César Luís Girardi

Valmor Rombaldi

Joceani Dal Cero

Paula M Nobile

Franc ¸ ois Laurens

Mondher Bouzayen

Vera Quecini
email: vera.quecini@embrapa.br

Genome-wide analysis of the AP2/ERF superfamily in apple and transcriptional evidence of ERF involvement in scab pathogenesis

Keywords:

The APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) superfamily of transcriptional regulators is involved in several growth, development and stress responses processes in higher plants. Currently, the available information on the biological roles of AP2/ERF genes is derived from Arabidopsis thaliana. In the present work, we have investigated genomic and transcriptional aspects of AP2/ERF genes in the economically important perennial species, Malus × domestica. We have identified 259 sequences containing at least one ERF domain in apple genome. The vast majority of the putative proteins display predicted nuclear localization, compatible with a biological role in transcription regulation. The AP2 and ERF families are greatly expanded in apple. Whole-genome analyses in other plant species have identified a single genomic sequence with divergent ERF, whereas in apple seven soloists are present. In the apple genome, the most noteworthy expansion occurred in sub-groups V, VIII and IX of the ERF family. Expression profiling analyses have revealed the association of ripening-involved ERF genes to scab (Venturia inequalis) pathogenesis in the susceptible Gala cultivar, indicating that gene expansion processes were accompanied by functional divergence. The presented analyses of AP2/ERF genes in apple provide evidences of shared ethylene-mediated signaling pathways in ripening and disease responses.

Introduction

Hormones are responsible for the control of plant growth and development and are also involved in mediating responses to several biotic and abiotic stresses. Besides its important role in regulating distinct physiological processes; such as seed germination, cell elongation, wounding, ripening, senescence and abscission, ethylene also regulates the plant responses to biotic and abiotic stresses [START_REF] Chen | Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses[END_REF]. Ethylene exerts its action through complex regulatory steps of its biosynthesis, perception and signal transduction, leading to dramatic changes in gene expression [START_REF] Chen | Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses[END_REF][START_REF] Kendrick | Ethylene signaling: new levels of complexity and regulation[END_REF]. The promoter sequences of several genes induced by ethylene were found to contain a cis-regulatory element known as the ethylene-responsive element (ERE) [START_REF] Broglie | Functional analysis of DNA sequences responsible for ethylene regulation of a bean chitinase gene in transgenic tobacco[END_REF]. Sequence analysis of various ERE regions identified a short motif, rich in G/C nucleotides, labeled the GCCbox, essential for ethylene-mediated responses. The ERE motif is recognized by a family of transcription factors: the ERE binding factors (ERF) [START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF].

The ERF is a large gene family of transcription factors that constitute a sub-group of the APETALA2 (AP2)/ERF superfamily, which also contains the AP2 and RAV families [START_REF] Riechmann | Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[END_REF]. The superfamily is defined by the presence of the AP2/ERF domain, a conserved DNA-binding sequence consisting of approximately 60-70 amino acids [START_REF] Weigel | The APETALA2 domain is related to a novel type of DNA binding domain[END_REF]. The AP2 family consists in proteins with two AP2/ERF domains, whereas the ERF family is constituted of proteins containing a single AP2/ERF domain, and the RAV family consists of proteins characterized by the presence of a B3 domain, a DNA-binding domain conserved in other plant-specific transcription factors, in addition to a single AP2/ERF domain. Previous studies have proposed a sub-division of the ERF family into two major sub-families; the ERF subfamily and the CBF/DREB subfamily [START_REF] Sakuma | DNA binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydrationand cold-inducible gene expression[END_REF]. More recent data suggest the existence of ten groups containing distinct conserved motifs within the family rather than distinct sub-families [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF].

The role of the ERF proteins in stress-related, important agronomical responses [START_REF] Xu | Functions and application of the AP2/ERF transcription factor family in crop improvement[END_REF] and the accumulation of genomic and expressed sequence data have prompted in-depth bioinformatic investigation of the ERF sub-family in several plant species, including Arabidopsis, rice, Populus, soybean and grapevine [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF][START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF][START_REF] Zhang | Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.)[END_REF][START_REF] Zhuang | Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa[END_REF]. In fruit species, ERFs are associated to flavor biosynthesis and texture modification during ripening [START_REF] Alba | Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development[END_REF][START_REF] Da Silva | Characterizing the grape transcriptome. Analysis of expressed sequence tags from multiple Vitis species and development of a compendium of gene expression during berry development[END_REF][START_REF] Janssen | Global gene expression analysis of apple fruit development from the floral bud to ripe fruit[END_REF][START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF][START_REF] Ziliotto | Transcriptome profiling of ripening nectarine (Prunus persica L. Batsch) fruit treated with 1-MCP[END_REF]. In apple (Malus × domestica Borkh. cv. Golden Delicious), two ERF transcripts, MdERF1 and MdERF2, demonstrated to be predominantly and exclusively expressed in ripening fruits, respectively, were isolated from ripening fruit [START_REF] Wang | MdERFs, two ethyleneresponse factors involved in apple fruit ripening[END_REF]. Similarly, [START_REF] Newcomb | Analyses of expressed sequence tags from apple[END_REF] identified expressed sequences of ethylene response factors in the tag collection and the pipeline annotation of the draft apple genome has found 274 sequences corresponding to AP2/ERF factors [START_REF] Velasco | The genome of the domesticated apple (Malus × domestica Borkh)[END_REF].

Due to its importance in plant reproductive development and abiotic and biotic stress responses, AP2/ERF transcription factors represent interesting gene pools to be investigated for breeding and genetic engineering purposes [START_REF] Xu | Functions and application of the AP2/ERF transcription factor family in crop improvement[END_REF]. Thus, we have performed an in-depth investigation of AP2/ERF superfamily in apple, demonstrating the presence of 259 sequences containing at least a single AP2 domain. The AP2, ERF and Arabidopsis soloist families have undergone differential expansion in apple. Groups V, VIII and IX of the ERF family were preferentially expanded. In apple, ERF genes are expressed in vegetative and reproductive tissues and an ERF gene, previously associated to ripening, has been demonstrated to be induced in Venturia inequalis-infected tissue. Thus, suggesting a role for ethylene and ethylene response genes in fungal pathogenesis in apple.

Materials and methods

Database searches and alignments

Sequences of Arabidopsis thaliana AP2/ERF genes were retrieved from the TAIR website and the amino acid sequence of the members described by [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF] were used as baits to search the apple genome at the Genome Database for Rosaceae (GDR) using the Batch BLAST search [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. In order to retrieve all AP2-containing sequences, the consensus amino acid domain sequence was also employed to search the NCBI database for Malus. The recovered sequences were checked by reverse BLAST analyses against the Arabidopsis genome and sequences failing to retrieve the original bait were eliminated. The presence and the significance of AP2 domains in the recovered apple sequences were determined by SMART analyses [START_REF] Letunic | SMART 6: recent updates and new developments[END_REF].

Phylogenetic analysis

The functionality of Malus genes in comparison to their Arabidopsis counterparts was assessed by genetic distance and phylogenetic studies. Amino acid sequence alignments were performed using ClustalX [START_REF] Thompson | The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF]. When necessary, alignments were manually adjusted using Lasergene MegAlign (DNASTAR, Madison, WI, USA). Phylogenetic analyses were performed using distance and parsimony methods in the software PAUP* 4.0b10 (http://paup.csit.fsu.edu/), using the software default parameters. Resampling bootstrap trees containing 1000 random samples were constructed using PSIGNFIT software (http://www.bootstrap-software.org/). Modular functional domains were employed for genetic distance studies for genes previously characterized as having divergent regions and conserved blocks.

