Brigitte Picard

Florence Lefèvre

Bénédicte Lebret

Meat and fish flesh quality improvement with proteomic applications

Keywords: biomarkers, sensory quality, skeletal muscle, technological quality, texture

HAL is

Introduction

Thanks to the sequencing of genomes, it is now possible to decode and locate many thousands of genes in a genome. This available information can be used for purposes of knowledge, diagnosis, or selection. The development of tools that accompanied this progress allows for the simultaneous analysis of thousands of genes (DNA chips), transcripts (transcriptomics), and proteins (proteomics; Figure 1).

The emergence of these functional genomic technologies enables the measurement of complex phenotypes, such as traits related to the quality of food products. Because meat quality traits result from genetic and environment interactions, working at the gene or protein expression level allows scientists to take both the genetic potential of animals and its modulation by the environment into account. Thus, biomarkers of gene expression appear more accurate than genetic markers to better understand biological processes determining phenotypes, and explain and predict meat quality variations [START_REF] Te Pas | Biomarkers as management tools for industries in the pork production chain[END_REF]. The principle is to identify genes and/or proteins whose expression or abundance is associated to the value of a phenotypic trait of interest, such as the quality of a product. These genes or proteins are thus considered as biomarkers that could be used to predict a given phenotype. In this review, we will focus on studies undertaken at the protein level, because proteins have the advantage to represent the fi nal result of a complex gene expression system where different isoforms may exist although corresponding to a single gene because of post transcriptional modifi cations. Moreover, proteins are thought to be the main effector of some quality variables of great interest such as fi rmness. The signifi cant interest of proteomic approaches in the fi eld of animal and meat science to improve knowledge on biological mechanisms determining phenotypes and identify biomarkers of traits of interest has been highlighted in recent reviews [START_REF] Hollung | Application of proteomics to understand the molecular mechanisms behind meat quality[END_REF][START_REF] Bendixen | Farm animal proteomics: A review[END_REF].

In this review we will focus on the recent proteomic studies conducted in relation with meat quality determination, evaluation, and improvement, in cattle, pig, and fi sh species by French INRA groups. In these three models, proteomics has been used to study specifi c questions according to each production sector.

Beef Quality: From Biomarkers to Phenotyping Tools

Although several biochemical factors are well known and a number of quantitative trait loci (QTL) have been determined, control of the variability in beef tenderness remains a major challenge for the beef industry. Beef tenderness presents a strong and uncontrolled variability that induces a consumer's dissatisfaction and partly explains the decrease in beef consumption. Moreover, this quality can be assessed only after slaughter by mechanical measurements such as the Warner-Bratzler test or by a sensory analysis panel. There is no technique for measuring tenderness on the living animal. So, the beef industry is waiting for tools to estimate the potential of tenderness from the live animal or carcass (Figure 2).

The strategy developed over the past 10 years has been 1) to search for biomarkers of tenderness by comparing extreme groups of animals on this criterion with genomic tools; 2) to validate the relationships between these markers and tenderness on large numbers of animals; 3) to precisely defi ne the infl uence of management factors on the expression of these

Implications

• Proteomics technologies have been used to better understand the development of complex phenotypic traits such as meat and fi sh fl esh quality and identify biomarkers of these quality traits.

• Many protein biomarkers of beef tenderness have been highlighted and are currently under validation on different muscle and animal types. Biomarkers of fi sh fl esh fi rmness, and of many sensory (tenderness) and technological (drip loss and pale, soft, and exudative defect) pork quality traits, have also been identifi ed.

• Expected outcomes are to provide control tools of meat quality evaluation usable in the livestock sector and meat industries in the near future. biomarkers; 4) to further analyze the biological functions involved in the development of tenderness; and fi nally 5) to use this knowledge to build tools used by industry to estimate the quality of an animal before or after slaughter.

Use of Genomic Tools for Identifying Biomarkers of Tenderness: Comparative Proteomics

