Monique Drielle

Gomes Ferreira

Jacqueline Araújo Castro

Raner José

Santana Silva

Fabienne Micheli

HVA22 from citrus: A small gene family whose some members are involved in plant response to abiotic stress

Keywords: in silico analysis, transmembrane domains, cis-elements, gene expression, drought, transgenic tobacco

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The HVA22 gene has been isolated for the first time from the aleurone layer of barley (Hordeum vulgare), giving its name [START_REF] Shen | Hormone response complex in a novel abscisic acid and cycloheximideinducible barley gene[END_REF]. HVA22 is present in several eukaryotes, such as in plants, fungi and humans, but has not been described in prokaryotes [START_REF] Shen | The stress-and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms[END_REF]. In plants, HVA22 is expressed in different tissues, such as seeds, shoots and roots and is induced under several environmental stress conditions (e.g. cold, salinity and drought) mediated by abscisic acid (ABA) [START_REF] Shen | The stress-and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms[END_REF]. HVA22 from barley is homologous to Yop1p in Saccharomyces cerevisiae, AtHVA22 in Arabidopsis thaliana, AtpCHVA22 in Aloe vera L., OsHVA22 in Oryza sativa and TB2/DP1 in human [START_REF] Yang | Specific binding to a novel and essential Golgi membrane protein (Yip1p) functionally links the transport GTPases Ypt1p and Ypt31p[END_REF][START_REF] Xiao | Evaluation of seven function-known candidate genes for their effects on improving drought resistance of transgenic rice under field conditions[END_REF][START_REF] Sharon | Cloning of HVA22 Homolog from Aloe vera and Preliminary Study of Transgenic Plant Development[END_REF][START_REF] Calero | Yop1p, the Yeast Homolog of the Polyposis Locus Protein 1, Interacts with Yip1p and Negatively Regulates Cell Growth[END_REF]. Approximately 355 HVA22 homologues were described in eukaryotes. In all homologous, the TB2/DP1 region present in the HVA22 protein structure is conserved [START_REF] Sharon | Cloning of HVA22 Homolog from Aloe vera and Preliminary Study of Transgenic Plant Development[END_REF][START_REF] Guo | An Abscisic Acid-Induced Protein, HVA22, Inhibits Gibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells[END_REF].

In Arabidopsis, five homologs of HVA22 were identified, designated as AtHVA22a, AtHVA22b, AtHVA22c, AtHVA22d and AtHVA22e. These genes showed differential expression in plant tissues, when treated with ABA and subjected to stress conditions [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF].

Studies of Yop1p showed that this gene may be involved in vesicular trafficking and transport of substances from the endoplasmic reticulum (ER) to the Golgi complex in yeast [START_REF] Yang | Specific binding to a novel and essential Golgi membrane protein (Yip1p) functionally links the transport GTPases Ypt1p and Ypt31p[END_REF][START_REF] De Antoni | Significance of GTP Hydrolysis in Ypt1pregulated Endoplasmic Reticulum to Golgi Transport Revealed by the Analysis of Two Novel Ypt1-GAPs[END_REF]. Yop1p/DP1 proteins interact with the reticulon protein Rtn4/NogoA, modulating in vivo the protein interaction and ER functions [START_REF] Voeltz | A Class of Membrane Proteins Shaping the Tubular Endoplasmic Reticulum[END_REF][START_REF] Hu | Membrane Proteins of the Endoplasmic Reticulum Induce High-Curvature Tubules[END_REF]. According to the environmental conditions, the lipid composition of the plasma membrane in yeast and plants could be modified by transport vesicles that deliver the newly synthesized lipids to the membrane [START_REF] Hamrouni | Effects of water-deficit on lipids of safflower aerial parts[END_REF]. However, the role of HVA22 in this process is still unknown and, more generally, the HVA22 gene family has been a poorly studied, even if its potential in response to abiotic stresses has already been highlighted [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF].

To our knowledge, in citrus plants, neither genome-wide characterization of the HVA22 family nor functional studies has yet been performed. The availability of the data from the recent sequencing of the genome of some citrus species (https://www.citrusgenomedb.org/) allowed for the genome-wide analysis of gene families as a pre-requisite for functional and/or pre-breeding studies. The present study aimed to characterize the family of HVA22 in mandarin (Citrus clementina) and sweet orange (Citrus sinensis) at nucleotide and protein levels, including promoter analysis and expression pattern.

It also provides specific information about the CcHVA22d gene; transgenic tobacco plant expressing this gene showed higher dehydration tolerance and less oxidative stress than nontransgenic ones.

Material and methods

In silico analysis of HVA22 citrus genes and proteins

The identification and structural analysis of the HVA22 genes (introns/exons) were performed using the Citrus Genome Database (https://www.citrusgenomedb.org/). Open reading frame (ORF) analysis was performed using the ORFinder software (http://www.ncbi.nlm.nih.gov/orffinder/). The isoelectric point (pI) and molecular weight (MW) were predicted using the pI/Mw tool (www.expasy.org). The predictions of the subcellular location of the protein and of the location of the cleavage site were performed by the TargetP 1.1 server (https://www.cbs.dtu.dk/services/TargetP). Analysis of the conserved family and domains was performed using Pfam (http://pfam.sanger.ac.uk/search/sequence) and ProScan software [START_REF] Quevillon | InterProScan: protein domains identifier[END_REF]. Transmembrane helices were predicted using TMPred software [START_REF] Hofmann | TMbase -A database of membrane spanning proteins segments[END_REF]. Hydropathicity level was identified using the Protparam program (https://web.expasy.org/protparam/). The putative phosphorylation sites (Ser / Thr / Tyr) and N-glycosylation sites (tipo Asn-X-Ser / Thr) were identified through the NetPhos 3.1 Server [START_REF] Blom | Sequence and structure-based prediction of eukaryotic protein phosphorylation sites[END_REF] and the NetNGlyc 1.0 server (http://www.cbs. dtu.dk/services/NetNGlyc/) respectively.

The protein motif analysis was conducted using the program MEME/MAST [START_REF] Bailey | MEME Suite: tools for motif discovery and searching[END_REF]. The maximum number of motifs was set to 20, the maximum motif length was set to 80 amino acids, the optimum motif width was constrained to between 6 and 300 residues, and the other parameters were used as default.

Analysis of the promoter regions and chromosomal location of HVA22 genes

The identification of cis-elements in the promoter regions (1500 bp upstream region from the translation start site) was performed using the plantCARE software (sphinx.rug.ac.be:8080/PlantCARE/cgi/index.html) [START_REF] Lescot | PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[END_REF]. The chromosomal location of the HVA22 genes was obtained by screening the GFF3 file of the genome of C. clementina and C. sinensis deposited in the citrus genome database using the HVA sequence ID.

Phylogeny

Phylogenetic analysis was performed based on the alignment of the amino acid sequence of the HVA22 proteins from C. sinensis and C. clementina with HVA22 proteins from Arabidopsis thaliana. The sequences were aligned with ClustalW2

(http://www.ebi.ac.uk/Tools/msa/clustalw2/) [START_REF] Thompson | improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]. The MEGA 6 program was used to construct a phylogenetic tree by using the neighbor-joining statistical method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF] reliably established by 1000 bootstrap samples.

In silico C. sinensis HVA22 gene expression

The expression of the genes of the C. sinensis HVA22 gene in different tissues (callus, leaf, flower and fruit) was analyzed using the RNA-seq expression data available in the Citrus sinensis Annotation Project database version 2.1 (CAP; http://citrus.hzau.edu.cn/orange/) [START_REF] Wang | Citrus sinensis Annotation Project (CAP): A Comprehensive Database for Sweet Orange Genome[END_REF]. The Heatmap function of the ComplexHeatmap library from the R program [START_REF] Gu | Complex heatmaps reveal patterns and correlations in multidimensional genomic data[END_REF] was used for the generation and visualization of the transcription level Heatmap.