Motif analysis and in silico characterization

Conserved motifs were further investigated by multiple alignment analyses using ClustalX and the MEME version 4.7 suite [START_REF] Bailey | Fitting a mixture model by expectation maximization to discover motifs in biopolymers[END_REF]. The presence and sequence conservation of recognizable functional domains was studied employing protein analysis and gene function tools from databases (European Bioinformatics Institute-European Molecular Biology Laboratory -EMBL-EBI; Expert Protein Analysis System -ExPaSy from Swiss Institute of Bioinformatics -SIB; Gene Ontology database -GO; Protein Families database -Pfam).

Prediction of cellular and molecular parameters

The physical characteristics; isoelectric point (IP) and predicted molecular weight, of apple ERF sequences were calculated from the deduced amino acid using default parameter of the Lasergene MegAlign software (DNASTAR, Madison, WI, USA). The proteins folding state was predicted by the FoldIndex program (http://bioportal.weizmann.ac.il/fldbin/findex). The prediction of the sub-cellular compartment location of the apple ERF-like proteins was performed using the plant algorithm of the software WoLF PSORT (http://wolfpsort.org/) [START_REF] Horton | WoLF PSORT: protein localization predictor[END_REF].

Gene expression analysis

Qualitative gene expression profiling was performed by in silico analyses of the Malus EST databases using virtual northern blot analyses. The gene of interest was used in queries against reference sequence databases, generating an alignment of the input gene to its orthologs. The resulting alignment was used to find sequences in the entire mRNA input that are specific to the gene (probe). The resulting alignments were collectively used to query the EST database again using BLAST. This heuristic was critical to avoid false-positives, or ESTs from a paralog of the input gene rather than the gene itself. The identity numbers of the ESTs matching the probes were recovered and the databases were used to find the names of the libraries from which those ESTs were derived. The description of the libraries used is available at The Malus × domestica Gene Index, at the Computational Biology and Functional Genomics Laboratory (Dana Farber Cancer Institute and Harvard School of Public Health, http://compbio.dfci.harvard.edu/tgi/gi/mdgi/searching/xpress search.html) and in Supplementary Table IV. The frequency of reads of each EST contig in a given library was calculated and normalized according to the total number of reads from the investigated library and the total number of reads in all libraries. A correlation matrix between EST contigs and libraries was then generated and gene expression patterns among ESTs and libraries were obtained by hierarchical clustering based on Spearman Rank correlation matrix using Cluster v.2.11 software [START_REF] Eisen | Cluster analysis and display of genome-wide expression patterns[END_REF], by substituting the clusters by their average expression pattern. Graphic outputs were generated using Tree View v.1.6 software (http://rana.lbl.gov/EisenSoftware.htm) and presented in colorscale.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. scienta.2012.12.017.

Plant material and V. inequalis inoculation

Lateral shoots of Malus × domestica 'Royal Gala' were propagated in MS medium [START_REF] Murashige | A revised medium for rapid growth and bioassays with tobacco tissue cultures[END_REF] pH 5.8 supplemented with 8.8 M of 6-benzylaminopurine, 30 g L -1 sucrose and solidified with 6 g L -1 agar, under 16-h photoperiod and 22 ± 2 • C temperature. After four weeks of culture, plants were transferred to rooting medium, containing MS salts and vitamins at pH 5.8 and supplemented with 5 M of indole-3butyric acid, 15 g L -1 sucrose and solidified with 6 g L -1 agar to induce rooting. Rooted plants were gradually ex vitro acclimated and four weeks later, the plants were inoculated with the fungus.

V. inequalis (apple scab) inocula were prepared from monoconidial isolates cultivated in Potato-Dextrose-Agar (PDA) medium. The plates were incubated at 16 • C under continuous light for 15 days and sporulating colonies were scraped for inocula preparation.

Conidia were resuspended in saline solution and the concentration was adjusted to 2.5 × 10 5 conidia/mL before plant spraying. The plants were sprayed with approximately 50 mL of conidia suspension until the formation of droplets. Negative control plants were sprayed with sterile saline solution without conidia. After inoculation, the plants were kept for 30 days under 20 • C, 90-100% relative humidity and 12-h photoperiod and symptom development was visually evaluated. Leaf samples were harvested after 30 days, immediately frozen in liquid nitrogen and stored at -80 • C until processed.

RNA extraction and cDNA synthesis

Total RNA was extracted as described by [START_REF] Boss | Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv. Shiraz grape berries and the implications for pathway regulation[END_REF] and the samples were treated with 100 U of RNase-free DNase (NEB Biolabs, USA) and further purified with chlorophorm wash and ethanol precipitation. The purity and concentration of the isolated RNAs were checked on 1% (w/v) agarose gels and by spectrophotometry. Reverse transcription was carried out using 2 L of total RNA and M-MLV Reverse Transcriptase RT-PCR (Promega) and oligo d(T)25 primers, as recommended by the manufacturer. The concentration of the cDNA was determined spectrophotometrically.

Semi-quantitative PCR analyses

Polymerase chain reactions were carried out in a final volume of 25 L, containing 2.5 L of 10× PCR buffer, 2.5 L of MgCl 2 (25 mM), 4.0 L of dNTP mix (2.5 mM), 1 L of each primer (10 mM), 1 L cDNA and 2.0 L Taq DNA polymerase. The sequence of the primers used is as follows: 5 ′ ATGACCTGGTGGCATATCAG3 ′ and 5 ′ CACCGTAGCAAACAACACAC3 ′ for MDP0000128979, 5 ′ TATGCTGGCAATTGGCGAGC3 ′ and 5 ′ ATGACCAATCCCGCAC-TCAC3 ′ for MDP0000226115 and 5 ′ GGCTGGATTTGCTGGTGATG3 ′ and 5 ′ TGCTCACTATGCCGTGCTCA3 ′ for a normalizer gene MdACT [START_REF] Wang | MdERFs, two ethyleneresponse factors involved in apple fruit ripening[END_REF]. The reaction linear range was determined by serial tests of cycle lengths and was determined to be linear at 27 amplification cycles. Digital analyses of band intensity on agarose gels were performed to quantify gene expression.

Results

The family of ethylene response factors in apple

Sequences containing AP2 domains were extensively searched in the apple genome and 259 were confirmed by reverse BLAST and SMART domain analyses to be members of the AP2/ERF superfamily (Table 1, Table SI). The number of identified sequences is smaller than that found by automated annotation of the apple genome [START_REF] Velasco | The genome of the domesticated apple (Malus × domestica Borkh)[END_REF] (274) due to the elimination of short sequences and of those with introns longer than 10 kb, which are likely resultant from assembly mistakes. The AP2 family in apple consists of 51 genes, from which 36 contain the characteristic double AP2 domain. The RAV family of proteins, containing the B3 domain in addition to the family characteristic AP2, consists of six members in apple as observed in other investigated plant species and its member number appears to be highly conserved throughout evolution. One hundred and ninety-five genes encoding proteins with a single AP2/ERF domain were assigned to the ERF family. In Arabidopsis and other higher plant species, a single locus (At4g13040) codes for a protein exhibiting divergent AP2 domain was identified, thus it was labeled the soloist [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][START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF]. In apple genome the group consists of seven closely related proteins containing the divergent AP2 domain, similar to that of At4g13040 (Figs. 1 and2). Apple ERF proteins were classified into 11 groups according to their similarity to Arabidopsis ERF sequences and conserved motifs outside the DNA-binding SII). Although the vast majority of the Arabidopsis motifs were present in apple sequences, specific conserved regions were identified in the proteins identified in Malus × domestica (Figs. S1-S10). The apple ERF family (195 members) is expanded in comparison to the Arabidopsis (147), poplar (202) and rice (180) families. The expansion of the apple family is significant for ERF proteins clustered in group V, VIII and IX (Table 1, Figs. S1-S10). Interestingly, groups V and IX are also expanded in grapevine and are transcriptionally associated to berry ripening processes [START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF]. In poplar, ERF groups V and IX are also expanded although no functional significance has been attributed to those groups [START_REF] Zhuang | Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa[END_REF]. The Xb-like group of ERF proteins is absent from the grapevine genome, however, in apple it consists of two proteins, similar to the numbers found in Arabidopsis (3) and Populus (4) [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][START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF].