Over the years, several proteomic analyses were performed in specifi c programs to better understand the mechanisms involved in tenderness or to provide biomarkers that can predict it. For that, the strategy has been to compare extreme groups of beef tenderness by proteomics [START_REF] Picard | Skeletal muscle proteomics in livestock production[END_REF](Picard et al., , 2011) ) and/or transcriptomics [START_REF] Bernard | New indicators of beef sensory quality revealed by expression of specifi c genes[END_REF]. For comparative proteomics, the proteins of muscles from two groups (very tender and not tender) were extracted and separated according to their isoelectric point by two-dimensional electrophoresis. The differences in spot volumes were analyzed by image analysis. Then the protein corresponding to the signifi cant differential spots were identifi ed by mass spectrometry (Figure 1). These studies established a list of biological markers of beef tenderness. The main results obtained in the Longissimus thoracis (LT) have demonstrated that fast glycolytic type proteins were more abundant in animals giving the less tender meat. Among these proteins, we identifi ed: Phosphoglucomutase (PGM), lactate dehydrogenase B (LDHB), and Triphosphate isomerase in Charolais and Salers, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the Limousin, the fast troponin T (TnTf) isoforms in Charolais and Blond d'Aquitaine, and the β Enolase in Limousin and Blond d'Aquitaine breeds. Additional experiments led to similar conclusions about the positive relationships between oxidative metabolism and tenderness [START_REF] Picard | Skeletal muscle proteomics in livestock production[END_REF]. Similar results have been observed for pigs. For example, D ' Alessandro et al. (2011) reported PGM was more abundant in the muscle of pigs that produced more tender meat. Several proteins involved in calcium metabolism were also identifi ed as positive markers for tenderness. For example, the amount of Parvalbumin peptides is considerably increased in tender muscles of Charolais and Limousin breeds. The calcium cycle proteins seem strongly involved in meat tenderness, in connection with the important role of calcium in meat ageing [START_REF] Ouali | Revisiting the conversion of muscle into meat and the underlying mechanisms[END_REF]. Accordingly, [START_REF] Bjarnadottir | Changes in protein abundance between tender and tough meat from bovine longissimus thoracis muscle assessed by iTRAQ and 2DE analysis[END_REF] recently found a relationship between Annexin 6, involved in the release of cal-October 2012, Vol. 2, No. 4 cium, and tenderness. A set of proteins of the family of heat shock proteins (Hsp) was revealed as markers of tenderness at the transcript or protein level in the different experiments. For example, [START_REF] Bernard | New indicators of beef sensory quality revealed by expression of specifi c genes[END_REF] have shown that gene expression of the DNAJA1 protein (protein Hsp40) was an appropriate indicator of meat toughness of Charolais young bulls. According to the hypothesis of [START_REF] Ouali | Revisiting the conversion of muscle into meat and the underlying mechanisms[END_REF], the anti-apoptosis DNA-JA1 could slow down the process of cell death during the early stages of transformation of muscle into meat. Other family members were also identifi ed as markers of tenderness in several programs (Hsp27, Hsp20, αB-crystallin, Hsp70). Proteins involved in oxidative stress, such as superoxide dismutase (SOD1) or Peroredoxin 6 (PRDX6), were found to be negatively related to tenderness. This is in contradiction with the data of D' Alessandro et al. (2011), which shows that SOD1 was more abundant in pork that was more tender and apparently had more protein degradation. A similar study conducted on Semitendinosus (ST) muscle showed differences in the identifi ed markers according to muscle type [START_REF] Guillemin | Different phenotypic and proteomic markers explain variability of beef tenderness across muscles[END_REF]; breed-specifi cities for proteomic markers of tenderness were also reported [START_REF] Chaze | Beef tenderness markers in the three main French beef breeds: Proteomic analysis on young bulls from the Qualvigene French program[END_REF].

Validation of Biomarkers: Prediction of Tenderness

Comparative analyzes to identify biomarkers were conducted primarily in young bulls of different breeds. Currently, we want to validate the relationships between markers abundance and tenderness level over large numbers of cattle in French systems with cows, heifers, steers, bulls or beef, hardy breeds, and dairy breeds. In order to simultaneously analyze different biomarkers for several muscle samples, we developed the Dot-Blot technique [START_REF] Guillemin | Validation of a Dot-Blot quantitative technique for largescale analysis of beef tenderness biomarkers[END_REF]. It consists of depositing the protein extracts on a membrane (PVDF) and hybridizing the membrane with an antibody specifi c for the desired protein. Dot-Blot was used on 111 samples from LT and ST muscles from the Charolais bovine breed to quantify 24 proteins previously identifi ed as biomarkers of tenderness by comparative proteomics [START_REF] Guillemin | Different phenotypic and proteomic markers explain variability of beef tenderness across muscles[END_REF]. The main results showed that biomarkers better discriminated tenderness (evaluated by sensory or mechanical analysis) in ST than in LT muscle. This could be the consequence of different compositions of these muscles in characteristics such as total lipid content (higher in LT) or collagen content which are also involved in tenderness. Multiple regressions highlighted PRDX6 (cis-peroxiredox-6), LDHB (lactate dehydrogenase B), Hsp70-1B, Hsp70-GRP75 (Heat Shock Protein), and MyHC (Myosin Heavy Chain) II (IIa + IIx) as proteins explicative of ST tenderness (WBSF; R 2 =0,86). In LT muscle, PRDX6, Hsp20, Hsp70-GRP75, and αB Crystallin (CRY-AB) were the most explicative of tenderness (R 2 =0.69; [START_REF] Guillemin | Different phenotypic and proteomic markers explain variability of beef tenderness across muscles[END_REF]. These predictive equations revealed that the PRDX6 protein is the main biomarker explaining the WBSF in both muscles (P=0.003). This enzyme is involved in the fi ght against oxidative stress which is caused by free radicals of oxygen, resulting in the formation of protein aggregates that impair tenderness [START_REF] Morzel | Muscle proteome and meat eating qualities of Longissimus thoracis of "Blonde d'Aquitaine" young bulls: A central role of HSP27 isoforms[END_REF]. Proteins of small Hsp family (Hsp27, Hsp20, and CRY-AB) are known to prevent the formation of these aggregates. Thus, the positive relationship between these Hsp and tenderness is quite concordant with the negative relationship between oxidative stress (PRDX6) and tenderness. We confi rm that Hsp is important in tenderness determination, with Hsp70 as negative markers, in contrast to Hsp20 [START_REF] Guillemin | Different phenotypic and proteomic markers explain variability of beef tenderness across muscles[END_REF]. Indeed, Hsp70 also sequester proapoptotic factors such as BCL-2 and inhibit apoptosis [START_REF] Beere | Stress management: Heat shock protein-70 and the regulation of apoptosis[END_REF]. These proteins also have chaperone functions, but not on protein structure. This can explain why they are negative markers of tenderness in ST muscle, in contrast to Hsp20. Indeed, [START_REF] Guillemin | Different phenotypic and proteomic markers explain variability of beef tenderness across muscles[END_REF] have shown that the ratio of the family of small Hsps/the family of Hsps70 appeared to explain variability of beef tenderness.