Generation of CcHVA22d transgenic tobacco plants

The coding sequence of CcHVA22d was obtained by RT-PCR from leaves of C. clementina cultivated at Embrapa CNPMF (Cruz das Almas, Bahia, Brazil (12°40′39″S latitude, 39°06′23″W longitude and 225m elevation) and submitted to drought as previously described [START_REF] Neves | Recurrent water deficit causes alterations in the profile of redox proteins in citrus plants[END_REF]. Briefly, total RNA was extracted using the RNAqueous kit according to the manufacturer instructions (Ambion, Inc.), cDNA was obtained using the Super Script Double Havana) as previously described [START_REF] Cidade | Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco[END_REF]. Several independent transgenic lines (T0 generation) derived from distinct transformation events were transferred to soil and grown under standardized greenhouse conditions [START_REF] Cidade | Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco[END_REF]. GUS histochemical assays [START_REF] Jefferson | GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF] and PCR screening using the CcHVA22d primers indicated above [START_REF] Cidade | Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco[END_REF] were used to discriminate between transgenic and non-transgenic (control) plants.

Analysis of relative dehydration rate of transgenic HVA22d plants

The relative dehydration rate was evaluated in control and transgenic (T0) tobacco plants by measuring the difference between the initial and final fresh weight (FW) of the leaf tissues in each evaluated period. For this purpose, uniform samples of leaf discs were extracted from the youngest, fully expanded leaf of adult plants, using a 1.0 cm diameter perforator, avoiding the central vein. Then, the leaf discs were submitted to dehydration at room temperature (~22ºC)

for 240 min, and fresh weight measurements were performed every 30 min. Each treatment consisted of three replicates with six leaf discs per replicate. Data were submitted to variance analysis and compared by Student's t-test with a probability of 1%.

Detection of H2O2 in transgenic HVA22d plants

To evaluate the oxidative stress in foliar tissues submitted to dehydration, the (control)

and transgenic tobacco leaf discs (T0) were submitted to DAB-HCl (3,3'-diaminobenzidene) histochemical staining assay as previously described [START_REF] Thordal-Christensen | Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction[END_REF]. The samples were subjected to vacuum infiltration with 1 mg/ml of DAB-HCl for 4 h. After this period, the leaf discs were boiled in 96% ethanol for 4 h and subsequently photographed in a magnifying glass to detect the brown coloration.

Results

The HVA22 family in sweet orange and mandarin

The annotation of the citrus genome database allowed the identification of 12 HVA22 genes, 6 in C. clementina (CcHVA22a, CcHVA22b, CcHVA22c, CcHVA22d, CcHVA22e and CcHVA22f) and 6 in C. sinensis (CsHVA22a, CsHVA22b, CsHVA22c, CsHVA22d, CsHVA22e

and CsHVA22f) (Table 1). The C. clementina HVA22 genes were located on chromosomes 3 (CcHVA22c), 4 (CcHVA22b, CcHVA22d and CcHVA22e), 8 (CcHVA22a) and 9 (CcHVA22f) (Table 1). In C. sinensis, the HVA22 genes were located on chromosomes 5 (CsHVA22c), 7

(CsHVA22b and CsHVA22e) and 8 (CsHVA22a) (Table 1). Colinearity analysis showed duplications events between CcHVA22c (chromosome 3) and CcHVA22b (chromosome 4) 1; Figure 1).

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Promoter sequence analysis of the citrus HVA22 genes

A 1500 bp fragment belonging to the upstream region of each HVA gene was analyzed to find plant specific cis-elements using the PlantCARE database (Supplementary material 3).

The TATA and CAAT-box elements were found in all citrus HVA22 promoter regions; the other cis-elements varied between sequence promoters (Fig. 2, Supplementary material 4).

Light-responsive cis-elements (BOX 4, G-BOX and sp1 elements) and drought-induced element (MYB) were found in great amounts in all promoter sequences (Fig. 2, Supplementary material 4). In smaller proportion, cis-elements were found that were responsive to i) hormone or inducers such as auxin, gibberellin, salicylic acid, methyljasmonate, and abscisic acid; and ii) biotic or abiotic stresses such as elicitors, anaerobiosis, defense and stress, and anoxia (Fig. 2, Supplementary material 4). Other cis-elements related to plant development such as seed-specific regulation, meristem expression, zein metabolism, circadian control, endosperm expression were also present in the promoters of the citrus HVA genes (Fig. 2, Supplementary material 4).

Analysis of the citrus HVA proteins

The amino acid amount of HVA22 proteins ranged from 94 to 267 in C. clementina and from 94 to 189 in C. sinensis (Table 2, Supplementary material 5). Most of the citrus HVA22 presented basic pI, except CcHVA22a, CcHVA22c, CcHVA22c*, CsHVA22a, and CsHVA22c (Table 2). The citrus HVA22 proteins showed transmembrane location with mainly 2-3 transmembrane domains (Table 2). The hydropathicity ranged from -0.272 to 0.419 in C. clementina and from -0.335 to 0.315 in C. sinensis (Table 2). Citrus HVA22 molecular weight (MW) ranged from 11.512 (CcHVA22f) to 31.072 kDa (CcHVA22b; Table 2). The conserved TB2/DP1 region was found in all the citrus HVA22 (Fig. 3; Supplementary material 5). Putative phosphorylation sites were found in all citrus HVA22 proteins, and Nglycosylation site were found in most of them (Table 3, Supplementary material 5). Most of the citrus HVA22 proteins also contained casein kinase phosphorylation sites; these sites were spread along the protein sequence and ranged from one to five (Supplementary material 5).

The protein sequence identity varied from 30% to 93% between CcHVA22 proteins and from 25% to 94% in CsHVA22 proteins (Supplementary material 6). The greatest degree of identity (100%) was observed between CcHVA22a and CsHVA22a, CcHVA22b and CsHVA22b, CcHVA22c/CcHVA22c* and CsHVA22c, CcHVA22d and CsHVA22d (Supplementary material 6). The percentage of identity between the two proteins resulting from the alternative transcripts of the gene was i) 93% for CcHVA22c; ii) 94% for CsHVA22a; iii) 95% for CsHVA22b; and iv) 100% of CsHVA22d (Supplementary material 6).

Phylogenetic relationships of the HVA22 genes of citrus and Arabidopsis

Phylogenetic analysis of the HVA22 sequences of citrus and A. thaliana showed that CcHVA22d, CsHVA22d and CsHVA22d* were clustered with the sequence AtHVA22d and AtHVA22e, while CcHVA22c, CcHVA22c* and CsHVA22c were clustered with AtHVA22a (Fig. 4). The other citrus HVA22 sequences did not clustered with A. thaliana sequences.

CcHVA22e, CsHVA22e, CcHVA22f and CsHVA22f were grouped in the same cluster, as well as CsHVA22b, CsHVA22b* and CcHVA22b, and CcHVA22a, CsHVA22a and CsHVA22a* (Fig. 4).

In silico CsHVA22 gene expression

The expression of the CsHVA22 genes was previously obtained and was available in the CAP database. Four tissues were analyzed: callus, flower, leaf and fruit. The CsHVA22d gene showed high expression levels in the fruit (> 15 Reads Per Kilobase Million / RPKM) and callus (17.54 RPKM) and low expression levels in flowers and leaves (1.64 and 1.57

RPKM respectively) (Fig. 5, Supplementary material 7). CsHVA22a and CsHVA22c showed a similar pattern of expression in different tissues with a general quite high expression (>10 RPKM for CsHVA22a; > 10 RPKM in fruits > 8 RPKM in flower > 7RPKM in callus and leaf for CsHVA22c) (Fig. 5, Supplementary material 7). CsHVA22e gene showed highest expression in flower (6.37 RPKM), followed by leaf (4.15 RPKM) and fruit (1.09 RPKM), in callus this expression was very low (<1 RPKM) (Fig. 5, Supplementary material 7). The CsHVA22b expression did not exceed 5 RPKM; the lowest level was observed in fruit (0.24 RPKM) (Fig. 5, Supplementary material 7). CsHVA22b and CsHVA22e showed similar expression pattern and were clustered in the heatmap analysis (Fig. 5). No expression was detected for CsHVA22f (data not shown).