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta. 2012.12.017.

Accuracy of protein AP2/ERF predictions

Genome databases employ ab initio protein prediction methods, which are prone to errors. In order to confirm the existence of the identified AP2/ERF proteins, we have checked intron junctions in comparison to the homologs in Arabidopsis and poplar. Subsequently, the predicted mRNAs of all identified proteins were used to query apple EST databases. We found no unrealistic introns and EST sequences matching (e-value lower than 1•e -25 ) all predicted mRNA were found in the databases, except for gene model MDP0000876858.

Cellular and molecular characteristics of apple AP2/ERF proteins

We have employed bioinformatic tools to determine the physical properties of the ERF-like sequences from apple, such as the molecular weight (MW), pH value of isoelectric point (pI) and folding index. The vast majority of the apple ERF-like sequences were predicted to have low molecular weight, ranging from 1.5 to 4.48 kDa and an average of 233 amino acids, going from 111 to 413 amino acids (Table SIII). In apple, most of the sequences sharing sequence similarity to Arabidopsis ERF proteins (74.2%, 49) were predicted to be basic to neutral, whereas only 25.8% (17) displayed predominance of acidic amino acids (Table SIII).

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta. 2012.12.017.

The folding states of ERF family proteins in apple were predicted by FoldIndex program [START_REF] Prilusky | FoldIndex©: a simple tool to predict whether a given protein sequence is intrinsically unfolded[END_REF]. The majority of the identified apple sequences sharing sequence similarity to ERFs were predicted to be unfolded (59.1%, 39) under physiological conditions (Table SIII). The predicted sub-cellular localization of the ERF proteins identified in Malus was investigated using the software WoLF pSORT [START_REF] Horton | WoLF PSORT: protein localization predictor[END_REF]. Most of the ERF-like sequences from apple (83.3%, 55) was predicted to be exclusively nuclear or with ambiguous sub-cellular location for the nucleus and organelle or cytoplasm (Table SIII) (Fig. 3).

Expression profiling of ERF genes

The expression profile of 195 ERF genes was preliminarily investigated by in silico analyses for reproductive and vegetative libraries. Moreover, the relative expression of the 10 groups constituting the sub-family was also investigated in scab infected tissues. The expression patterns of the genes encoding for members of the ERF family, presented as groups, are shown in Figs. 4 and5. Transcripts corresponding to the vast majority of the identified genes were expressed in apple leaves, flowers and fruits, except for gene models MDP0000451365 (group III), group V sequences MDP000031419, MDP0000525933, MDP0000276536, MDP0000551969, MDP0000246184, MDP0000-127054, MDP0000297646 and MDP0000243375, MDP0000467753 and MDP0000778140 (group VIII) and group IX sequences MDP0000447570, MDP0000133854, MDP0000829925, MDP0000-155543, MDP0000789227, MDP0000880063, MDP0000187369 that were expressed in buds, roots and vascular vessel tissues. The expression profile of most ERF genes is developmentally and/or environmentally controlled in apple (Figs. 4 and5). Within ERF groups, the expression patterns were also divergent, especially for groups I, VI L, VII, VIII and IX (Figs. 4 and5). Transcripts corresponding to the sequences MDP0000127123 and MDP0000316843, from group Xb, were exclusively present in fruits (Figs. 4 and5). Most ERF genes from groups VII and IX expressed in the fruits were also highly expressed in V. inequalis infected tissues (Figs. 4 and5). Thus, we have further investigated the transcriptional profile of ripening-associated ERFs in scab pathogenesis using monosporic V. inequalis cultures inoculated to in vitro propagated plants and semi-quantitative PCR.

Thirty days after inoculation, plant leaves exhibited scab symptoms in the leaves and stem and a three-fold induction of MDP0000128979 (group VII) expression (Fig. 6). Phylogenetic analyses demonstrated that MDP0000128979 correspond to fruitripening associated MdERF1 [START_REF] Wang | MdERFs, two ethyleneresponse factors involved in apple fruit ripening[END_REF]. In silico analyses suggested the association of the induced transcription of group IX genes to scab response and fruit ripening (Fig. 5), however transcription of MDP0000226115 was only slightly altered in scab infected tissues (Fig. 6).

Discussion

Identification of the AP2/ERF family transcription factors in apple

A comprehensive search for AP2/ERF transcription factors in Malus × domestica genome has revealed the presence of 259 AP2-motif containing sequences. The number is smaller than the 274 AP2/ERF found by the automated annotation of the apple genome [START_REF] Velasco | The genome of the domesticated apple (Malus × domestica Borkh)[END_REF] due to the elimination of short sequences and of those containing unrealistic introns. The apple superfamily is larger than the rice and Populus ones, with 180 and 202 genes, respectively (Table 1). The larger size of the superfamily in apple is mainly due to expansion of the AP2, ERF and At4g13040-like families, since the number of genes in the RAV family is conserved throughout evolution (Table 1). In apple genome, recent gene duplication appears to be involved in the expansion of the At4g13040-like genes, since it is represented by a single sequence with divergent AP2 domain in all investigated plant species, whereas in apple, it is represented by seven sequences. The sub-set of AP2 proteins containing a doubled AP2 domain in apple is more than two times larger than it is in Arabidopsis and has approximately 40% more members than the one in Populus trichocarpa. Interestingly, the expansion pattern of the families is conserved in the woody perennial species investigated; poplar, grapevine and apple [START_REF] Zhuang | Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa[END_REF][START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF].

The ERF family in apple consists of 195 sequences phylogenically classified into the ten groups (I-X) identified in Arabidopsis by [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF]. The distribution of apple ERF sequences in groups is similar to that of other investigated species. As observed in woody perennial Vitis vinifera and Populus trichocarpa, groups V and IX are expanded in apple genome, although both also display a lower number of genes in group I, which does not occur in apple. A recent study in Vitis vinifera has proposed that the lower number of ERF genes in group I in woody perennial is likely to be due to its functional redundancy in controlling wax accumulation [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] to the genes found in expanded group V [START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF]. The differential expression and the expansion of both groups I and V suggest that in apple it does not occur. In contrast, the low levels of expression of group IV genes in apple may indicate functional redundancy to other ERF gene group. The group Xb in apple is exclusively expressed in fruits and has a number of genes similar to that found in Arabidopsis and poplar. The group is absent from grapevine. The largest ERF group in apple is group IX, as observed in poplar and grape genomes, it is almost twice the size of the group in Arabidopsis and rice (Table 1). As observed in grapevine [START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF], apple ERF-IX sequences exhibit species-specific protein domains (Supplementary Table SIII) and distinct expression patterns. Interestingly, members of group IX displayed a particular clustered organization along chromosomes 7 and 16 (Fig. 2), similar to the one observed in the poplar and grapevine genome [START_REF] Zhuang | Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa[END_REF][START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF], although in this species the tandem repetition does not exceed four genes. The coding sequences of proteins involved in signal transduction and transcriptional regulation are frequently duplicated in higher plant genomes, due to their role in plant fitness responses [START_REF] Paterson | Insights from the comparison of plant genome sequences[END_REF].

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta. 2012.12.017.