Influence of Management Factors on the Expression of Biomarkers of Tenderness

Our objective was to determine the precise effect breeding systems have on the abundance of protein biomarkers. First, a thorough analysis of the regulation of gene expression of DNAJA1 (coding protein Hsp40) was performed to show how its expression may vary according to muscle type and animal behavior. The main results showed that the greatest abundance of Hsp40 was observed in the youngest animal and the most oxidative muscles. No effect was detected for dietary treatment (pasture vs. maize based diet) or growth path (compensatory growth after a restriction period). Moreover, its abundance was not modifi ed by pre-slaughter stress [START_REF] Cassar-Malek | Expression of DNAJA1 in bovine muscles according to developmental age and management factors[END_REF].

20

Animal Frontiers

The effect of muscle type (LT vs. ST) and animal type (bulls vs. steers) on the abundance of a list of 24 proteins was studied in Charolais cattle (67 young bulls and 44 steers). We detected muscle type effect on 14 of the 24 analyzed proteins and an animal effect on 15 of the 24 proteins. The main results showed that the family of small Hsp (27, 20, αB cristallin) varied according to these two factors. They are more abundant in young bulls than in steers and in LT than in ST muscle. On the contrary, Hsp70/ GRP75 was more abundant in steers and showed no effect of muscle type. Proteins of calcium dependent proteolysis (M and μ-calpains) were more abundant in steers with no effect of muscle type. Contractile and metabolic proteins were more variable according to muscle than animal type (Guillemin et al., 2011a). This approach is being completed for various biomarkers and management factors. Altogether, this data will help provide advice on farming practices that could allow for optimal expression of tenderness biomarkers.

Biological Functions Governing Beef Tenderness

From the list of 24 proteins, we used bioinformatic tools to search for proteins interacting with other proteins identifi ed as markers of tenderness (Guillemin et al., 2011b). This work highlighted cellular pathways strongly involved in the tenderization processes: apoptosis, Hsp functions, and oxidative stress resistance. We also demonstrated that the role of these pathways on the tenderization process differs according to muscle type. Moreover, this analysis revealed new data. For example, three proteins, never studied in tenderness, appeared to be at a crossroad of the tenderness interactome: SUMO4, H2AFX, and TP53. Direct relationships with tenderness is improbable. However, these proteins could be responsible for the balance between pathways, like apoptosis or stress response. So, studying their relationship with tenderness could complete our knowledge on tenderness and help to better explain tenderness variability.

Development of Analytical Tools

From these data, the goal is to develop a routine application for the beef sector. The principle is the opposite approach of the Dot-Blot technique. It consists in depositing the interested antibodies on the membrane and hybridizing with the protein extract. This technology is called "antibody chip". This technology is already used for muscle proteins with medical applications in disease diagnostics [START_REF] Sakanyan | High-throughput and multiplexed protein array technology: Protein-DNA and protein-protein interactions[END_REF]. Our challenge is to develop this technology for phenotyping bovine for tenderness. The advantages are that it is a fast, high-sensitive immunological technique that enables the simultaneous quantifi cation of several proteins in samples obtained by biopsy live animals or carcasses. The expected outcomes are to provide the beef sector with a tool for "paddock" use to estimate the tenderness potential of a live animal or a meat cut. If we succeed in this challenge, this tool will improve the competitiveness of this industry by allowing it to provide the consumers with controlled quality beef.

