large catchment area and strong links with the national rail operator may explain these exceptions. The other ports on the Rhine and elsewhere in Europe, although they may be well connected to the rail system and sometime dispose on their own installations (cf. Dourges in the North of France) haven t developed similar offers (Beyer, 2011 and2012). So pre-or post-shipping are carried out by road haulage. From the seaports themselves, rail or barge services remain alternative and competing routes to the hinterland. However the recent development of hinterland strategies to a more complex and integrated system [START_REF] Notteboom | Inland Terminals within North American and European Supply Chains[END_REF]& 2012) and the ambitious public policies to reduce road transport gives an opportunity to reconsider how rail/waterway combination may be a realistic option to be implemented. Our research will focus on the Greater Paris region where important shifts have occurred and may induce a possible change in the modal coordination: the rapid development of the container traffic from Le Havre to the Ile-de-France, a proactive political programme on modal shift stated at national and regional level (Grenelle 1 and 2 Acts, the Ecotax etc.) (CAS, 2012) that won t be by far not reached as the rail freight volumes dramatically dropped during the past decade, the port reforms of 2008 that gave the maritime ports and France s two largest river ports (Paris and Strasbourg) a leadership and promotion role for multimodal transport, the specific access constraints to the central urban area and lastly, the recent property transfer from the rail port infrastructure to the port authority. The carriers themselves consider a modal shift with interest to reduce their carbon emissions (by conviction or for commercial reasons) and in order to avoid increasing road congestion in metropolitan areas but only when run at the same costs level [START_REF] Van Schijndel | Congestion and multimodal transport: a survey of cargo transport operators in the Netherlands[END_REF] (Tsamboulas, 2005). All those points encourage to reconsider the place of the rail within a global port strategy. The research objective is to consider how those trends could enhance the cooperation between rail and river despite of the economical and territorial constraints. The methodology followed relies on an inductive geographical and regional planning approach centred on a case study. It gives a preeminent place to consideration on spatial development and territorial management aspects so that the transportation cost-advantage criteria have been less developed for themselves in the text. After a short presentation of the overall context, the argument of the paper is successively devoted to a theoretical approach based in a typology of possible combinations between rail and river transport that can be applied to the Parisian case. A following section presents the current situation and infrastructural constraints that shapes the alternative transport offer in the Greater Paris. The fourth part relies on in-depth interviews with major economical actors in the field that allow to draw two contrasted situations and possible organizational logistics patterns changes: one for aggregates and the other for the container. The conclusion highlights the opportunities but also the limits of a possible cooperation between rail and river transport. This paper is part of more wide-ranging research into the development of river-based logistics in major French conurbations in the framework of the ANR Fluide project (2010-13).

The different types of complementarity between railways and waterways

In the economic theory, the modal choice and the modal combinations depend primarily on the generalized cost. This value takes into account the transport costs itself, the cost generated by the possible modal transfer and the value of time which differs for each type of good. A transport combining several modes must have a lower or at least an equivalent generalized cost compared to a concurrent solution to be taken into account. Hence, the generalized cost defines relevant areas that can partially overlap other transport solutions. In the latter case, the shipper disposes on alternative opportunities. It s not the place here to discuss the advantages or disadvantages of each transport mode and their possible combinations. Concerning the multimodal transport adding barge and rail services may present the advantages of being cheaper for long and massive transport but suffer from far higher delay and relatively high break-bulk costs. Furthermore, rail presents generally a lower functional reliability on the contrary to barge services. The number of transhipments increases the risk of having technical or organizational problems especially if the final destination is not located alongside a quay or directly served by a rail terminal. As mass transport, barge and even more rail are by far less flexible than the road. So a realistic market for a train-barge solution can only occur for high volume, regular flows and distant trip (above 200 km). For our Parisian example, we identified two important good types that could be concerned by the approach as massive and regular traffics: the sea containers coming from the Havre with a final destination further than the Ile-de-France, aggregate from the Parisian Basin and neighbouring regions. If the difficulty to combine rail and river transport is due to economic reasons, the historical situation is also playing a relevant role. The economic and technical characteristics of the two modes are fairly similar and mean they share the same areas of commercial viability. These involve large consignments (heavy and bulk freight for the agricultural sector and heavy industry and quarry products) which have more recently been joined by container shuttle services from sea ports. Consequently, there is some overlapping between the sizes of their unit loads. These are 1250 to 1400 tonnes for a full train and 1000 to 1500 tonnes for boats on the Seine, even if the range is large, extending from 380 tonnes in the case of self-propelled Freycinet barges to 4,000 tonnes for pushed barge trains. Although cost favours the waterways (0.2 cents/t.km on average compared to 0.6 cent/t.km for rail (data from Ports de Paris 2013), the catchment area for river transport is limited by its very low network density. The decision is usually to use a single mode for the main leg and the road for additional access and distribution legs if the consignee or shipper does not have a private siding or a quayside site.