Transcription factors often exhibit regions outside the DNAbinding domain that are involved in regulation of the transcriptional activity, protein-protein interactions, and nuclear localization [START_REF] Liu | Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in droughtand low-temperature-responsive gene expression, respectively, in Arabidopsis[END_REF]. These motifs, frequently associated to specific functions or regulation patterns, are characteristic of large transcription factor families in plants, such as MYB, WRKY, NAC, Dof, GATA, and GRAS [START_REF] Eulgem | The WRKY superfamily of plant transcription factors[END_REF][START_REF] Kranz | Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana[END_REF][START_REF] Lijavetzky | Genome-wide comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families[END_REF][START_REF] Ooka | Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana[END_REF][START_REF] Reyes | The GATA family of transcription factors in Arabidopsis and rice[END_REF][START_REF] Tian | Genome-wide analysis of the GRAS gene family in rice and Arabidopsis[END_REF]. The vast majority of the ERF-like sequences identified in apple shared one or more motifs outside the AP2/ERF domain with their Arabidopsis counterparts, as observed in rice, Populus, soybean and grapevine [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF][START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF][START_REF] Zhang | Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.)[END_REF][START_REF] Zhuang | Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa[END_REF]. The functionally characterized gene MdERF1 (A1LM1), associated to fruit ripening in apple [START_REF] Wang | MdERFs, two ethyleneresponse factors involved in apple fruit ripening[END_REF] clustered in the most abundant group VII, whereas MdERF2 (A1LM2), which is exclusively expressed in ripening fruits was clustered in group IX.

Cellular and molecular characteristics of apple ERF-like proteins

Proteins biochemical and physical properties are responsible for several characteristics associated to their biological function, such as molecular structure, ligand binding and subcellular location. Similar to Arabidopsis, rice, Populus, soybean and grapevine [START_REF] Nakano | Genome-wide analysis of the ERF gene family in Arabidopsis and rice[END_REF][START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF][START_REF] Zhang | Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.)[END_REF][START_REF] Zhuang | Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa[END_REF], apple ERF-like proteins are predicted to have low molecular weight. In apple, most of the ERF-like sequences were predicted to be basic to neutral, as observed for Arabidopsis and Populus, although the proteins from the annual and perennial model plants exhibit slightly lower pI values. These observations are consistent with the presence of a basic region mediating sequence-specific DNA-binding in families of transcriptional regulators from evolutionarily distant organisms [START_REF] Miller | Structural basis for DNA recognition by the basic region leucine zipper transcription factor CCAAT/enhancer-binding protein alpha[END_REF][START_REF] Panne | Crystal structure of ATF-2/c-Jun and IRF-3 bound to the interferon-beta enhancer[END_REF]. Thus, the predicted chemical nature of the proteins identified in apple is compatible with a functional role in transcriptional control.

The predicted sub-cellular localization of the ERF proteins identified in Malus was investigated using the software WoLF pSORT [START_REF] Horton | WoLF PSORT: protein localization predictor[END_REF]. The sub-cellular location is an important clue for the biological function of identified sequences, especially for those hypothesized to be involved in gene expression regulation. The majority of the ERF-like sequences from apple were predicted to be exclusively nuclear or exhibiting ambiguous sub-cellular location for the nucleus and organelle or cytoplasm. At this point, the incomplete nature of some of the sequences and the limitations of bioinformatic analyses prevent us from establishing how accurate the predictions are in vivo. Further functional analyses will be necessary to determine the sub-cellular location of the apple ERF-like sequences.

Expression analyses of ERF genes

The expression profiling of ERF genes in apple along with the knowledge on their molecular function in model plant species is an important tool to uncover the biological role of these transcription factors in developmental and stress-response processes. Traditionally, the expression of ERF genes has been associated with the molecular response to ethylene [START_REF] Dietz | AP2/EREBP transcription factors are part of gene regulatory networks and integrate metabolic, hormonal and environmental signals in stress acclimation and retrograde signalling[END_REF]. However, genome-wide transcriptional profiling of AP2/ERF genes in several plant species has demonstrated that they are regulated by a number of physical-chemical stimuli. A sub-set of ERF genes exhibit a classical ethylene responsive pattern and have been shown to be involved in the ripening process in climacteric fruits. Although several of the investigated ERF genes were highly expressed in fruits, only those from group Xb were exclusively found in fruits. In Arabidopsis, Nicotiana tabacum and Solanum lycopersicum transcripts of group II and III ERFs have been associated to abiotic stress responses [START_REF] Gilmour | Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced gene expression[END_REF][START_REF] Tsutsui | DEAR1, a transcriptional repressor of DREB protein that mediates plant defense and freezing stress responses in Arabidopsis[END_REF], whereas in Vitis vinifera, their expression is constitutively high, suggesting a role in a default defense system [START_REF] Licausi | Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[END_REF]. In apple, genes from these groups exhibit an expression pattern more closely related to stress responses, as observed in Arabidopsis, tobacco and tomato. The genes from ERF group IX, the larger group in apple, exhibit distinct regulation patterns, with a sub-set being highly induced in fruits and in scab infected tissues, thus indicating that the ripening and the V. inequalis-induced stress pathway share common transcriptional regulators but whether the targets of these transcription factors is also shared or independent remains to be elucidated. In a previous study to characterize ripening-associated genes in apple, the expression of MDP0000128979 (MdERF1) and MDP0000226115 (MdERF2), analyzed by northern blot and RT-PCR, was demonstrated to occur exclusively in fruit tissues [START_REF] Wang | MdERFs, two ethyleneresponse factors involved in apple fruit ripening[END_REF].

Our results indicate that ERF genes are also expressed in vegetative tissue and that MDP0000128979 (MdERF1) transcription is induced in plants exhibiting scab symptoms, confirming the transcriptional association between the ripening factor and V. inequalis pathogenesis in apple. These results are in accordance to other reports demonstrating a role for ethylene in biotic stress responses in apple [START_REF] Akagi | Resistance of Malus domestica fruits to Botrytis cinerea depends on endogenous ethylene biosynthesis[END_REF] and other plant species [START_REF] Gutterson | Regulation of disease resistance pathways by AP2/ERF transcription factors[END_REF][START_REF] Broekaert | The role of ethylene in host-pathogen interactions[END_REF]. In general, analyses of ERF expression patterns suggest some extent of functional specialization in apple.

Fig. 1 .

 1 Fig. 1. Phylogenetic analyses of AP2/ERF superfamily in Malus × domestica. Neighbor-joining tree for full length sequences were aligned with ClustalX. Sub-families AP2, RAV and the Arabidopsis-like soloist sequences in apple genome are represented by gray shading. Roman numerals represent ERF family sub-groups.

Fig. 2 .

 2 Fig. 2. Chromosome location of ERF genes in apple genome. M. × domestica chromosomes are represented according to the evolution model proposed by Velasco et al. (2010).

Fig. 3 .

 3 Fig. 3. Phylogenetic relationships among the Arabidopsis ERF genes, from group IV (D) in Arabidopsis and Malus ERF families. Bootstrap values from 1000 replicates are shown above the branches. The phylogenetic tree and a schematic diagram of the protein structures of groups I-X are shown as Supplementary Figs. S1-S10. Each colored box represents the AP2/ERF domain and conserved motifs, as indicated in legends below the trees. The amino acid sequences of the conserved motifs are summarized in Supplementary Table IV. Classification by Nakano et al. (2006) is shown above each tree and by Sakuma et al. (2002), indicated in parentheses. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 4 .

 4 Fig. 4. Expression profile of ERF genes from groups IV, VII and V in leaf, flower, fruit and scab-infected tissues. Apple gene models are listed as available at Genome Database for Rosaceae (GDR). Hierarchical clustering is based on Spearman Rank correlation. Graphic outputs are represented as color scale. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 5 .