Pig Meat Quality: Understanding the Development and Identifying Biomarkers of Complex Phenotypic Traits

Pork is the predominant meat consumed in the world (Figure 3), either as fresh meat or processed products, exhibiting a great diversity in both the type of raw material (entire parts [hams] or minced meat [sausages]) and processing techniques used (cooking or dry-curing). Therefore, pork quality covers technological and sensory dimensions, including many traits like post-mortem (p.m.) pH, drip loss, color, intramuscular fat (IMF) content, tenderness, juiciness, and fl avor. These pork quality traits result from interactions between genetic backgrounds, rearing and slaughtering conditions of animals, and meat processing. Even though many factors infl uencing pork quality have been identifi ed, its variability remains high and muscle properties underlying good eating quality are still unclear. Thus, like for other animal species, identifying markers of quality traits is of signifi cant interest in the pork industry. As a result, applications of proteomic approaches is increasing, generally based on two-dimensional electrophoresis and mass spectrometry for protein identifi cation, in order to improve knowledge on biological mechanisms determining pork quality and identifying biomarkers of phenotypes. Studies consider either one important quality trait (e.g., IMF, color, shear force) in a differential animal design for a given trait, or many traits simultaneously (e.g., technological and sensory traits) using an experimental design leading to a range of variation for these traits. Most of the work concerns the Longissimus (LM), but the Semimembranosus (SM, ham muscle) has also been considered in relationships with the importance of cooked and dry-cured hams production.

Differential Proteomic Profiles Associated to Contrasted Levels for Quality Traits

Traits related to both technological and sensory qualities of pork [i.e., IMF content; pale, soft, and exudative (PSE) meat defect; or shear force] have been studied using comparative proteomics approaches to improve biological knowledge and identify potential biomarkers.

The IMF content is an important component of pork quality, and is highly variable among pig populations. To better understand its biological determinism and thereby help the design of genetic schemes for increasing IMF, contrasted groups for LM IMF content (1.36 vs. 4.58%) were compared by proteomic analyses [START_REF] Liu | Differentially-expressed genes in pig Longissimus muscles with contrasting levels of fat, as identifi ed by combined transcriptomic, reverse transcription PCR, and proteomic analyses[END_REF]. The expression of proteins of glucose and protein metabolic processes, cell communication, metabolites binding, and the response to stimulus functional categories were associated with IMF variation. Associated to transcriptomic data obtained on the same animals at similar and earlier age, these results indicate that variability in pig IMF content might arise essentially from differences in early adipogenesis and adipocyte development, thereby revealing biological processes to be considered for further studies on IMF control.

Pale, soft, and exudative meat, a major problem regarding pork quality, actually corresponds to various defects depending on both genetic and non-genetic factors, and originates from various physiological and biochemical mechanisms. In the SM, PSE zones result in low cohesiveness of meat that becomes unsuitable for cooked ham production, leading to important problems in pork industry. Using proteomic analyses, [START_REF] Laville | Characterisation of PSE zones in semimembranosus pig muscle[END_REF] showed a decrease in protein solubility and p.m. proteolysis of myofi brillar proteins, and lower quantities of small Hsp (Hsp 27, CRY-AB) in the PSE zones compared with normal SM. The reduced protein solubility and abundance of Hsp 27 and proteins of oxidative metabolism were also observed in the SM of pigs of nn genotype at the RyR1 locus (halothane gene) that leads to the genetic PSE defect, compared with NN pigs [START_REF] Laville | Differences in pig muscle proteome according to HAL genotype: Implications for meat quality defects[END_REF], as well as in pigs exhibiting pale vs. dark color in the SM (61.3 vs. 43.2 L* value;[START_REF] Sayd | Proteome analysis of the sarcoplasmic fraction of pig semimembranosus muscle: implications on meat color development[END_REF]. These studies open the way to markers of PSE defect by quantifi cation of chaperones proteins like small Hsp. In addition to the quantifi cation of protein expression, proteomics has been used recently to quantify protein phosphorylation changes in p.m. porcine muscle in relation with pork quality. [START_REF] Huang | Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences[END_REF] reported that fast pH decline muscles exhibited the greatest phosphorylation level (1 hour p.m.) and the least (24 hours p.m.), whereas slow pH muscles showed the reverse case. Studying another important defect of pork, the acid meat resulting from Rendement Napole-(RN-) genotype, Lametsch et al. (2011) reported greater phosphorylation levels of key enzymes of glycogenolysis and glycolysis during p.m. metabolism in RN-compared with wild-type pigs. This illustrates that proteomic approaches are relevant to characterize post-translational modifi cations of proteins, like phosphorylation levels that could be interpreted as metabolic fi ngerprints related to biological processes determining phenotypic traits such as meat quality.