This competition is long-standing. The initial symbiotic relationship which existed between about 1830 to 1860 when railway lines were built to prolong waterway networks rapidly gave way to a ferocious battle for the control of markets as the power of locomotives increased as it occurred from 1850 onwards in Europe and in America (Moulton, 1912) [START_REF] Merger | La concurrence rail-navigation intérieure en France 1850-1914[END_REF]. In addition, their high network density provided the railway companies with a decisive advantage (Léon, 1903). In France, their monopoly within the national territory provided a basis for them to restrict or even strangle waterway transport. Only State intervention made it possible to improve the situation for the waterways, first via the Freycinet Plan of 1877, then through the 1909 Act which compelled the railway companies to connect their lines to inland ports [START_REF] Dounet | Le raccordement des voies ferrées et des voies d eau[END_REF]. The transport coordination policy that was implemented in the 1920s relied on market sharing and controlling operator pricing rather than operational continuity [START_REF] Neiertz | Coordination des transports et « rationalité économique en France de 1918 à nos jours[END_REF]. The creation of the SNCF in 1938 barely changed the situation, and in fact even increased the pre-eminence of rail where waterways could compete. The renovation and electrification of the railway network after 1945 reinforced SNCF s dominant position at a time of constantly decreasing public interest in waterways until the creation of VNF (Voies Navigables de France) in 1991 [START_REF] Sueur | Mariniers et mémoire de la batellerie artisanale, Chasse-marée[END_REF]. In this conflict, the potential for transfer between waterways and rail was very marginal. For the railways, as for waterways, intermodality was a question of cooperating with road transport.

What can be a disadvantage for intermodal competition can, however, be an advantage in the case of a logistic chain where the two modes succeed each other, both with regard to the unit volumes that can be transported and complementarity between the areas covered by services. The transfer from rail to inland waterway transport or vice-versa demands effective transhipment technologies when the product is not processed during its transfer from one mode to the other. In the case of physical transhipment we have decided to consider the two most representative categories of products, containers and quarry products:

• At the sites with the greatest amount of traffic, container handling is performed with a gantry crane. Reach-stackers are used at secondary ports. These may be fitted with telescopic beams with negative reach to be able to pick up a container that is below the quay and make the transfer from the barge to the train in a single operation (as at the Dourges intermodal yard). • In the case of dry bulk cargoes, a conveyor belt can be used for the transfer. This can be mounted on a truck and positioned underneath wagons in order to collect sand and gravel through gravity before discharging them into the barge. For transfers in the other direction, a bucket crane is used to collect the cargo and empty it into the wagons (this technique is slower and less precise than the first). In both cases physical proximity is absolutely essential. If direct transfer between the two transport vehicles is impossible additional costs are entailed.