Dehydration analysis and H2O2 detection in CcHVA22d transgenic tobacco plants

Genetic transformation of tobacco leaf explants was made by Agrobacterium tumefaciens co-cultivation method using the 35S::CcHVA22d::pCAMBIA2301 construct.

After regeneration, the T0 transformed lines were confirmed by GUS histochemical test and PCR; five transgenic lines were selected according to both confirmations (Fig. 6A, Supplementary material 8). The L2 and L5 lines showed a more intense blue coloration corresponding to GUS test, followed by the L1, and then the L3 and L4 lines (Fig. 6A). All transgenic lines showed a 534 bp band on electrophoresis gel corresponding to the gene amplification (Supplementary material 8). Transgenic CcHVA22d tobacco plants were evaluated under short-term dehydration conditions (Fig. 6B). All transgenic lines lost significantly less water than WT from 30 min to 240 min after leaf disc dehydration (Fig. 6B).

Among the transgenic lines, L3 and L5 showed the lowest dehydration rates (Fig. 6B). The dehydration tolerance of transgenic tobacco lines were correlated to oxidative stress levels by detecting H2O2 in the same leaf discs using the DAB-HCl histochemical test (Fig. 6C). The brown coloration indicator of H2O2 presence was less intense in the transgenic line leaf discs than in the WT ones (Fig. 6C).

Discussion

Here, we analyzed the HVA family from citrus. The gene amount was the same in both C. clementina and C. sinensis (6 genes; Fig. 1, Table 1) and close to the number of HVA22 genes observed in Arabidopsis (5 genes; [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF]). Interestingly, the 100% of identity was observed between the same ID genes from both C. clementina and C. sinensis (e.g.

CcHVA22a and CsHVA22a; Supplementary material 6). These sequence pairs from both citrus species, also clustered together in the phylogenetic analysis (Fig. 4), indicating they could be considered as orthologues. Only few HVA22 gene duplication events were observed in citrus (CcHVA22c/CcHVA22b in chromosomes 3/4 and CsHVA22c/CsHVA22b in chromosome 5/7; Supplementary material 1). In Arabidopsis, AtHVA22d were located on chromosome 4 and AtHVA22e on chromosome 5, and evidences indicated that these two genes arose from a same ancestral gene via duplication events and reorganization [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF][START_REF] Blanc | Extensive Duplication and Reshuffling in the Arabidopsis Genome[END_REF][START_REF] Chen | Coalescent processes and relaxation of selective constraints leading to contrasting genetic diversity at paralogs AtHVA22d and AtHVA22e in Arabidopsis thaliana[END_REF].

Gene duplication could be considered as the primary source of new gene generation, participating of the genome complexity increase. It also contributed to evolution of novel gene function associated, among others, to floral structure production or stress adaptation [START_REF] Panchy | Evolution of Gene Duplication in Plants[END_REF]. In addition to sequence variations, the analysis of the gene duplication and the evolution of paralogs need to take in consideration other characteristics such as gene expression [START_REF] Chen | Coalescent processes and relaxation of selective constraints leading to contrasting genetic diversity at paralogs AtHVA22d and AtHVA22e in Arabidopsis thaliana[END_REF].

Divergent expression patterns observed for some duplicate genes could be due to subfunctionalization and/or neofunctionalization [START_REF] Panchy | Evolution of Gene Duplication in Plants[END_REF]. Here, CsHVA22b and CsHVA22c

showed similar expression pattern (expression in all tissues) but with a lower intensity for the CsHVA22b gene, mainly in fruits in which the expression is almost null (Fig. 5). This result could be consistent with subfunctionalization model via duplication-degenerationcomplementation processes, in which one of the paralogs (CsHVA22b) loss a subfunction of the original gene (degeneration; loss of function in fruit). In this model, both duplicates have been kept to maintain the ancestral functionality (complementation) [START_REF] Chen | Coalescent processes and relaxation of selective constraints leading to contrasting genetic diversity at paralogs AtHVA22d and AtHVA22e in Arabidopsis thaliana[END_REF][START_REF] Panchy | Evolution of Gene Duplication in Plants[END_REF]. Moreover, the complete loss-of-function and/or the absence of gene expression (e.g. CsHVA22f, data not shown; AtHVA22f [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF]) could be associated to pseudogenization, i.e. presence of undeleted nonfunctional duplicates [START_REF] Panchy | Evolution of Gene Duplication in Plants[END_REF]. On the other hand, it has been shown that whole-genome duplication has contributed to important agronomic traits in several crops [START_REF] Panchy | Evolution of Gene Duplication in Plants[END_REF], and for this reason the impacts of gene duplication will be important to future plant studies mainly the agronomically important ones. Interestingly, the duplicated genes in citrus corresponded to the orthologues between C. clementina and C. sinensis (CcHVA22c/CsHVA22c and CcHVA22b/CsHVA22b) (Supplementary material 1 and 6). Considering that C. clementina is descendant from C. sinensis, it could be suggested that these duplication events occurred upstream this crossing via the sweet orange parental [START_REF] Wu | Genomics of the origin and evolution of Citrus[END_REF]. The identification of sequence variations (e.g. polymorphism) in orthologs and/or paralogs could be usefully used in intra-or interspecific diversity studies and/or breeding programs of citrus [START_REF] Wu | Genomics of the origin and evolution of Citrus[END_REF][START_REF] Curk | Nuclear Species-Diagnostic SNP Markers Mined from 454 Amplicon Sequencing Reveal Admixture Genomic Structure of Modern Citrus Varieties[END_REF][START_REF] Curk | Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers[END_REF][START_REF] Wu | Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication[END_REF][START_REF] Ahmed | Genotyping by sequencing can reveal the complex mosaic genomes in gene pools resulting from reticulate evolution: a case study in diploid and polyploid citrus[END_REF].

The citrus HVA22 genes presented alternative transcription; thus 12 citrus genes encoded 16 proteins (Fig. 1, Table 1, Table 2). In general the amount of introns was low among the homologues and this may indicate a shorter period to respond to stimuli caused by a stress condition [START_REF] Jeffares | Rapidly regulated genes are intron poor[END_REF]. This small amount of introns has already been observed for other citrus genes that respond to abiotic stresses [START_REF] Castro | Alternative oxidase (AOX) constitutes a small family of proteins in Citrus clementina and Citrus sinensis L. Osb[END_REF]. It has been reported that the majority of alternatively spliced RNAs bind to ribosomes and consequently are translated [START_REF] Reyes | Alternative start and termination sites of transcription drive most transcript isoform differences across human tissues[END_REF]. Moreover, most of the alternative proteins did not present neither disrupted structures nor functions [START_REF] Reyes | Alternative start and termination sites of transcription drive most transcript isoform differences across human tissues[END_REF].