Aimed at understanding the development of pork tenderness and identifying potential markers, [START_REF] Laville | Comparison of sarcoplasmic proteomes between two groups of pig muscles selected for shear force of cooked meat[END_REF] compared two contrasted groups for LM WBSF of cooked meat. They showed that low WBSF group (i.e. tender meat) exhibited an overabundance of proteins of lipid metabolism, including adipocyte-fatty acid binding protein (FABP4), thus suggesting greater number of intramuscular adipocytes in these muscles. A greater FABP4 protein level was associated with greater IMF content in agreement with [START_REF] Damon | Number of intramuscular adipocytes and fatty acid binding protein-4 content are signifi cant indicators of intramuscular fat level in crossbred Large White X Duroc pigs[END_REF], and could explain the increased tenderness. [START_REF] Laville | Comparison of sarcoplasmic proteomes between two groups of pig muscles selected for shear force of cooked meat[END_REF] also reported a greater abundance of proteins involved in folding and polymerization, indicating increased protein synthesis in low WBSF group.

Associations between Proteomic Profiles and Variations in Technological or Sensory Traits for Identification of Biomarkers

Experimental designs are aimed at associating between, or within, breed or rearing condition variations in pork quality and the muscle proteomic profi le in order to understand underlying biological processes and identify biomarkers of quality. In an experiment associating two contrasted pure breeds (local Basque, corresponding to premium quality products, and conventional Large White) reared in various production systems (conventional, alternative, or extensive), associations between biochemical, physico-chemical, and sensory traits, and transcriptomic and proteomic profi les of LM revealed biological mechanisms and metabolites, transcripts, and proteins associated to the variations of many pork traits [START_REF] Salmi | Multivariate analysis to compare pig meat quality traits according to breed and rearing system[END_REF][START_REF] Lebret | Towards the identifi cation of molecular markers of pork quality[END_REF][START_REF] Damon | Comparison of muscle transcriptome between pigs with divergent meat quality phenotypes identifi es genes related to muscle metabolism and structure[END_REF]. As an example, sarcoplasmic proteome analyses revealed that protein oxidation generated during meat ageing and cooking, which might impair tenderness, water holding capacity, and technological properties of raw meat, relied on proteins involved in antioxidant protection (selenium binding protein and mitochondrial superoxide dismutase; SOD) and on iron containing proteins (myoglobin isoforms and serotransferrin; [START_REF] Promeyrat | Early post-mortem sarcoplasmic proteome of porcine muscle related to protein oxidation[END_REF]. Proteins of antioxidant pathways were negatively associated with drip and cooking losses and positively associated with tenderness, whereas opposite associations were found between proteins of energy metabolism and these traits [START_REF] Sayd | Use of sparse PLS method for the integration of proteomic and phenotypic data related to pork quality[END_REF]. In agreement, greater levels of antioxidant enzymes such as SOD1 were found in tender meat from Casertana pigs compared with tougher meat from Large White pigs [START_REF] Marrocco | Meat quality of the longissimus lumborum muscle of Casertana and Large White pigs: Metabolomics and proteomics intertwined[END_REF]. Combining proteomics and transcriptomics results, these authors hypothesized that antioxidant enzymes could play a role in protecting the proteolytic enzymes cathepsines and calpains during 22 Animal Frontiers p.m. proteolysis, thereby enhancing meat tenderization in Casertana pigs. In addition to studies performed on samples taken early p.m., proteome degradation during meat ageing in relation with technological and sensory pork quality traits has also been investigated by proteomics approaches [START_REF] Lametsch | Identifi cation of protein degradation during post-mortem storage of pig meat[END_REF][START_REF] Te Pas | Postmortem proteome degradation profi les of longissimus muscle in Yorkshire and Duroc pigs and their relationships with pork quality traits[END_REF]. Post-mortem proteolysis of actin and metabolic enzymes has thus been demonstrated [START_REF] Lametsch | Identifi cation of protein degradation during post-mortem storage of pig meat[END_REF], providing new insights on the biochemical phenomena occurring during meat ageing and tenderization. Altogether, these results improve knowledge on pork quality variation and the protein targets identifi ed can be considered for further development of biomarkers of quality. However, validation of potential markers for use in various breeds or pork chains may be a diffi cult task. When associating LM protein abundance and pork quality variations using multiple regressions analyses, [START_REF] Kwasiborski | Pig Longissimus lumborum proteome: Part II. Relationships between protein content and meat quality[END_REF] found that the abundance of 1 or 2 proteins could explain up to 85% of variability of traits like ultimate pH (muscular creatine kinase and dimericdihydrodiol dehydrogenase, 83%), drip loss (pyruvate kinase isoform M1, 65%), or thawing loss (actin interactin protein, 85%). Nevertheless, protein-trait associations displayed signifi cant gender and breed differences, indicating that the identifi cation of 'robust' (i.e., generic) proteomic markers of pork traits for wide use in pork industries deserves further research.