 5 Fig. 5. Expression profile of ERF genes from group IX in leaf, flower, fruit and scab-infected tissues. Apple gene models are listed as available at Genome Database for Rosaceae (GDR). Hierarchical clustering is based on Spearman Rank correlation. Graphic outputs are represented as color scale. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 6 .

 6 Fig. 6. Expression profile of apple ERF sequences from groups VII and IX in Venturia inequalis-infected vegetative tissue. (A) Mock-inoculated control; (B) plant sprayed with scab conidia; (C) scab symptoms on leaves and petioles 30 days after inoculation and (D) relative expression of MDP0000128979 (group VII) and MDP0000226115 (group IX) in fungus-infected and control plants. The bars represent percentage standard error.

Table 1

 1 Summary of the AP2/ERF superfamily of Malus × domestica and model species Arabidopsis thaliana, Vitis vinifera, Populus trichocarpa and Oryza sativa. The total data for AP2, At4g13040, RAV and ERF families are presented in bold letters.

	Classification	Group	Species
			Arabidopsis thaliana	Malus × domestica	Vitis vinifera	Populus trichocarpa	Oryza sativa
	AP2 family		18	51	20	26	29
		Single AP2/ERF	4	15	0	0	5
		Double AP2/ERF	14	36	20	26	24
	At4g13040	Divergent ERF	1	7	1	1	1
	RAV family	AP2/ERF and B3	6	6	6	6	5
	ERF family		122	195	122	169	145
		I	10	10	5	5	9
		II	15	13	8	20	16
		III	23	22	22	35	27
		IV	9	23	5	6	6
		V	5	19	11	10	8
		VI	8	6	5	11	6
		VI -L	4	4	2	4	3
		VII	5	8	3	6	15
		VIII	15	31	11	17	15
		IX	17	45	40	42	18
		X	8	12	10	9	12
		Xb -L	3	2	0	4	10
		Total	147	259	149	202	180

Acknowledgments

The authors would like to acknowledge the excellent technical assistance of Daniela Dal Bosco and Iraci Sinski in plant propagation and acclimation. We also thank Renata Gava for fungus cultivation and plant inoculation. This research is funded by an Embrapa grant number 02.07.05.001.00 Macroprograma 2 -Agrofuturo to C.L.G. VQ is recipient of a productivity research grant from CNPq (307031/2010-1).


Keywords: fatty acid, polyol, polyurethane, thiol-yne coupling

Oleic acid and diacid were converted into propargylic esters followed by thiol-ene/yne coupling (TEC/TYC) functionalizationin presence of mercaptoethanol. The multi-radical addition on fatty esters leads to the formation of lipidic polyols (OH1 and OH2), as judged by 1 H NMR and mass spectroscopiesas well as by steric exclusion chromatography (SEC).

The crosslinking reaction between TEC/TYC-based polyols and MDI isocyanate reactant was monitored by FTIR experiment and reaction parameters were optimized. By differential scanning calorimetry (DSC), relatively high glass transitions are measured corresponding to structure with little or without dangling chain. Moreover, the thermal stability of the resulting plant oil-based polyurethane materials (PU1 and PU2)were found to be fully consistent with that of other lipidic polyurethanes respectinga three-step process.Thanks to TYC methodology, fatty diacid produces lipidic polyol without dangling chain and lipidic thermoset PU with relatively high Tg.

Phuoc-Dien.Pham@univ-montp2.fr, Vincent.Lapinte@univ-montp2.fr, y.raoul@novance.com, Jean-Jacques.Robin@univ-montp2.fr RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) TITLE RUNNING HEAD.Lipidic polyols for polyurethane materials.

CORRESPONDING AUTHOR FOOTNOTE.Institut Charles Gerhardt Montpellier

UMR5253 CNRS-UM2-ENSCM-UM1, EquipeIngénierieet Architectures Macromoléculaires, Université Montpellier II cc1702, Place EugèneBataillon. 34095 Montpellier Cedex 5, France.

Tel: 33-4-67-14-48-32; fax: 33-4-67-14-40-28. Vincent.Lapinte@univ-montp2.fr

Introduction

With growing concerns over sustainable development and environmental impact of petrochemical polymer products, researchers are paying more attention to bio-based materials.The main bio-based materials are vegetable oils, polysaccharides, sugars and woods 1 that are chemically modified for the development of chemicals, plasticizers, 2 polymers, 3 blends, 4 composites, and coatings. 5 In particular, vegetable oils and their derivatives (fatty alcohols, esters and acids) have been extensively modified to generate bio-based materials 6 such as polyamides, 7 non-isocyanate polyurethanes, 8,9 surfactants, 10 and epoxy resins. [START_REF] Gandini | Epoxy Polymers[END_REF][START_REF] Stemmelen | [END_REF][13] Polyurethanes (PU)sare very interesting polymeric materials allowing a huge variety of industrial applications from paints, coatings, adhesives, sealants and insulation foams. They are synthesized from polyol and poly(isocyanate) reactants producingcarbamate (urethane) bonds. The conversion of unsaturated oils into polyols has been widely investigated to prepare polyurethaneswithdifferentphysical and chemicalproperties.There are severalapproaches to introduce the hydroxyl group into the vegetableoils structure.To date, the synthesis of lipidic polyolshas been mainly accomplished from epoxy vegetable oils or fatty estersintermediate (Table 1). The ring-opening of oxirane by various nucleophiles like alcohol (methanol, 14 propylene glycol 15 ) or carboxylic acid (lactic acid 16 ) was described. An alternative approach consists intheoligomerization of fatty ester by ring-opening of epoxy groups in presence of catalyst based on aluminum. 17 Ozonolysis of oils and further reduction of terminal acid groups also lead to lipidic polyols. 18 Cadiz et al. explored another strategy by hydrosilylation of methyl 10-undecenoate with phenyl tris(dimethylsiloxy)silane (PTDS). 19 Additionally, polyols were prepared by radical pathway: thiol-ene coupling (TEC) using mercaptoethanol in presence of raw oil 20 or fatty esters. 21,22 One drawback of plant oil-based PU is the low Tg value (<40°C) due to both the low functionality of polyols and the presence of dangling chains which act as plasticizers. Herein, we report a new synthetic approach for the facile synthesis of lipidic polyols viathiolene/yne coupling (TEC/TYC) using 2-mercaptoethanol (Scheme 1). 23 The TYC splits in two cycle mechanism. 24 In the first cycle, the thiyl radicals generated from thiol groups were addedon the ethynyl groups to form a vinyl sulfide radical, which could attract a hydrogen atom from a thiol group, producing the vinyl sulfide and generating another thiyl radical at the same time. In the second cycle, a thiyl radical adds on the double bond of the vinyl sulfide, generating a dithioether radical, which attracts a hydrogen atom from a thiol groups and forms the disubstituted product.This synthetic strategy leadsto high-functional polyols since oneethynyl group turns into two thioether groups. By this methodology, propargylic fatty acid (PFE) and diacid (PFD) were converted into lipidic polyols (OH1) and (OH2), respectively.Cadiz et al. havealreadysynthesized lipidic polyols by thiol-yne coupling after bromination, dehydrobromination of 10-undecenoic and 9-octadecenoic acids. 25,26 However the ethynyl group is in internal position, along the fatty chain whereas in our case itis in terminal position via propargylic precursor. and cooling schedules used were as follows: the samples were heated at 10 °C.min -1 and cooled at the same rate to room temperature. LC/ESI-MS chromatograms were acquired in the positive ion mode with the capillary voltage set at 3000 V and cone voltage at 30 V while the dry temperature was set at 350 °C and dry gas flow was maintained at 13 mL.min -1 . The mass acquisition range was from m/z 50 to 1000.The thermo-oxidative stability of the polyurethane materials was examined using a Q50 thermogravimetricanalyzer (TGA) from TA Instruments ® . The experiments consisted in registering the weight loss of the sample under nitrogen flow (60 mL.min -1 ) as a function of temperature from the ambient up to 550 °C.