Here, the percentage of identity between the two proteins resulting from the alternative transcripts of the gene was high (from 93 to 100%; Supplementary material 6), suggesting that the alternative proteins may have real biological function. At the protein level, the citrus HVA22 sequences showed an average of 156 amino acids, close to the 130 amino acids from barley HVA22 [START_REF] Shen | Hormone response complex in a novel abscisic acid and cycloheximideinducible barley gene[END_REF][START_REF] Shen | The stress-and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms[END_REF]. The barley HVA22 protein showed a pI of 9.2 [START_REF] Shen | Hormone response complex in a novel abscisic acid and cycloheximideinducible barley gene[END_REF][START_REF] Shen | The stress-and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms[END_REF], close to the one of most of the citrus HVA22 proteins. The average of the citrus HVA22 MW was about 18 kDa, close to the MW of the HVA22 from Aloe vera (15 kDa; [START_REF] Sharon | Cloning of HVA22 Homolog from Aloe vera and Preliminary Study of Transgenic Plant Development[END_REF]). Some of the citrus HVA22 proteins contained casein kinase II phosphorylation sites (Supplementary material 5).

Analysis of the amino acid sequence of HVA22 in barley demonstrated that this protein also contained two of casein kinase II phosphorylation sites (SKVD sequence), one between region 36 and 39 and another between region 54 and 59, the latter being conserved in several homologues [START_REF] Shen | Hormone response complex in a novel abscisic acid and cycloheximideinducible barley gene[END_REF][START_REF] Shen | The stress-and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms[END_REF]. Studies have shown that CK2 has action on several cellular functions, including maintenance of cell viability [START_REF] Shen | The stress-and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms[END_REF]. The protein phosphorylation mediated by kinases and phosphatases plays essential roles in plant response to many stresses and environmental signals (such as drought) and are convenient for the rapid regulation of protein activity in various signaling pathways [START_REF] Wu | A Casein Kinase II Phosphorylation Site in AtYY1 Affects Its Activity, Stability, and Function in the ABA Response[END_REF].

All the citrus HVA22 proteins presented transmembrane location due to several transmembrane domains (Table 2) located in the TB2/DP1 region (Fig. 3; Supplementary material 5). TB2-like 1 is a membrane protein belonging to the YOP1/TB2/DP1/HVA22 family that may play an important role in the intracellular membrane trafficking in retinal ganglion cells from animals [START_REF] Sato | Deleted in Polyposis 1-like 1 Gene (Dp1l1): A Novel Gene Richly Expressed in Retinal Ganglion Cells[END_REF]. The TB2/DP1 region from barley and its orthologue in yeast, Yop1p, were integral proteins of the endoplasmic reticulum membrane (ER), inserted into the lipid bilayer whose function is to maintain its structure [START_REF] Calero | Yop1p, the Yeast Homolog of the Polyposis Locus Protein 1, Interacts with Yip1p and Negatively Regulates Cell Growth[END_REF]. It has been shown that this transmembrane region played an important physiological role in barley but also in yeast and human [START_REF] Calero | Yop1p, the Yeast Homolog of the Polyposis Locus Protein 1, Interacts with Yip1p and Negatively Regulates Cell Growth[END_REF][START_REF] Voeltz | A Class of Membrane Proteins Shaping the Tubular Endoplasmic Reticulum[END_REF][START_REF] Sato | Deleted in Polyposis 1-like 1 Gene (Dp1l1): A Novel Gene Richly Expressed in Retinal Ganglion Cells[END_REF]. In barley aleurone cell, HVA22 negatively regulated vacuolization and programmed cell death, inhibited vesicular trafficking which promotes membrane turnover in stressed cells, nutrient mobilization in seed germination and seedling growth regulation, as well as cell wall components supplying in plant expanding regions (e.g. roots) allowing a greater absorption of water and minerals [START_REF] Guo | An Abscisic Acid-Induced Protein, HVA22, Inhibits Gibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells[END_REF][START_REF] Voeltz | A Class of Membrane Proteins Shaping the Tubular Endoplasmic Reticulum[END_REF][START_REF] Brands | Function of a plant stress-induced gene, HVA22. Synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic[END_REF][START_REF] Gilroy | Through form to function: root hair development and nutrient uptake[END_REF]. In Arabidopsis, the analysis of the three transmembrane domains (TM1, TM2 and TM3) of AtHVA22d by fusion of the truncated domains with GFP (DTM1:GFP, DTM2:GFP, and DTM3:GFP) showed that TM2 was important for protein stability and correct subcellular localization of the protein in vivo [START_REF] Guo | An Abscisic Acid-Induced Protein, HVA22, Inhibits Gibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells[END_REF]. Even if the expression and localization of DTM1:GFP and DTM3:GFP were similar to those of the full length protein (AtHVA22d:GFP), the authors did not excluded the possibility that these two domains are also required for the protein function [START_REF] Guo | An Abscisic Acid-Induced Protein, HVA22, Inhibits Gibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells[END_REF]. Transgenic Arabidopsis containing HVA22d silenced by RNAi showed reduction of flower, pollen and seed development associated to enhanced autophagy (e.g. accumulation of vacuoles and cytoplasm invagination) [START_REF] Chen | Autophagy is enhanced and floral development is impaired in AtHVA22d RNA interference Arabidopsis[END_REF]. Similar result was observed in yeast and it has been suggested that HVA22 homologs play a negative role in autophagy, and may be part of a protective mechanism used to suppress unnecessary catabolism while anabolic activities are reduced, as observed in plant submitted to environmental stresses (e.g. high salinity, drought, low temperature) [START_REF] Chen | Autophagy is enhanced and floral development is impaired in AtHVA22d RNA interference Arabidopsis[END_REF].

Citrus sinensis HVA22 gene expression showed that CsHVA22a, CsHVA22c and CsHVA22d presented a high expression compared to the other genes (Fig. 5). The CsHVA22d gene was mainly expressed in fruits and callus (Fig. 5). In Arabidopsis, the HVA22 genes presented differential expression pattern according to plant tissues and to biotic stresses (e.g.

ABA, cold stress, dehydration and salt stress) [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF]. The AtHVA22a gene, which is phylogenetically close to CsHVA22c/CcHVA22c (Fig. 4), showed a constitutive high expression in all stress conditions as well as in most of the plant tissues analyzed (rosette leaves, leaves, inflorescence stems and flower buds) [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF]. Similar expression pattern was observed for CsHVA22c (Fig. 5). The AtHVA22b expression is specifically induced by ABA and suppressed by cold stress [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF], but in our analysis, this sequence did not clustered to any citrus sequence (Fig. 4). The AtHVA22c gene showed a constitutive expression in all stress conditions analyzed -similar but lower in intensity than AtHVA22a expression -and in stems and flower buds [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF]. AtHVA22c is phylogenetically isolated from the other genes (Fig. 4); the closest sequence was CsHVA22b, which showed a low expression in the different analyzed citrus tissues (Fig. 5). The AtHVA22d gene was highly expressed under ABA, cold stress, dehydration and salt stress, as well as in flower buds, while AtHVA22e was mainly expressed under cold stress, dehydration and salt stress, and in stems [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF]. AtHVA22d and AtHVA22e

were phylogenetically close to CsHVA22d/CcHVA22d (Fig. 4) and the CsHVA22d expression was high in callus and fruit (Fig. 5). The CsHVA22e/CcHVA22e and CsHVA22f/CcHVA22f sequences did not clustered to any Arabidopsis sequences in the phylogenetic analysis (Fig. 4); the CsHVA22e expression was low in all tissues (similar pattern to the CsHVA22b one; Fig. 5) and the CsHVA22f expression was not detected (data not shown). The phylogenetic and expression analyses of the citrus HVA sequences showed a separation of CsHVA22d/CcHVA22d (close to AtHVA22d and AtHVA22e) from the other citrus sequences (Fig. 4 and5). As discussed above, the CcHVA22b and CcHVA22c were shown to be duplications from the same ancestral, and this result may be correlated to the phylogenetic separation between the AtHVA22d/AtHVA22e group (also close to barley HVA22) from the AtHVA22a/AtHVA22b/AtHVA22c group, suggesting that these two groups had diverged before monocots/dicots divergence [START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF].