Fish Quality: Towards a Better Understanding of Flesh Texture

In fi sh, fl esh quality is dependent on environmental factors, mainly water and food quality for product safety and food composition for fl esh nutritional quality. Nevertheless, amongst sensory quality, fl esh texture is mainly determined by biological factors such as muscle organization, protein content, and composition. In fi sh, the best quality is fi rm and cohesive fl esh with good water holding capacity. These traits are mainly determined by proteins' nature and properties, so proteomic tools appear especially of interest to study fi sh fl esh quality. However, very few studies were undertaken to identify fl esh quality biomarkers, probably due to the small number of research teams working in that topic.

Post-mortem Changes

The fi rst studies analyzed muscle proteome p.m. changes in relation to fl esh softening. Cod muscle proteome observed in 2D-PAGE during 8 days p.m. revealed a limited degradation compared to mammal muscles, with 9 spots in which intensity increased and 2 spots in which intensity decreased [START_REF] Kjaersgard | Proteome analysis elucidating post-mortem changes in cod (Gadus morhua) muscle proteins[END_REF]. In this study, protein identifi cation was not reported and very little genomic data are available illustrating the limitations of proteomic approaches for species in which the genome was not sequenced. More recently, the evolution of rainbow trout muscle protein was studied during 5 days p.m. by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel band intensity was found to be related to fl esh fi rmness. Proteins from both myofi brillar [i.e., α-actinin, actin, Myosin Light Chain (MyLC) 1 and 2, MyHC fragment] and sarcoplasmic (creatine kinase, glycogen phosphorylase, triose phosphate isomerase) fractions closely correlated with fi rmness (i.e., [START_REF] Godiksen | Contribution of cathepsins B, L and D to muscle protein profi les correlated with texture in rainbow trout (Oncorhynchus mykiss)[END_REF]. In these cases, proteomic approaches allowed scientists to explore new targets of post-mortem proteolysis and to study the possible implications of the different proteolytic systems in fl esh quality.

Pre-slaughter Stress Effect

Pre-slaughter stress is diffi cult to avoid in aquatic species because of fi shing, and it was shown that even a short time stress of 15 min crowding (high density stocking) impairs product quality [START_REF] Lefèvre | Rearing oxygen level and slaughter stress effects on rainbow trout fl esh quality[END_REF]. Some studies reported changes in muscle proteome analyzed by twodimensional polyacrylamide gel electrophoresis. Pre-slaughter stress associated with intense muscle activity was shown to affect both proteins involved in muscle metabolism (i.e., triose phosphate isomerase, enolase, pyruvate dehydrogenase) and structural proteins (i.e., desmin, cap-Z, MyHC fragment). Moreover, desmin appeared persistently underrepresented in stressed fi sh, suggesting that muscle integrity was affected [START_REF] Morzel | Modifi cations of trout (Oncorhynchus mykiss) muscle proteins by preslaughter activity[END_REF]. In Atlantic salmon, 40 min crowding stress altered the abundance of 27 protein spots, including both structural proteins (i.e., actin, MyLC, MyHC, tropomyosin) and sarcoplasmic proteins, especially enzymes involved in energy production (i.e., creatine kinase, enolase, phosphoglycerate kinase), suggesting accelerated p.m. metabolism [START_REF] Veiseth-Kent | Changes in muscle and blood plasma proteomes of Atlantic salmon (Salmo salar) induced by crowding[END_REF]. A 15 min crowding stress applied to high-or low-stress-responsive rainbow trout before slaughter (Figure 4) led to an over-representation of spots identifi ed as desmin, MyLC3, myeloperoxidase, malate dehydrogenase, apolipoprotein A1, nucleoside diphosphate kinase (Nme), and parvalbumin but with no difference between low-responsive and high-responsive fi sh strains. Principal component analysis of proteomics data and fl esh texture parameters showed that desmin and FABP-H were positively correlated with raw fl esh fi rmness, whereas myeloperoxidase and apolipoprotein A1 were negatively correlated. Firmness of cooked fl esh was positively correlated to apolipoprotein A1, Nme, and parvalbumin.