The volumes carried by a rail or barge train are similar. This means it is fairly easy to avoid the need for an intermediate stockpiling site for bulk cargoes. In the case of containers, it may be necessary to find additional containers to make up a full load as a full rail train carries 80 containers but a barge train can carry up to 200. However, the technical interface and organizational co-ordination needs to be very good for this. When the modes are used one after the other, the cost of the transfer must of course be considered. When asked about the sums involved, VNF quoted the figure of 3 per tonne of cereals and 1 tonne for aggregates. The cost of handling a container is about 25. So, in the case of bulk materials the cost of transfer does not constitute a major obstacle to combining large volume modes. But the latter may be combined with storage (in silos, tips or stacks), repackaging, mixing or an industrial process, from all of which the firms present in the ports draw the maximum amount of profit. In the last cases the functional transfer creates a break which means that it is no longer possible to talk about a single transport operation that takes the form of a transfer in its simplest form. From the organizational standpoint, the relationship between rail and waterways transport allows six possible forms of linkage we met in our researches (Fig. 1): coexistence with a degree of cooperation, competition, succession, as an alternative mode based on technical complementarity, whose systematic presence is known as synchromodality.

Figure 1. The different types of relationship between rail and waterways River ports are evident point of contact and ensure that both modes will continue to be used as an alternative to each other (Fig. 1c) because of spatial factors (for example rail provides access to zones that cannot be reached by barge), or temporal factors (rail takes over from waterways for containers that require urgent delivery, provides an alternative when water levels are too high or too low, or following an accident). In the case of the successive use (Fig. 3d) of the two consolidated inland modes, the cargo is transferred from one mode to the other. We have already seen that the volumes required to fill rail or barge trains may be similar. If this is not the case, smaller flows can be consolidated or be broken up further if their distribution is required. The concept of synchro-modality has been introduced in the Dutch Logistic sector [START_REF] Verweij | Synchronmodal transport: efficient en duurzaam transport via netwerregie[END_REF], which increases the availability and robustness of services. It has been developed for traffic leaving Benelux ports for this sort of situation. TNO (2011) defines synchromodality as the supply of services of the various modalities synchronized to a cohesive transport product, which meets at any moment the transport demand in term of price, punctuality, reliability and/or sustainability and present more an coherent alternative modal choice as a successive combination of both modes.

Rail connexion on the agenda of the Ports of the Greater Paris Region

Like their maritime and foreign counterparts, the river ports of the Greater Paris Region are seeking to make the provision of trimodal services a key aspect of their development (Meuriot & Meignen, 2012). Complementarity of this type, though frequently mentioned in marketing statements has barely been implemented in reality (Blum, 2010). Inland ports have primarily been interested in developing their river traffic exclusively and until now have paid little attention to rail transport for which, incidentally, they were not responsible. As most of the firms using rail had their own sidings at the port, services were often created through direct contacts between shippers and the railway sector. Until recently, even the rail tracks were the responsibility of the rail infrastructure manager. The upheavals that affected the rail market in river ports led to a reorganization of the industry that was particularly deleterious to captive traffic, and rail also failed to take much advantage of the opportunities for growth (combined continental transport and maritime containers). Technical barriers and industrial relations difficulties with regard to the organization of the work of the barge operators and railway workers have led to further reductions in traffic.

Its 12 million inhabitants and 5 million jobs mean that the metropolis of Paris generates a large volume of freight traffic (211 million tonnes in 2010) (DREIF, 2012), in order to satisfy the consumption and production needs of what is France s most important economic region. The road completely dominates the transport market, carrying 89% of the traffic, far ahead of waterways (6.7%) and rail (4.3%). The risk of congestion and environmental concerns have prompted the State, in the framework of the Grenelle environment summit, to lay down new goals for modal transfer, namely to increase the proportion of freight traffic to be carried by modes other than the road from 14% to 25% by the year 2020, i.e. an increase in traffic of 85% on the railways and 15% on the inland waterways. These comprehensive directives are implemented in the region by the Master Plan (Schéma Directeur) and the Urban Travel Plan (Plan de Déplacement Urbain) for the Ile-de-France Region which have made modal transfer a central priority for the Ile-de-France Eco-region . Several recommendations have been made. Freight capacity should be maintained, even during peak periods, on the radial railway lines that meet in Paris or on the Grande Ceinture circular railway line around the city, and all sites with railway sidings should be kept, even those in central Paris. For river transport, the idea is to support the development of new intermodal hubs in the Paris region in order to handle containers and aggregate, while retaining the port sites in the centre of the conurbation (Région IdF, 2009). A study of the links between the two modes will therefore benefit from analysis that considers the nesting of territorial scales. This will guide our analysis of complementarity between the railways and waterways from the regional scale to the changes affecting port sites. underlines a new interest in this perspective [START_REF] Constant | Ports de Paris va créer un OFP pour dynamiser sa desserte ferroviaire[END_REF]. Greater Paris provides us with a second useful analysis zone, which also corresponds to the area covered by Ports de Paris and the regional authority of Ile-de-France. Modal split to river transport is stable, with the waterways carrying more than 20 million tonnes in 2010.