The regulation of gene expression depends of cis-elements present in the promoter sequences of each gene (Fig. 2, Supplementary material 5). The G-BOX element, present in almost all citrus HVA22 promoters, is known to be associated to the response to several environmental factors and/or internal stimuli such as light, anaerobiosis and hormones [START_REF] Shen | Modular nature of abscisic acid (ABA) response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in barley[END_REF]. In both citrus species, ABA responsiveness elements were found such as the ABRE element containing the ACGT motif, as also observed in barley HVA1 sequence [START_REF] Shen | Modular nature of abscisic acid (ABA) response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in barley[END_REF][START_REF] Velasco | Gene structure and expression analysis of the drought-and abscisic acid-responsive CDeT11-24 gene family from the resurrection plant Craterostigma plantagineum Hochst[END_REF]. In barley, the HVA1 promoter contained distal and proximal coupling elements (CEs), which have a synergistic effect on the absolute expression level as well as on ABA induction [START_REF] Shen | Modular nature of abscisic acid (ABA) response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in barley[END_REF]. These cis-elements were specifically recognized by the HvABI5 bZIP transcription factor, which together with another transcription factor, HvVP1, transactivated the promoter [START_REF] Casaretto | The transcription factors HvABI5 and HvVP1 are required for the abscisic acid induction of gene expression in barley aleurone cells[END_REF][START_REF] Casaretto | Transcriptional regulation by abscisic acid in barley (Hordeum vulgare L.) seeds involves autoregulation of the transcription factor HvABI5[END_REF]. The citrus HVA22 promoters contained cis-elements related to anaerobic induction, anoxic specific inducibility and drought inducibility (Fig. 2, Supplementary materials 4 and 5) that could be related to root function in stress situation such as flooding or, on the opposite, water loss.

Interestingly, it had been shown that the homolog of Arabidopsis HVA22 in yeast, Yop1, and the homolog of Arabidopsis root hair defective 3/RHD3 in yeast, SEY1, presented synthetic enhancement, and could be involved in vesicular traffic regulation in root stressed cells [START_REF] Brands | Function of a plant stress-induced gene, HVA22. Synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic[END_REF].

In barley and Arabidopsis, HVA22 genes showed differential expression under drought stress [START_REF] Shen | The stress-and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms[END_REF][START_REF] Chen | AtHVA22 gene family in Arabidopsis: phylogenetic relationship, ABA and stress regulation, and tissue-specific expression[END_REF], and here we showed that CcHVA22d expressed under constitutive promoter in transgenic tobacco plants conferred them dehydration tolerance (Fig. 6). In rice, the ABRC HVA1-promoter like motif (which contain the ABRE element and a CE) was found in genes related to biotic stress responses, disease resistance and defense such as NPR1 or WRKY transcription factors [START_REF] Ross | Computational Prediction and Experimental Verification of HVA1-like Abscisic Acid Responsive Promoters in Rice (Oryza sativa)[END_REF]. In citrus HVA22 promoters, several cis-elements were related to elicitor response or defense (Fig. 2, Supplementary materials 4 and 5), corroborating the idea that the plant response pathways to biotic and abiotic stresses significantly overlap, and that HVA1-like enhancer modules participate to a complex regulatory network signalized by ABA [START_REF] Ross | Computational Prediction and Experimental Verification of HVA1-like Abscisic Acid Responsive Promoters in Rice (Oryza sativa)[END_REF]. The citrus HVA22 promoter also contained cis-element related to plant development mainly seed regulation, endosperm expression and zein metabolism (Fig. 2) that may be correlated to the well-known function of HVA barley gene in seed maturation and dormancy [START_REF] Shen | The stress-and abscisic acid-induced barley gene HVA22: developmental regulation and homologues in diverse organisms[END_REF][START_REF] Guo | An Abscisic Acid-Induced Protein, HVA22, Inhibits Gibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells[END_REF]. The accumulation of HVA22 mRNA in aleurone layer during the late stage of seed maturation was associated to high levels of endogenous ABA required for dormancy [START_REF] Guo | An Abscisic Acid-Induced Protein, HVA22, Inhibits Gibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells[END_REF]. The citrus promoters also contained cis-elements responsive to gibberellin (Fig. 2). In barley, a treatment with gibberellic acid (GA) was responsible for dormancy break associated to decrease of HVA22 transcripts [START_REF] Shen | Hormone response complex in a novel abscisic acid and cycloheximideinducible barley gene[END_REF][START_REF] Guo | An Abscisic Acid-Induced Protein, HVA22, Inhibits Gibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells[END_REF]. It has also been shown that GA and ABA regulate vacuolation associated to PCD in barley aleurone cells, and that overexpression of HVA22 inhibited GA-induced PCD [START_REF] Guo | An Abscisic Acid-Induced Protein, HVA22, Inhibits Gibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells[END_REF]. Finally, the citrus HVA22 promoters contained cis-elements responsive to light (Fig. 2, Supplementary materials 4 and 5), similar to those observed in the promoter of the chalcone synthase, which is induced by light / UV light [START_REF] Shen | Functional dissection of an abscisic acid (ABA)-inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cis-acting element[END_REF].

According to its expression pattern (high expression in fruit and callus; Fig. 5) and to the presence of drought inducible cis-elements in its promoter (e.g. MYB; Fig. 2, Supplementary materials 4 and 5), CcHVA22d was selected for further analysis of its involvement in drought tolerance using Agrobacterium mediated-plant transformation (Fig. 6). Transgenic CcHVA22d tobacco lines showed lower dehydration rate associated to lower accumulation of H2O2 than WT plants (Fig. 6B and 6C) suggesting that CcHVA22d conferred a higher tolerance to drought and less suffered oxidative stress. This result also supports the involvement of citrus HVA22 genes in response to environmental stresses; more detailed phenotypic characterization of CcHVA22d transgenic plants is under way by our research team.

Conclusion

To our knowledge, this is the first citrus HVA22 family characterization. Six HVA22 genes were identified in each citrus species analyzed; orthology was observed between the C. clementina and C. sinensis genes. Phylogenetic and expression analyses of the citrus and Arabidopsis HVA22 sequences revealed that CsHVA22d sequence clustered with AtHVA22d and that both were expressed in reproductive organs (e.g. flowers, fruits). According to the cis-elements present in the citrus HVA22 promoters, the gene expression may be regulated by several internal and external factors. Other regulation levels were also predicted, such as alternative splicing and post-translational modifications including CK2 phosphorylation, which is involved in plant response to environmental stress. The citrus HVA22 proteins were transmembrane and contained YOP1/TB2/DP1/HVA22 region associated to regulation of vesicular traffic in stressed cells. Moreover, the CcHVA22d gene showed to be involved in dehydration tolerance and oxidative stress reduction.

  Stranded cDNA Synthesis kit according to the manufacturer instructions (Invitrogen), and the primers. CcHVA22d cDNA was cloned into the pCAMBIA2301 vector (Cambia) under the control of the Cauliflower mosaic virus (CaMV) 35S promoter (35S-P) and upstream the terminator (35S-T) sequences. The resulting vector contained the 35S-P::CcHVA22d::35S-T expression cassette as well as the neomycin phosphotransferase II (nptII) selectable marker gene and the beta-glucuronidase (GUS) uidA as reporter gene. During cloning, the CcHVA22d sequence was confirmed by sequencing on the ABI3130XL sequencer according to the Genetic Analyzer User Guide (Applied Biosystems). The Agrobacterium tumefaciens strain EHA 105 containing the pCambiaCcHVA22d vector was used to transform tobacco plants (Nicotiana tabacum cv.

Figure 1 .

 1 Figure 1. Structure of HVA22 genes from C. clementina and C. sinensis. The exons and the introns were represented in orange and in black, respectively. (*) indicated the alternative transcript of the HVA gene with the same name from the same species.

Figure 2 .