Effect of Plant-based Diet

Currently, one of the main challenges in fi sh culture is implement a vegetable-based diet without affecting production effi ciency and product quality. Indeed, in a recent study, reduced raw fl esh fi rmness of rainbow trout fed an all-vegetable diet was associated with a decrease in MyHC and changes in several glycolysis enzyme bands in SDS-PAGE analysis of muscle proteins [START_REF] Lefèvre | Sélection génétique sur la réponse au stress et stress à l'abattage: Conséquences sur le protéome musculaire et lien avec la qualité de la chair chez la truite Arc-en-ciel[END_REF].

The identifi cation of biomarkers of fl esh quality in fi sh species is much less documented than in terrestrial species. Interestingly, identifi ed biomarkers are different than those found for meat quality, suggesting that the determinants of quality would be distinct. All the studies mentioned above are based on gel separation of proteins, limiting biomarker identifi cation to soluble proteins. Matrix proteins, while diffi cult to study, also seem to be major determinants of fl esh texture.

Conclusion

The approaches developed during the past years, with fi nancial support from professionals and national and international research policies, has led to substantial progress in our understanding of genes and proteins involved in the determination of meat quality traits, with a major focus on the technological and sensory traits, particularly tenderness. The results confi rm the complexity of the determination of phenotypic traits determined by genetic and environment interactions. However, they improve our understanding of the biological mechanisms that determine meat quality and provide elements (markers) to move from knowledge to the development of tools for fi eld evaluation of these complex traits.

Figure 1 .

 1 Figure 1. Different steps of proteomic analysis.

Figure 2 .

 2 Figure 2. The beef industry is currently in need of a tool to estimate the tenderness of live cattle (source: Rick Harrison).

Figure 3 .

 3 Figure 3. Pork at Palua Tikus Market in the coastal town of Penang, Malaysia (source: fl ickr.com/shebalso).

Figure 4 .

 4 Figure 4. A rainbow trout harvest. Only 15 min of crowding stress of rainbow trout can lead to increased fl esh fi rmness (source: fl ickr.com/spikefi /).

About the Authors

Dr. Brigitte Picard is a meat scientist. She obtained a Ph.D. degree in biochemistry in 1990 and is currently developing research on muscle growth and cattle meat quality. She is the head of an INRA team of 26 people with currently six thesis and two post-doc students. She manages programs on markers of beef tenderness (biological integrative approach associating biochemical studies, functional genomics [proteomics] and modelling). Her competences are: muscle physiology, analysis of proteins (electrophoresis mono and twodimensional, western-blot, ELISA, and immunohistochemistry), primary cultures of muscle cells (myoblasts and satellite cells), and the proteomic analysis of muscle.


Hatem Boubakri
email: hatem.boubakri@cbbc.rnrt.tn

Anne Poutaraud

Mohamed Ali Wahab

Celine Clayeux

Raymonde Baltenweck-Guyot

Damien Steyer

Christophe Marcic

Ahmed Mliki

Isabelle Soustre-Gacougnolle

Thiamine modulates metabolism of the phenylpropanoid pathway leading to enhanced resistance to Plasmopara viticola in grapevine

Keywords: Thiamine, Plasmopara viticola, Stilbenes, Phenylpropanoid pathway genes, Lignin, Flavonoids, Grapevine, Induced resistance, Real-Time q-PCR, HPLC, UPLC-MS

Background: Previously, we have reported the ability of thiamine (vitamin B1) to induce resistance against Plasmopara viticola in a susceptible grapevine cv. Chardonnay. However, mechanisms underlying vitamins, especially, thiamine-induced disease resistance in grapevine are still largely unknown. Here, we assessed whether thiamine could modulate phenylpropanoid pathway-derived phytoalexins in grapevine plants, as well as, the role of such secondary metabolites in thiamine-induced resistance process to P. viticola. Results: Our data show that thiamine treatment elicited the expression of phenylpropanoid pathway genes in grapevine plants. The expression of these genes correlated with an accumulation of stilbenes, phenolic compounds, flavonoids and lignin. Furthermore, the total anti-oxidant potential of thiamine-treaded plants was increased by 3.5fold higher level as compared with untreated-control plants. Four phenolic compounds are responsible of 97% of the total anti-oxidant potential of thiamine-treated plants. Among these compounds, is the caftaric acid, belonging to the hydroxy-cinnamic acids family. This element contributed, by its own, by 20% of this total anti-oxidant potential. Epifluorescence microscopy analysis revealed a concomitant presence of unbranched-altered P. viticola mycelia and stilbenes production in the leaf mesophyll of thiamine-treated inoculated plants, suggesting that stilbenes are an important component of thiamine-induced resistance in grapevine.

Conclusion:

This work is the first to show the role of thiamine, as a vitamin, in the modulation of grapevine plant secondary metabolism contributing to an enhanced resistance to P. viticola, the most destructive fungal disease in vineyards.