Half of this figure consisted of freight flows within the region, 74% of which were construction materials (DRIE, 2012). Container traffic on the river has been increasing steadily since its launch in 1995, and grew by 51% between 2008 and 2012 when 161,712 units were handled. Several hubs benefit from this dynamism: Gennevilliers, which has a capacity of 120,000 TEU, Bonneuil-sur-Marne (15,000 TEU), Limay (10,000 TEU) and Evry, which opened in 2010 (10,000 TEU). The trend for rail transport is much less encouraging. As we have seen, rail transport is generally speaking the weak link in logistics chains. Its share of the region s traffic fell from 14 to 10 million tonnes between 2004 and 2009 (DREIF, 2012).

The rail terminals in Ile-de-France such as La Chapelle, Noisy-le-Sec and Valenton are large by national standards and are therefore operating much below their full capacity. There are many reasons for this: the national economy has become less focused on bulk goods, rail pricing has been modified and Fret-SNCF has changed its strategies, but most important of all is the continuous reduction in freight traffic in favour of passengers. Freight slots are thus organized around passenger transport, and are therefore scheduled at night or during off-peak periods. These times do not always suit shippers and coincide with the times set aside for maintenance works. The mixed use of the network has a clear impact on freight traffic. Thus, the largest traffic capacities are to the east thanks to the Grande Ceinture. This line is entirely dedicated to freight traffic between Sartrouville and Villeneuve-Saint-Georges and connects the western network (Normandy), the northern network (Picardy, Benelux, Great Britain), the eastern network (Lorraine, Germany) and the south-eastern and southwestern networks (Fig. 1). To the west, a short link between Sartrouville and Achères shares the same track with the Paris-Rouen-Le Havre line and one branch of the RER A, and the southern section of track between Versailles-Chantiers and Juvisy is shared with the RER C and TGV services (as is the section between Massy and Juvisy). Only the Achères-Versailles section has been partly abandoned by passenger services. Access to this track is an advantage for nearby ports (Fig. 2). It is of interest to ports to have access to a rail network which does not carry passengers as it permits a completely different train frequency which is not constrained by passenger peak periods. While Gennevilliers is some distance away, Bonneuil-sur-Marne benefits from direct access to this track. Linkage between railways and waterways is thus s considered in the context of a regional logistics plan and the more distant prospect of a plan to supply Le Grand Paris (which would, in particular, involve the creation of a new metropolitan port for Paris at the southern mouth of a future Seine-North canal). This would, incidentally require a rapid re-organization of urban logistics in which Paris would act as a forerunner at European level, and new dynamism at bimodal facilities that would make it possible to provide a concrete response to the issues of urban congestion. At the scale of its hinterland, the port of Le Havre suffers from its mediocre rail access which limits modal transfer and restricts the extension of its hinterland in particular for containers. The State is currently giving prioirty to several projects: the construction of an intermodal hub on the GMP site at Le Havre due to be completed in 2014 with a handling capacity of 500,000 TEU, doubling the rail track between Mantes and Paris and, finally, the modernization of the Serqueux-Gisors route which provides a good quality alternative link between the maritime terminals at Le Havre and the Valenton hub in the South-East of the Paris region.