 2 Figure 2. Cis-elements present in the promoter region of citrus HVA genes. A) Cis-elements from the analysis of the C. clementina promoter. B) Cis-elements from the analysis of the C. sinensis promoter. The cis-elements were analyzed in the 1500 bp upstream from the translation start site using the plantCARE database.

Figure 3 .

 3 Figure 3. Conserved motifs in citrus HVA proteins obtained by the MEME program.Conserved amino acid sequence in the TB2/DP1/HVA22 region.

Figure 4 .

 4 Figure 4. Phylogenetic tree obtained with the HVA22 proteins of A. thaliana, C. sinensis and

Figure 5 .

 5 Figure 5. Heatmap of the CsHVA22 gene expression in different tissues. Pattern generated through R software.

Figure 6 .

 6 Figure 6. CcHVA22d transgenic tobacco plants analysis. A. GUS histochemical test. B. Dehydration rate analysis. (*) indicates that all the transgenic lines are statistically different from the WT plant. C. H2O2 detection using DAB-HCl. L1 to L5: transgenic CcHVA22d lines; WT: wild-type (non transgenic plants).

Acknowledgments

MDGF and RJSS were supported by the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB). JAC was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). FM received o Productivity grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This work was supported by CNPq Universal project (n°471742/2013-9) coordinated by FM. We thank Abelmon da Silva Gesteira (Embrapa CNPMF, Brazil) for providing Citrus clementina leaves. We thank the Núcleo de Biologia Computacional e Gestão de Informacões Biotecnologicas (NBCIGB) from UESC for providing the infrastructure for the bioinformatics analysis. This work was developed in the frame of the International Consortium in Advance Biology (CIBA; https://www.ciba-network.org/).

Abbreviations

ABA: abscisic acid; ER: endoplasmic reticulum; MW: molecular weight; ORF: Open reading frame; pI: isoelectric point; RPKM: Reads Per Kilobase Million.

Authors' contribution

MDGF executed all the analyses; MDGF, JAC and FM designed the analyses; MDGF, JAC and FM wrote the manuscript; JAC participated to the transgenic plant generation; RJSS participated of the phylogeny analysis; FM was responsible for the financial support of the research and for the advising of MDGF, JAC and RJSS.

Tables

Protein Phosphorylation sites N-glycosylation sites

CcHVA22a S4, S44, S64, S95, S105, S124, S130, S134, S179, S183, S185, T38, T96, T145, T157, T167 N155 CcHVA22b S24, S38, S43, S44, S77, S94, S124, S132, S134, S147, S202, S204, S205, S238, S252, S255, T26, T74, T79, T141, T150, T225, T236, T246, Y197, Y209, Y230, Y258, Y265 -CcHVA22c S7, S33, S41, S56, S76, S125, S156, S163, T39, T59, T64, T132, T143, T164, Y31, Y96, Y137, Y160, Y165, Y167, Y171 N141

CcHVA22c* S15, S23, S38, S58, S107, S138, S145, T21, T41, T46, T114, T125, T146, Y78, Y119, Y142, Y147, Y149, Y153 N112 CcHVA22d S13, S34, S36, S51, S72, S118, S119, T121, T123, Y144 -CcHVA22f S11, S26, T6, T38, Y1, Y15 -CcHVA22e S28, S34, S36, S51, S59, S110, S111, S131, S133, T45, T155, Y100, Y106, Y135 N112 CsHVA22a S4, S44, S64, S95, S105, S124, S130, S134, S179, S183, S185, T38, T96, T145, T157, T167 N155

CsHVA22a* S4, S44, S64, S95, S105, S124, T38, T96 -CsHVA22b S6, S36, S44, S46, S59, S114, S116, S117, S150, S167, T53, T62, T137, T148, T158, Y109, Y142, Y170, Y177 -CsHVA22b* S11, S19, S21, S34, S89, S91, S92, S125, S139, S142, T28, T37, T112, T123, T133, Y84, Y96, Y117, Y145, Y152 -CsHVA22c S7, S41, S56, S76, S125, S156, S163, T39, T59, T64, T132, T143, T164, Y31, Y96, Y137, Y160, Y165, Y167, Y171 N141 CsHVA22d S13, S34, S36, S51, S72, S118, S119, T121, T123, Y144 -CsHVA22d* S15, S17, S32, S53, S99, S100, T102, T104, Y125 -CsHVA22e S19, S25, S27, S78, S80, S81, S101, S103, T28, T36, T125, Y76, Y105 N82 CsHVA22f S11, S26, T6, T38, Y1 -


Minrui Xu

Frédéric Richard

Matthieu Corbet

Philippe Marion

Jean-Marc Clacens
email: jean-marc.clacens@univ-poitiers.fr

Upgrading of furfural by Knoevenagel condensation over functionalized carbonaceous basic catalysts

Keywords:

Commercial carbonaceous materials (Darco KB-G, Norit SX2 and Vulcan XC72) were functionalized with phenyl primary amine using diazonium coupling. The chemical grafting procedure yielded carbon-based basic catalysts, in which the introduced amine group was readily accessible to the reactants. These basic solids were characterized by different technics (elemental analysis, N2 adsorption-desorption, TGA, SEM, FT-IR and XPS). Basic solid Darco-0.5PDA exhibited high activity to the Knoevenagel condensation of furfural and malononitrile under mild experimental conditions (40 °C, Patm).

Introduction:

Homogeneous catalyst applications are usually limited in industrial chemical processes because massive wastes are produced and catalyst separation with raw materials and products is inconvenient during industrial production processes. As a result, the development of heterogeneous catalysts by immobilizing homogeneous catalysts onto solid supports is still a continuing need [1]. Various strategies were developed for immobilizing homogeneous catalysts onto inorganic supports and organic polymers supports [2,3]. Nevertheless, not only the tedious synthesis for the post immobilization process but also the low catalyst loadings and the resulting low efficiencies contribute to the high cost of supported catalysts [4].

Moreover, these catalysts often suffer from important active species leaching while they are used in liquid phase reactions [5]. To remedy the deficiency of supported catalysis, scientists have worked on direct functionalization of solid support via covalent bond building between the functionalities of the solid support and organocatalytic species. Among the approaches of functionalization being investigated, the chemistry of diazonium is becoming more and more attractive since this promising method enables to graft different organic moieties onto various solid supports such as carbon substrate, gold substrate or hydrogenated silicon in aqueous or organic media [6][7][8]. Compared to silica supports, carbonaceous materials generally exhibit good chemical stability while the Si-O bond of silica supports can easily be broken when acid or basic media are involved. Besides, activated charcoal based supports usually have very high specific surface area and strong adsorption capacity, which are crucial for a catalysis application. Diazonium cations can be formed by reacting an aromatic amine with sodium nitrite and a mineral acid in aqueous media [9,10], while they can also be generated in organic media using an alkyl nitrite like tert-butyl nitrite (t-BuNO) or iso-pentyl nitrite (i-PeNO) in a mixture of orthodichlorobenzene (ODCB) and acetonitrile (CH3CN) [11][12][13][14]. still under debate. Recently, Lipinska et al. proposed an overall reaction mechanism for multiwalled carbon nanotubes (MWCNT) in the presence of isopentyl nitrite [15]. The authors confirmed that the chemical reduction grafting procedure followed a radical chain mechanism, in which the reaction takes place through electron transfer from the carbonaceous material into the diazonium species with the loss of N2 and the formation of an aryl radical that reacts with the carbonaceous support to form a covalent bond [16].

This spontaneous surface modification via diazonium chemistry makes the functionalization of carbonaceous materials simple, flexible and efficient for various applications such as negative electrode for lithium-ion batteries [17] and biomedical detection agents [13,14].