Different experiments were heated at 10 °C.min -1 .

Conversion of oleic diacid into fatty diacid chloride(DCl)

In a 500 mL two-necked flask equipped with a refluxcondenser and a dropping funnel, 53.5 g (171.2mmol) of oleicdiacidwere dissolved in 150 mL of dry chloroform. The chloroform solution (200 mL) containingthionyl chloride was added dropwiseover a period of 1 h. The mixture was heated gently on anoil bath held at 60°C for 16 hours. The mixture was evaporated under pressure in order to remove any excess of thionyl chloride. The product wasused without any further purification in 97% yield.

Synthesis of propargylic esters

Propargylic alcohol (7.71 g, 137.4mmol) and triethylamine (13.9 g, 137.4mmol) were dissolved in dry dichloromethane (100 mL). The solution was cooled in an ice bath under N 2 for 15 min. At 0 °C, dichloromethane solution (100 mL) of diacid chloride (20 g, 57.2mmol) was added dropwise to the previous solution over 1 h. The stirring was continued for one additional hour. Then, the mixture refluxed for 16 h. The solution was washed with water (3x 200 mL) and brine (2x100 mL), dried over anhydrous MgSO 4 , filtered, and evaporated under reduced pressure to yield a brown oil (19.2 g, 86 %).

Propargylic fatty ester (PFE)

Results and discussion

The various approaches described in the literature to synthesize lipidic polyols are summarized in Table 1.Herein, we focus the discussion on the lipidic polyols used in thermoset polyurethanes in presence of MDI isocyanate. Three lipidic structuresare mentioned: i) the triacylglycerols (TAG) (2, 3, 5 and 7), ii) the linear fatty acid derivatives (FA) (1, 8 and 9), and iii) other structures like three-arm stars (6) and oligomers (4). It is interesting to note that the presence of dangling chain was observed in almost all structures, except for 5 and 6. Otherwise, some structures carry primary alcohol groups (1, 5, 6, 7, 8 and 9) while others carry secondary ones (2, 3 and 4). This comparative study also highlights the presence of ester groupin polyol structures apart from4,6,8 and 9. Herein, we proposed two types of polyol based on oleic acid and diacid. From the first one, a triol (OH1) bearing an ester group and a dangling chain was elaborated whereas a pentaol (OH2) bearing a diester structure without dangling chain was synthesized using fatty diacid.

Synthesis of propargylic fatty ester and diester

The synthesis of propargylic fatty ester (PFE) and diester (PFD)starts from activated species of oleic (di)acids. The oleic chloride (OACl) is marketed whereas the corresponding diacid

(

Scheme 2 Photo-inducedthiol-ene/yne reaction for an efficient synthesis of polyols

To access the highly functional fatty polyols, thiol-ene and thiol-yne couplings could be both associated. In our case, the key step is the functionalization of propargylic fatty (di)esters (PFE and PFD) into fatty polyols (OH1 and OH2) by photo-inducedthiol-ene/yne coupling (Scheme 1). Mercaptoethanol was both added on internal double bond by thiolenecouplingand terminal propargylic unsaturation by thiol-yne coupling. The photo-induced radical addition of thiol into unsaturation carried out at room temperature in presence of DMPA photoinitiator. Thanks to a simple work-up, the highly functional moieties OH1 (triol) and OH2 (pentaol) were successfully synthesized in good yields (66-68%). The 1 H NMR spectra confirmed the chemical structure of both polyols as illustrated in Figures 1 and2 17% of oligomers during the epoxy ring-opening process by lactic acid followed by esterification in the case of 2,described in Table 1. 15 Additionally, Pétrovicet al.estimated 18-25% of oligomers for 3duringoxirane ring-opening reaction of epoxidizedvegetable oil in presence of methanol. 14 In our case, the formation of oligomers duringthiol-unsaturation coupling was detected by GPC using DMAc and polystyrene standards (Figure 3). We noted that the difference between propargylic ester and diester structure is good enough to be detected by this technique and obviously the modification of PFE and PFD by thiol-ene/yne coupling. First, the synthesis of OH1 polyol happened withoutoligomer formationsince no trace was observed at higher retention time and a singleGPC tracewas detected with a polydispersity index of 1.03.For OH2 polyol, very little oligomers likely dimers appeared as evidenced by the polydispersity index of 1.12. In addition, the shift of the polyoltraces related than those of PFD and PFEprecursors confirmed the"clicking" of three and five mercaptoethanol units into fatty structures. GPC analysis of OH1 and OH2 show an increase in molecular weight from 400 and 500 g.mol -1 to 1400 and 2400 g.mol -1 . The large difference between targeted and experimental molecular weights could be explained by the use of inappropriate polystyrene standards.

Figure 3

The structure of OH2polyol, alcohol highest functionality of both synthesized,was also

Preparation of PU materials based on lipidic polyols

The polyol structure obviously impacts the density of polyurethane network. Therefore, the Tg values and the thermal properties of the thermosets are influenced by the lipidic polyol structures described in Table 1. It is widespread that the dangling chains of the fatty polyols affect the physical properties of the resulting materials and act as plasticizers decreasing the their Tg values. 18 Apart from 5 and 6, the polyols described in the literature carry one (1, 8 and 9), three (2, 3and 8) or numerous (4) dangling chains as illustrated in Scheme 3.The design of our vegetable oil based polyols considers this aspect. Thus, thanks to fatty acid and diacid precursorscoming from oleic chain,OH1 and OH2 polyols were provided with one or without dangling chain using a simple and versatile thiol-ene/yne coupling approach.

Scheme 3

The comparative study gathers the thermoset polyurethanes coming from MDI and various polyols as compiled in Table 2. The relationship between the polyol structure and the Tg value of the resulting material was discussed. The Tg valueswere affected by the number of alcohol groups ofeach fatty chain. This value is directly related to the number of double bonds ofprecursors fatty chains. Note that the fatty chains are classified according to the number of carbon atoms (n) and of double bonds (m)such as Cn:m.For 1, 3 and 5 polyols, several types of vegetable oils are employed carrying various contents in oleic (C 18:1), linoleic (C 18:2) and linolenic (C 18:3) chains. 14,15,18 For instance, 3-based PU using canola oil (iodine index of 114) or linseed oil (iodine index of 182) have got a Tg value of 32 and 77 °C, respectively. 14 In short, as expected, the high level of unsaturations (iodine index closed to 220) favors the high Tg value in relation to the crosslinking density. The Tg values also depend on the strategy of incorporation of alcohol groups on fatty chains (TEC, ring-opening (RO) of oxirane). For instance, even if in all cases, fatty precursors are vegetable oils (TAG structure), PU based on 2(RO),3(RO)and 7(TEC) offered a large panel of Tg values ranging from 25 °C for 7 until 96 °C for 2. 14,16,20 Thus, the nature of the functional alcohol unit (lactic acid for 2andmethanol for 3) added on epoxidized vegetable oil influenced the Tg value.Interestingly, the three-arm star structure (5 and 6) having a terminal alcohol group exhibited low Tg values ranging from 22 and 39 °C. 18,19 This result can be explained by the flexibility of the alkyl chainbearing alcohol group and the long distance between two reactive alcohol functions producing a network with wide mesh.