Infrastructure constraints mean that the hinterland of Le Havre for containers consists of two parts each of which is geographically and modally coherent: one dense zone that extends to the south-east of the Paris region that is served by the river, and more distant destinations that are scattered throughout the area that are served by the train. The situation here corresponds to what we have called complementarity earlier in this paper. The growing integration of the Haropa Economic Interest Grouping enhances the possibilities afforded by the combination of modes for serving a maritime hinterland. On an operational level, cooperation with regard to railway services has brought the ports of Le Havre and Rouen closer together, in order to organize a joint call for tenders for the maintenance and operation of their infrastructure. They entrusted the service to Europorte by its subsidiary Socorail and Colas Rail, the same partners that were selected by Ports de Paris in 2012 to manage its port facilities (Table 1). We therefore now have the necessary information in order to conduct a re-evaluation of the complementarity between railways and waterways.

Combining high volume modes: the views of the major players

In-depth interviews with the major operators in the Paris region * enabled us to identify the principal markets, namely the construction sector (in particular aggregates) and container transport. These two sectors have specific geographic characteristics which can be used to identify synergies between rail and waterways which have been ignored hitherto. In the case of aggregate (sand and gravel), Paris is a major zone to be served and attracts flows. This is because most worksites and most of the demand are located around the centre of the metropolis. Most of the concrete mixing plants and stockpiling sites are located alongside waterways which encourages supply by barge. The materials they need can therefore be delivered directly without the need for intermediate points to amass or store materials. This question arises more in the case of sourcing from more distant quarries which are not near waterways. These can use two gateways on the edge of the urban area, Achères/Limay in the north-west and Montereau in the South-East. These provide varied supplies, brought in equally by waterway and rail, which is not hindered by competition with passenger services in these locations. Grand Ceinture at Valenton. In the case of waterways, it is possible to avoid problems due to barge size by carrying out transhipment downstream of Paris (boat/boat, or boat/train). This is the distribution hub role planners wish to assign to the future port of Achères. It will need to be designed to provide feeder services to the region s ports and serve the Paris region s market as well as possible. The barge trains leaving Le Havre will not encounter any major obstacles. Other sites will be able to perform the role of collection points for forwarding containers in transit, which is currently performed in a marginal way by Bonneuil (and perhaps Montereau in the near future). For the south-western sector, Achères could also perform this function, sending containers by rail to the Centre Region if the western part of the Grande Ceinture is reinstated or by road if it is not. But according to the current situation, no complementarity situation could be observed on the Seine River. The superimposition of the two approaches also reveals port structures which complement each other and which mirror each other upstream and downstream of the dense zone of Paris. This symmetry is also present in their position at a confluence, the Seine with the Yonne (Montereau) and the Seine with the Oise (Achères). In both cases, the presence of two multimodal port areas upstream and downstream from Paris, and Achères (30 km from the centre of Paris) and Montereau (70 km from the centre of Paris) give a foretaste of the Paris region s two major 21 st century port hubs. The two sites have a considerable amount of reserve capacity and repeat, a century later, the shift away from the centre that was made possible by the ports of Gennevilliers and Bonneuil. It is also important to gauge accurately the quality and efficiency of their intermodal interconnection before initiating development.

Figure 4. The linkage between railways and waterways for containers: a regional view.

Conclusion. Rail and waterways: new possibilities for cooperation.

In 2011, the return of the rail tracks in ports to the control of Ports de Paris was undeniably an invitation to consider possible types of complementarity between the two modes which had previously tended to oppose each other. As the principal manager of the point of contact between the two social and technical worlds, it was incumbent on the port authority to play a crucial role in facilitating intermodality. The issue of the Paris region s ports and the potential they offer for rail transport directly raises a broader theoretical and practical question which is rarely dealt with in the French context, that of intermodality between the waterways and railways. Several combinations are possible most of them on the Rhine, but they have been implemented in an extremely unequal manner. These range from competition for markets which are frequently similar in terms of clientele and products to functional cooperation as regards the services offered by the two modes. Some factors are nevertheless essential in order for such services to develop, and the most important of these is regularity. This is one of the advantages of waterways over road transport which is becoming increasingly unreliable. It is imperative for rail to regain this advantage. The ageing of the network, the mixed nature of the traffic and high traffic loads make it more difficult to achieve this goal for routes that involve the Greater Paris Region than areas immediately adjacent to it. In the light of this, the type of cooperation between the two modes which is the easiest to envisage is one that keeps rail traffic in the outer parts of the metropolis. One factor which strikes us as important is the commercial dynamism of the railway companies which attempt to create new logistics solutions in close collaboration with their clients. The competitive dimension undeniably stimulates this niche market, but their efforts are not visible yet on the global downsizing of the sector.