Precisely, by using the chemical grafting procedure in aqueous media, Martin and Ramirez-Castro grafted aminophenyl groups (Ph-NH2) onto graphite flakes [17] and carbon black [18] respectively. Moreover, functionalized carbonaceous materials are wildly used in acid catalysis. Using this direct functionalization method, Stellwagen et al. grafted phenylsulfonic acid groups (Ph-SO3H) onto carbon nanofibers and these introduced acidic groups were efficient for the transesterification of triolein and methanol [19]. However, examples of functionalized carbon by diazonium grafting used in basic catalysis are still limited.

Knoevenagel condensation is one of the most valuable reactions for the C-C bond formation between a carbonyl group and an activated methylene group generally catalyzed by aminocatalysts [20] or solid bases [21] under mild experimental conditions (room temperature and atmospheric pressure). Recently, several catalysts were developed to perform this kind of reaction. Concerning homogeneous catalysts, List et al. designed cinchona-derived primary amine and reported the first example of asymmetric Knoevenagel condensation in DMSO at 20 °C [22]. Hilvert et al. carried out an enzymatic Knoevenagel condensation by using an artificial retro-aldolase [23]. When it comes to the design of heterogeneous catalysts, some recent works demonstrated that ionic liquids (ILs) and metal-organic frameworks (MOF) were also efficient for this reaction. Maschmeyer et al. showed that bromozincate based ionic allowed good yields (up to 88%) for the Knoevenagel condensation between benzaldehyde and malononitrile [24]. In addition, the use of porous zeolite imidazolate framework (ZIF-8) based catalysts in the Knoevenagel condensation between benzaldehyde and malononitrile in toluene at room temperature was already reported [25,26]. Nowadays, smart amine functionalized materials are drawing more and more attention in basic catalysis. For instance, Modak et al. designed a porphyrin based microporous organic polymer as heterogeneous catalyst [27]. This bi-functional solid contains both free porphyrin and Fe(III)-porphyrin framework which enabled to catalyze the Knoevenagel condensation of various aromatic with high yields (between 62 and 99 mol%). Furthermore, Mondal et al. managed to functionalize NH2 groups onto mesoporous silica through co-condensation of 3-aminopropyltriethoxysilane along with tetraethylorthosilicate [28]. In the Knoevenagel condensation of different aromatic aldehydes with malononitrile, this amino-functionalized basic catalyst exhibited both good activity and stability in catalytic runs. It is highly desirable to use the Knoevenagel reaction for the upgrading of furfural known as a platform molecule from lingo-cellulosic biomass [29]. A recent computational study revealed that the product of the Knoevenagel reaction between furfural and malononitrile, (2-furanylmethylene)malononitrile, noted as FMMN, was highly applicable for photovoltaic solar cell applications [30].

We report hereafter the functionalization of three commercial carbonaceous materials with phenyl primary amine moiety (Ph-NH2) and their application in basic catalysis through the Knoevenagel condensation between furfural and malononitrile.

Experimental part:

Catalyst synthesis

Commercially available carbon black (Vulcan XC-72) and activated charcoal (Darco KB-G and Norit SX2) obtained from Carbot Co. were dried for 16 h at 120 °C before use. The aryl amine precursor p-phenylenediamine (99%), denoted as PDA is purchased from Sigma-Aldrich and the isopentyl nitrite (97%) is purchased from Alfar Aesar. The diazonium coupling was performed according to Ramirez-Castro's procedure [18] with slight modifications. Typically, 0.5 g of p-phenylenediamine (4.62 mmol, 1 eq.) were dissolved in 100 mL of acetonitrile with vigorous stirring. 1 g of carbonaceous support is then dispersed in the solution followed by the dropwise addition of nitrile isopentyl (0.62 mL, 4.62 mmol, 1 eq.). The reaction mixture was stirred for 16 h at 25 °C and vacuum filtered on an EMD Millipore support with a nylon filtration membrane (0.22 µm, 47 mm, hydrophile). The functionalized carbon powder was washed by successive aliquots of acetonitrile (200 mL -7 times) until the filtrate is colourless. Finally the functionalized carbon was dried for 16h at 120 °C.

Catalyst characterizations

Available in ESI

Catalytic test procedure for the Knoevenagel reaction between furfural and malononitrile

The Knoevenagel condensation of furfural was carried out in a batch reactor fitted with a magnet stirrer and heated at 40 °C. The catalyst (200 mg) was introduced in a mixture composed by freshly distilled furfural (10 mmol, Sigma-Aldrich, 99%), malononitrile (10 mmol, Sigma-Aldrich, >99%), heptane (10 mmol, Sigma-Aldrich, 99%) and 2methyltetrahydrofuran (10 mL, 99%, Alfar Aesar) as solvent. Heptane, inert in these conditions, was used as an internal standard for quantitative analysis carried out by Gas Chromatography. Each liquid sample obtained after various reaction times (between 30 and 360 min) was analyzed by using a Varian 430 chromatograph equipped with CPSIL-5CB capillary column (length: 50 m; diameter: 0.25 mm, film thickness: 0.4 µm) and a flame ionization detector. The oven temperature was programmed from 70 °C to 150 °C (5 °C/min), then from 150 °C to 250 °C (10 °C/min) and maintained at 250 °C during 10 minutes. All detected products were identified by co-injection of commercial samples and/or by using Varian 3800 chromatograph coupled with a 1200 TQ mass spectrometer.

Results and discussion

Characterization of the catalysts

Spontaneous functionalization of three commercial carbonaceous supports (Darco, Norit and Vulcan) was carried out in two consecutive steps to have a maximal number of basic sites equal to 4.6 mmol g -1 (Scheme 1). The three functionalized solids are noted as Darco-0.5PDA, Norit-0.5PDA and Vulcan-0.5PDA where the number 0.5 represents the weight of pphenylenediamine involving in the synthesis per gram of carbonaceous support, depending on the type of support. As expected, all basic solids modified by this chemical grafting procedure possessed nitrogen in various weight content, depending on the support, as indicated in Table 1. The highest value was obtained in presence of Darco (5.14 wt% of nitrogen). From the amount of nitrogen content, the basicity of each solid (expressed in mmol g -1 ) can be estimated supposing that all nitrogen was in the form of -NH2 basic group. Thus, the Darco 0.5PDA sample exhibited the highest value of basicity, close to 3.7 mmol g -1 (Table 1). These observations suggested that these carbonaceous supports were successfully modified by the grafting procedure.

The shape of isotherms obtained from N2 adsorption of both carbonaceous supports and functionalized solids presented a type IV isotherm (Figure S1). The carbonaceous supports and their corresponding functionalized solids showed different specific surface areas and porous volume. For example, bare Darco exhibited the highest values for both textural characteristics (SBET = 1345 m 2 and Vp = 1.08 cm 3 g -1 ), which explained in part its higher nitrogen content, as reported in Table 1. Indeed, the specific surface area of Darco-0.5PDA was approximately 2.5 times higher than that of Norit-0.5PDA and more than 4 times higher than that of Vulcan-0.5PDA. Irrespective to the carbonaceous support, the functionalization by grafting -NH2 groups led to a decrease of both specific surface area and total pore volume but a slight increase of the pore diameter average (Table 1).

The grafting ratio of each functionalized solid can be calculated by dividing the number of basic sites to the theoretical maximal number of basic sites (4.6 mmol g -1 ). The highest value, close to 79%, was obtained when Darco was used as support. As highlighted in Figure 1, a linear correlation can be found between the grafting ratio of each functionalized solid and the specific surface area of each carbonaceous support. Darco presented the largest specific surface area and as a result its corresponding modified solid (Darco-0.5PDA) exhibited the highest grafting ratio compared to other functionalized solids. Consequently, it seems that the grafting ratio of the functionalized solid could be directly proportional to the specific surface area of its bare carbonaceous support before the surface saturation. This trend indicates that using a carbonaceous support, whose specific area is large enough to obtain a considerable grafting ratio, is favourable to maximize the number of catalytic basic sites.