As mentioned above, the presence of dangling chain in polyol structure decreasedthe Tg value by plasticization the Tg value. Besides the oligomer nature of 7, low Tg values were measured (-3 and 57 °C) likely caused by the dangling chain. 27 By TEC functionalization, the same problem occurred with modified TAG (7) and modified fatty acid (8). 20,21 The Tg values varied between 25 and 56 °C. Surprisingly, the structure 1 exhibited the highest Tg value (114 °C) with 3 alcohol groups (2 secondary and 1 primary) with one danglingchain. 15 In order to enhance the alcohol density in polyol structure without dangling chain, Cadiz et al. reported the thiol-yne functionalization of 10-undecynoic and 9-octadecynoic acids after bromation of double bond, deshydrobromation into alkenic bond followed byesterification of acid group. 25 The resulting PU thermosets have a Tg value up to 59 °C. In our methodology, even if we kept TYC approach, a shorter strategy was used to incorporatean alkenyl group in fatty acid (PFE) and diacid (PFD) structures. Unlike Cadiz, thiol-ynecoupling led the introduction of terminal alkenyl group by esterification instead of bromation/deshydrobromation of internal double bond. in PU1 means that the network was complete.Furthermore, the stretching vibration of C=O urethane appeared at 1708 cm -1 and overlapped with the C=O ester band at 1736 cm -1 . The N-H deformation as well as C-N stretching vibration occurred at 1520 and 1215 cm -1 , respectively. In addition, the broad band at 3500 cm -1 corresponding to O-H stretching, shifted to lower frequencies at 3350 cm -1 , characteristic of N-H stretching.

Figure 5

The thermal behaviors of PU1 and PU2 coming from OH1 and OH2 were investigated by DSC technique as shown in Figure 6. First, the glass transition temperatures of lipidic polyols (beforecrosslinking) were estimated.The Tg of OH1 and OH2 polyols were found to be -70 and -58 °C, respectively (Figure 6, left). The larger number of alcohol groups reduced the mobility of fatty chain and increased the Tg value. Secondly, the same experiments were conducted on resulting thermoset PUs (Figure 6, right). The influence of [NCO]/[OH] ratio on the crosslinking density and the Tg value was also investigated and reported in Table 2. For both polyols, the best results were observed for slight excess in isocyanate: 1.1/1 related than 1/1 as widely described in the literature.It is noteworthy that higher Tg value was measured for PU2in comparison to PU1 (72and45 °C, respectively). This differencecould be explained by the presence of dangling chain in the structure of OH1 in opposition to OH2 as well as the densification of the networkresulting in higher functionality in alcohol groups in the latter case.

Figure 6

The thermal stability of PU1 and PU2thermosets was investigated bythermogravimetric analysis (TGA) in nitrogen stream, and the results were collected in Figure7. As previously reported, the thermal stability of polyurethanesis relatively low. Our results were in accordance with other thermoset PUs coming from lipidic polyols and synthesized by TEC or TYC methodology. At weightlossof 5%, the degradation temperatures of PU1 and PU2 were 295 and 282 °C, respectively whereas those of Cadiz are 269-290 °C for TEC-based polyol (8) and 245 °Cfor TYC-based polyol (9) (Table 2). 21,25 Moreover, the well-known thermal degradation ofpolyurethanes undergoes in a three-step process. 21 As described, our productsdegraded in two overlapping steps: i) the first step around 325°C resultsin the degradation of weak urethane bondsthrough the dissociation to isocyanate and alcohol and ii) the second step occurs at 343°C and corresponds to the decomposition and the char formation of crosslinked polymer networks. Finally, the third decomposition took place at 428-468°Cand corresponds to the gasification of any remaining components.

Conclusion

In this contribution, propargylic fatty acid and diacid were synthesized in good yields. We showed that thiol-ene/yne coupling is an effective approach to synthesize lipidic polyols without oligomers. Foroleicdiacid, polyol without dangling chain was prepared and produced polyurethane materials with Tg valueof 72°C after optimization of the crosslinking parameters (temperature and [NCO]/[OH]). Comparing our results to those reported in the overview regarding toplant oil-based crosslinkedpolyurethanes,we showeda typical thermal stability in a three-step process of plant oil-based thermoset polyurethanes.Thus, through the synthesis of these lipidic polyols, we illustratedthe opportunity of TYC/TECfunctionalization in the synthesis of high functional monomers and branched polymers using bio-based building blocks.

Lipidicpolyolsusing

  Thiol-ene/yne strategyforcrosslinkedPolyurethanes Pham P. D., a Lapinte V., a* Raoul Y., b Robin J.-J. a a : Institut Charles Gerhardt Montpellier UMR5253 CNRS-UM2-ENSCM-UM1 -EquipeIngénierie et Architectures Macromoléculaires, Université Montpellier II -Bat 17 -cc1702, Place EugèneBataillon 34095 Montpellier Cedex 5. b : Société Interoléagineuse d'Assistance et de développement (S.I.A.), 11, rue de Monceau CS 60003 75378 Paris cedex 08, France.

Figure 1 Figure 2

 12 Figure 1

  explored by LC-ESI/MS technique. As expected, the isomolecular pic [M+H] + was detected at m/z of 779.40 in accordance with the m/z theoretical value of 779.33. Another pic was observed at m/z of 567.3 and corresponds to [M-211] + , outcome of the breaking of the C-O bond's breaking of one of the two estergroup.

Figure 4

 4 Figure 4

Figure 5 .

 5 Figure 5. The total disappearance of -N=C=O stretching of the isocyanate moiety at 2240 cm -1

Figure 7

 7 Figure 7

1 .Scheme 2 .Scheme 3 .

 123 Scheme captions

Figure captions Figure 1 . 1 H

 captions11 Figure captions

Figure 2 . 1 H

 21 Figure 2. 1 H NMR spectra of PFD and OH2 in CDCl 3 .

Figure 3 .

 3 Figure 3. GPC traces in DMAcof PFE and PFD precursors and OH1 and OH2 polyols.

Figure 4 .

 4 Figure 4.LC ESI/MS spectrum of PFD.

Figure 5 .

 5 Figure 5. FTIR spectra of OH1, MDI and PU1(NCO/OH= 1.1).

Figure 6 .

 6 Figure 6.DSC thermograms of thermoset polyurethanes (right) and the OH1 and OH2

Figure 7 .

 7 Figure 7.TGA chromatograms of PU1 (left) and PU2 (right) under nitrogen.

Table 1 .

 1 Comparison of polyols synthesized in this work and in the literature.

	Reference	Lipidic	Structure	Strategy	Reactant
		precursor
	(1)	various VO	FA	ROEG and	propylene glycol
				esterification
	(2)	soybean	TAG	ROEG	lactic acid
	(3)	various VO	TAG	ROEG	methanol
	(4)	methyloleate oligomer	ROEG and	LiAlH 4
				reduction
	(5)	various VO	TAG	ozonolysis and	O 3 and NaBH 4
				reduction
	(6)	methyloleate	TAG	hydrosylilation	PTDS and
				and reduction	AlLiH 4
	(7)	rapeseed oil	TAG	TEC	HS-(CH 2 ) 2 OH
	(8)	methyloleate	FA	allylation + TEC	HS-(CH 2 ) 2 OH
	(9)	methyloleate	FA	dehydrobromation	HS-(CH 2 ) 2 OH
				+ TYC
	OH1	oleate acid	FA	esterification +	HS-(CH 2 ) 2 OH
				TYC
	OH2	oleatediacid	FA	esterification +	HS-(CH 2 ) 2 OH
				TYC

TAG: triacyl glycerol; FA: fatty acid; VO: vegetable oil; ROEG: ring-opening epoxy group; PTDS: phenyl tris(dimethylsiloxy)silane.