Although ports are generally considered to be trimodal hubs, spatial constraints are forcing us to reconsider how they are used and develop new modal combinations. The present-day context also alters perspectives and encourages logistical innovation. This is particularly true in the case of services to dense areas of conurbations.

Cooperation between the two high volume modes is not easily achieved and can only concern a few products, and it is under the special constraints imposed by the metropolis that its benefits are greatest. In the context of Greater Paris, we have above all identified one case where rail and waterways transport have started to work together: the transport of aggregate from areas that are not served by waterways to concrete mixing plants on the banks of the Seine. On the contrary to Basel or Duisburg, the organization of consolidated container transport routes from the terminals at sea ports is not working yet. In this case, barge transport provides a way of gaining access to the capital or overcoming the difficulties caused by the saturation of railway lines, but no synchromodal complementarity have raised in the current organization. Is this partial solution destined to remain a niche activity or might it be possible to build on its initial success and implement cooperation at different levels? The organization advocated by VNF in the framework of the Seine-North Europe project and the multimodal corridors proposed by the European Commission encourage this, but the economic equation is hardly favourable for a generalization even if the road price would jump significantly. The case of Paris shows that the combination of rail and barge matches for specific metropolitan markets where high spatial constraints and favourable socio-political environment are observed. In this case, the river leg of the transport chain allows to deliver the very core areas being in less direct competition with other infrastructure users. The other way round from two Rhine ports. The strategic positioning of the port administration is important whereas combining two massive modes is hardly spontaneous. Such an transport option requires several types of investments as Ports de Paris shows: planning the port as a system of interconnected and intermodal terminals, developing locally adapted infrastructures, creating a transport subsidiary devoted to regional rail services. The just-starting Parisian experience has to be capitalised and with the benefit of hindsight further research have to be undertaken to see if the technical and economic conditions are met to duplicate such modal transfer in other major river metropolises.

Bibliographie

  rail/zone of competition)

  2011 represents an important turning point in the management of port rail networks because ownership of the three principle port sites (Gennevilliers with 27.2 km of track, Bonneuil-sur-Marne with 14.3 km and Limay with 8.6 km) passed from Ports de Paris to RFF (Réseau Ferré de France, the French national railway infrastructure manager) (Ports de Paris, 2012). In addition, the Grenelle 2 Act of 12 July 2010 applied the measures that were taken in 2005 for the major maritime ports to the two autonomous river ports (Paris and Strasbourg) (RFF& PAP, 2012). The transfer represented a real challenge for Ports de Paris which had to acquire new technical and legal expertise and which took over an annual financial burden of approximately 1 million Euros in order to modernize a system that was on its last legs. While three of the ports are operating, only six of the thirty or so active sites run by Ports de Paris enjoy rail access. Ultimately, the Paris Port Authority will need to develop a comprehensive project in partnership with the two port communities which bring together the firms present at Gennevilliers and Bonneuil. The port project of creating its own rail subsidiary by 2015 to gain 50% more rail traffic (1 Mt)

Figure 2 .

 2 Figure 2. Rail freight traffic in Ile-de-France and location of saturated routes

Figure 3 .

 3 Figure 3. A regional view of the link between railways and waterways for aggregates

Table 1 .

 1 Rail traffic of the major ports in the Paris region (in t.)

	Ports/Year	2010	2011	2012
	Bonneuil	286079	391559	665424
	Gennevilliers	747701	894673	742013
	Limay	90597	134576	272335
	Ports de Paris (Total)	1124377	1420808	1679772

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CAS Centre d analyse stratégique (2012), Pour une complémentarité du rail, de la route et du fleuve au service du transport de marchandises, 12 p.