Infrared analysis was carried out in order to confirm the presence of grafted Ph-NH2 moieties on the surface of functionalized solids. The IR spectra (in the region between 1000 and 1700 cm -1 ) of functionalized solids and bare support Darco) showed significant difference (Figure 2). As expected, characteristic IR bands of aniline were observed in the spectrum of Darco-0.5PDA. However, a slight shift towards higher values, especially for the band at 1496 cm -1 (aromatic C=C stretching vibration), can be attributed to a modification of the chemical environment of aromatic ring due to its grafting onto Darco [START_REF] Larkin | Infrared and Raman Spectroscopy[END_REF]. The C-N stretch of aromatic primary amine is expected to be observed between 1350 and 1250 cm -1 and its position is commonly used to distinguish between saturated and aromatic amines [START_REF] Smith | Infrared Spectral Interpretation: A Systematic Approach[END_REF]. The band at 1267 cm -1 can be hence assigned to the aromatic amine C-N stretching vibration. In addition, the bands at 1616 and 1598 cm -1 in the IR spectra of aniline can be attributed to the scissoring vibration of the primary amine NH2 group. On the contrary, for both Norit-0.5PDA and Vulcan-0.5PDA samples, negligible differences were showed on the IR spectra, probably due to the lower amount of grafted species (Figure S3). The X-ray photoelectron spectroscopy (XPS) analysis confirmed that Ph-NH2 molecules were successfully grafted onto the carbonaceous supports. Indeed, the XPS spectra of the three functionalized solids showed a single N 1s peak at 399.9 eV, which is attributed to the -NH2 groups presented in the functionalized solids (Figure 3) [9]. Obviously, this peak was absent on the unmodified Darco (Figure S4). The C 1s core-level spectra of all carbonaceous supports and functionalized solids showed a strong graphite component at around 284.3 eV (Figure S5) [9]. Other peaks at higher binding energies (between 286 and 292 eV) were attributed to the presence of surface oxides components [10]. The peak at 285.9 eV, relatively intense in the case of Darco-0.5PDA, can be attributed to the presence of C-N species. These observations indicated clearly that the functionalization led to the formation of new C-N bonds.

SEM images of Darco showed that the surface of this carbonaceous support was heterogeneous. Both smooth and rough parts could be observed on the surface. The chemical surface modification by grafting Ph-NH2 moieties on the support seemed not to change its morphology as the surface of modified Darco-0.5PDA remained heterogeneous (Figure S6).

The presence of nitrogen detected by the ESD detector (Energy Dispersive X-ray Spectroscopy) was found in both smooth and rough parts on the surface of Darco-0.5PDA and this interesting result indicated that grafted basic molecules were well dispersed randomly on the total surface of carbonaceous support (Figure S6).

The thermogravimetric analysis (TGA) of Darco and Darco-0.50PDA illustrated two weight losses at 449-475 °C and 497-498 °C corresponding to the carbonaceous support material combustion (Figure S7). Another weight loss at 438 °C was only observed on Darco-0.5PDA, and can be attributed to the grafted species combustion. Assuming that the decomposed grafted specie is amino phenyl, the 22.8% weight lost corresponds to 2.45 mmol g -1 ; which is in fair good agreement with the calculated value of basicity deduced from nitrogen elemental analysis (Table 1).

Catalytic properties of functionalized solids

The catalytic properties of functionalized solids were evaluated in the Knoevenagel condensation. This reaction involves both furfural and malononitrile as reactants; (2furanylmethylene)malononitrile (FMMN) is expected as the only organic product (Scheme 2).

The reaction was carried out under mild conditions (40 °C, under atmospheric pressure) during 6 hours using 200 mg of basic solid.

A kinetic study was performed for each functionalized solid. The yield in FMMN (the only product observed in the reaction mixture) was spotted in function of the reaction time (Figure 4), showing that each basic solid was active in this reaction. Mass balances were accurate to within 5% in all catalytic tests. Apparently, Vulcan-0.5PDA was rapidly deactivated since the product yield remained practically constant (about 14 mol %) after 30 minutes. On the contrary, in the case of Darco-0.5PDA and Norit-0.5PDA, the yield of FMMN increased continuously with the reaction time. As the basicity of Darco-0.5PDA was higher than Norit-0.5PDA (Table 1), the former exhibited the highest activity for Knoevenagel reaction between furfural and malononitrile. It is important to notice that the yield of FMMN could be nearly doubled when Norit-0.5PDA was replaced by Darco-0.5PDA with the same quantity of catalyst (200 mg).

In order to evaluate the bonding strength of the grafted species on Darco, an additional experiment was carried out. Firstly, the Darco-0.5PDA sample (200 mg) was added in the reaction mixture and the composition of the reaction mixture was followed during one hour, the yield in FMMN equal to 15 mol% was very close to the yield already obtained for the same catalyst under same experimental conditions. After that, the catalyst was eliminated by filtration, the constant yield in FMMN during the remaining reaction time indicated that no catalytic species leached out during the reaction as shown in Figure 4 (red dotted line).

It was also checked that using 0.45 mmol of aniline as catalyst yielded 20 mol% of FMMN after 6 hours. It proved that any leaching of catalytic species could lead to an increasing yield.

It is very interesting to remark that the functionalized Darco-0.5PDA overcome the phenomenon of leaching which is a common disadvantage of carbon based catalysts [19,[START_REF] Mo | [END_REF]34]. The recycling and reuse of Darco-0.5PDA was further performed in four consecutive runs at 40 °C under atmospheric pressure. After each run, the catalyst was filtered, washed by the reaction solvent (2-MTHF) and dried at 100 °C for 4 hours. Darco-0.5PDA was then reused without any other purification steps. After each run, a strong decrease of the yield of FMMN was observed indicating that a strong deactivation of Darco-0.5PDA occurred (Figure S8). This phenomenon could be explained by a strong adsorption of reactants or product on active sites. As an evidence, further investigations are needed to understand the deactivation mode and thus to propose a reactivation procedure allowing to restore the catalytic properties of Darco-0.5PDA.

Conclusion

Aromatic primary amine moieties were successfully grafted onto three carbonaceous solid supports (Darco, Vulcan and Norit) by diazonium coupling under mild conditions. Among these 3 solid supports, Darco having the highest specific surface area allowed to obtain the best grafting ratio of Ph-NH2 groups equal to 79%. These three functionalized solids were all active in the model reaction but with different activity. Darco-0.5PDA was the most active catalyst owing to its highest basicity. Under our experimental conditions, the leaching of grafted species was not observed. This is the first example of diazonium modified carbonaceous materials used as basic catalyst.

Figure captions

Fig. 1 .Fig. 3 .Fig. 4 .Fig. 2 . 22 Fig. 3 .

 1342223 Fig. 1. Correlation between the grafting ratio of the functionalized solids and the specific

Acknowledgements

Minrui Xu is grateful to Solvay and ANRT (Agence National de Recherche et de Technologie) for the financial support of a CIFRE PhD grant. Jean-Marc Clacens and Frédéric Richard acknowledge financial support from the European Union (ERDF) and "Région Nouvelle Aquitaine". Julie Rousseau is acknowledged for SEM experiments and Christine Canaff is acknowledged for XPS experiments. Fig.4. Kinetic profiles of Knoevenagel condensation of furfural catalyzed by amino functionalized solids and leaching evaluation for Darco-0.5PDA 24 Scheme 1.

Schematic reaction pathway for grafting aminophenyl groups to carbonaceous materials [8]

b: basicity calculated from elemental analysis c: specific surface area calculated by the BET method.

d: total pore volume determined at P/P0 = 0.99 e: average pore diameter deduced from the BJH method using the desorption branch Scheme 2.

Knoevenagel condensation of furfural with malononitrile