  NMR (CDCl 3 , 300 MHz)  (ppm): 0.79-0.95 (3H, H 1 ), 1.15-1.44 (20H, H 2-7 , H 12-15 ), 3x200 mL), and brine (2x200 mL), dried over anhydrous MgSO 4 , filtered, and evaporated under reduced pressure to yield a dark yellow oil. The product was purified by column chromatography using AcOEt/CH 2 Cl 2 3/7, as eluent, to afford a viscous light yellow product (6.59 g, 66%). .25 (C 3 ), 32.77 (C 17 ), 33.56-35.27 (C 9 ,C 11 , C 21 , C 22 ), 44.43 (C 10 ), 45.32 (C 20 ), 60.54, 60.86 (C 19 , 2 x C 23 ), 64.77 (C 25 ), 173.15 (C 18 ).The PU materials were prepared by mixing OH1 or OH2 with4,4'-methylenebis(phenyl isocyanate) (MDI) at 60 °C in an aluminum mold. The [NCO]/[OH] ratio ranged from 1/1 to 1.1/1. The vacuum was applied to remove bubbles. The mixture was cured at 60 °C for 24h and post-cured at 110 °C for 24h. The resulting PU materials werepeeled off from the mold for characterization tests.

	1.52-1.72 (2H, H 16 ), 1.87-2.11 (4H, H 8 , H 11 ), 2.27-2.39 (2H, H 17 ), 2.45-2.50 (2H, H 21 ),
	4.60-4.72 (4H, H 19 ), 5.26-5.41 (2H, H 9 , H 10 ). 23.35 g, 81% yield.
	13 C NMR (CDCl 3 , 100 MHz)  (ppm):14.04 (C 1 ), 22.64 (C 2 ), 24.74 (C 16 ), 27.10 and 27.16 Polyol derivating from PFE (OH1)
	(C 8 , C 11 ), 28.99-29.72 (C 4-7 , C 12-15 ), 31.87 (C 3 ), 33.88 (C 17 ), 51.63 (C 19 ), 74.61 (C 21 ), 77.74 1 H NMR (CDCl 3 , 400 MHz) (ppm): 0.81-1.02 (3H, H 1 ), 1.15-1.76 (29H, H 2-16 ), 2.27-2.44
	(C 20 ), 129.62 and 129.89 (C 9 , C 10 ), 172.72 (C 18 ). (2H, H 17 ), 2.53-3.23 (11H, H 20 , H 21 , H 22 , H 24 ), 3.63-3.91 (6H, H 23 , H 25 ), 4.06-4.43 (2H, H 19 ).
	Propargylic fatty diester (PFD)
	Polyol derivating from PFD (OH2)
	1 H NMR (CDCl 3 , 400 MHz)  (ppm): 1.14-1.65 (27H, H 2-16 ), 2.21-2.36 (4H, H 2 , H 17 ), 2.64-
	2.93 (18H, H 21 , H 22 , H 24 ), 2.99-3.16 (2H, H 20 ), 3.43 (OH), 3.61-3.80 (10H, H 23 , H 25 ), 4.13-
	4.39 (4H, H 19 ).
	13 C NMR (CDCl 3 , 100 MHz)  (ppm): 24.27 (C 3 , C 16 ), 26.06-28.88 (C 4-8 , C 12-15 ), 32.86 (C 2 ,
	Thiol-en/yne coupling (TEC/TYC) between mercaptoethanol and propargylic fatty C 17 ), 33.61, 34.36, 35.41, 32.77 (C 9 ,C 11 , C 21 , C 22 ), 44.51 (C 10 ), 45.33 (C 20 ), 60.50, 60.84 (C 19 ,
	esters 4 x C 23 ), 64.84 (C 25 ), 173.20 (C 1 , C 18 ).
	In a 50 mL flask, PFD (5 g, 12.86 mmol) was reacted with 2-mercaptoethanol (15.07 g, 193 ESI-MS (m/z): [M+H] + theo: 779.33 and found: 779.40, [M-(C 7 H 15 O 3 S 2 )] + theo: 567.30and
	mmol) in presence of the radical initiator DMPA (1.65 g, 6.43 mmol) under N 2 found: 567.3.
	atmosphere.An amount of DMF(2.4 mL) was added todissolve the photoinitiator. The FTIR (cm -1 ): 3375 (OH), 2931 and 2856 (C-H stretching ), 1731(C=O ester stretching ).

1

H FTIR (cm -1 ): 3312 (O-H), 2923 and 2853 (C-H stretching ), 1744 (C=O ester stretching ). 1 H NMR (CDCl 3 , 300 MHz)  (ppm): 1.17-1.41 (16H, H 4-7 , H 12-15 ), 1.61-1.71 (4H, H 3 , H 16 ), 1.88-2.08 (4H, H 8 , H 11 ), 2.24-2.40 (4H, H 2 , H 17 ), 2.45-2.49 (2H, H 21 ), 4.60-4.70 (4H, H 19 ), 5.26-5.38 (2H, H 9 , H 10 ). 19.2 g, 86 % yield. 13 C NMR (CDCl 3 , 100 MHz)  (ppm): 24.27 (C 3 ,C 16 ), 26.10 (C 8 , C 11 ), 27.97, 28.01, 28.08 (C 4-7 , C 12-15 ), 32.80 (C 2 , C 17 ), 50.61 (C 19 ), 73.86 (C 21 ), 76.85 (C 20 ), 128.73 (C 9 , C 10 ), 171.51 (C 1 , C 18 ). FTIR (cm -1 ): 3294 (O-H), 2926 and 2854 (C-H stretching ), 1739 (C=O ester stretching ). reaction carried out under UV irradiation (365 nm) at room temperature for 8 h. DMF was then removed under reduced pressure. The mixture was dissolved in CHCl 3 (200 mL), then washedwith water (13 C NMR (CDCl 3 , 100 MHz) (ppm): 13.52 (C 1 ), 22.05 (C 2 ), 24.23 (C 16 ), 26.02-29.01 (C 4-8 , C 12-15 ), 31FTIR (cm -1 ): 3389 (O-H), 2930 and 2858 (C-H stretching ), 1736 (C=O ester stretching ). General procedure for crosslinked polyurethanes(PU1 and PU2)

  DCl) is easily synthesized in 97% yield (see experimental part).Thus, the treatment ofOACl and DClwith propargylic alcohol in presence of triethylamine in CH 2 Cl 2 afforded PFE and

	PFDinagood yield (81 and 86%,respectively)(Scheme 2). The complete reaction was
	monitored by 1 H NMR experiment using in both cases the shift of CH 2 -C=O signal (H 17 )from
	2.88 to 2.34 ppm as well as the appearance of the propargylic signals corresponding to H 19
	and H 21 at 4.65 and 2.45 ppm.

Table 2 .

 2 Comparison of polyurethanes synthesized in this work and in the literature.

	Reference [NCO]/[OH]	Tg	T 5% loss
			(°C)	(°C)
	(1) (2) (3) (4) (5) (6) (7)	1.1 / 1 1.05 / 1 1.02 / 1 1.02 / 1 1.02 / 1 1.02 / 1 1.05 / 1	69 -114 c 96 b 30 -77 b -3 -57 d 22 & 36 39 25 a	---273 e ---337 -352 330 & 337 e 322 f ---
	(8)	1 / 1	8 -56	269 -290
	(9)	1 / 1	42 -59	245
	PU1	1 / 1	36	297
		1.1 / 1	45	295
	PU2	1 / 1	68	279
		1.1 / 1	72	282
	PU materials were curing at 110°C apart from 65 °C (a), 80°C (b) and 100°C (c). d : Tg
	value determined by DMA experiment.

e : deduced from figure.

Table captions Table 1 .

 captions1 Comparison of polyols synthesized in this work and in the literature.

Table 2 .

 2 Comparison of polyurethanes synthesized in this work and in the literature.

Acknowledgement

The authors gratefully acknowledge S.I.A. for their financial support.