Background

Grapevine (Vitis vinifera) is one of the most important horticultural fruit crops cultivated in the world. Unfortunately, all cultivars are susceptible to several diseases; fungi and oomycetes are the major pathogens that compromise the cultivation and economic profit from this plant. Downy mildew caused by the obligate biotrophic oomycete Plasmopara viticola, is one of the most destructive grapevine diseases. This disease reduces fruit quality and yield, either by direct infection of berries or as a result of the reduction in photosynthesis and plant vigor caused by leaf infections [START_REF] Kortekamp | Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen[END_REF]. The protection of grapevine cultivars against this pathogen requires multiple applications of chemicals, starting from bud burst until ripening. Nevertheless, the disease control by fungicides leads to the emergence of resistant strains of P. viticola [START_REF] Chen | At least two origins of fungicide resistance in grapevine downy mildew populations[END_REF], environmental pollution, and toxic residues on food. In the last decade, a new technology for disease control, which involves the induction of hostdefense mechanisms, was developed as an alternative to chemical fungicides. This activation of the plant's own defense system, known as induced resistance (IR), could be achieved by the application of inducers that mimic pathogen invasion [START_REF] Sticher | Systemic acquired resistance[END_REF]. IR in grapevine against P. viticola is characterized by the generation of H 2 O 2 [START_REF] Trouvelot | A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death[END_REF], enhanced expression of pathogenesis-related (PR) proteins with antimicrobial activity, such as chitinases and glucanases [START_REF] Trouvelot | A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death[END_REF][START_REF] Hamiduzzaman | β-Aminobutyric acid-induced resistance against downy mildew in grapevine acts through the potentiation of callose formation and jasmonic acid signaling[END_REF], phytoalexin production [START_REF] Slaughter | Beta-aminobutyric acid-induced resistance in grapevine against downy mildew: involvement of pterostilbene[END_REF][START_REF] Aziz | Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew[END_REF], and callose deposition [START_REF] Trouvelot | A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death[END_REF][START_REF] Hamiduzzaman | β-Aminobutyric acid-induced resistance against downy mildew in grapevine acts through the potentiation of callose formation and jasmonic acid signaling[END_REF].

Recently, the involvement of the phenylpropanoid pathway in IR mechanisms in grapevine was shown using a pharmacological approach [START_REF] Hamiduzzaman | β-Aminobutyric acid-induced resistance against downy mildew in grapevine acts through the potentiation of callose formation and jasmonic acid signaling[END_REF]. Phenylpropanoid pathwayderived defense responses such as the synthesis of flavonoids, lignin, stilbenes and phenols have been shown to be associated with β-aminobutyric acid (BABA)-IR to P. viticola in grapevine [START_REF] Hamiduzzaman | β-Aminobutyric acid-induced resistance against downy mildew in grapevine acts through the potentiation of callose formation and jasmonic acid signaling[END_REF][START_REF] Slaughter | Beta-aminobutyric acid-induced resistance in grapevine against downy mildew: involvement of pterostilbene[END_REF]. A marked expression of phenylpropanoid pathway-derived phytoalexins including different stilbenic forms has been also reported to be associated with IR to P. viticola in grapevine by using chitosan oligomers [START_REF] Aziz | Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew[END_REF]. In addition, β-1,3-Glucan-IR to P. viticola in grapevine was accompanied by a substantial accumulation of phenolic compounds [START_REF] Trouvelot | A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death[END_REF], which are secondary metabolites that encompass several structurally diverse classes of natural products biogenetically arising from the phenylpropanoid pathway [START_REF] Lattanzio | Role of phenolics in the resistance mechanisms of plants against fungal pathogens and insects[END_REF].

Phenolics constitute the main class of natural antioxidants present in plants and may function as reducing agents, free-radical scavengers, singlet oxygen quenchers, and potential complexers of pro-oxidants [START_REF] Qingming | Antioxidant activities of malt extract from barley (Hordeum vulgare L.) toward various oxidative stress in vitro and in vivo[END_REF]. Phenolics seem to inhibit disease development via different mechanisms involving the inhibition of extracellular fungal enzymes (cellulases, pectinases, laccase, xylanase, etc.), inhibition of fungal oxidative phosphorylation, nutrient deprivation (formation of metal complexes, protein insolubilization), and antioxidant activity in plant tissues [START_REF] Scalbert | Antimicrobial properties of susceptible wheat leaves following stem rust tannins[END_REF][START_REF] Stadnik | Inhibition of phenylalanine ammonia-lyase suppresses the resistance induced by benzothiadiazole in wheat to Blumeria graminis F.SP. tritici[END_REF].

Low-molecular-mass secondary metabolites with antimicrobial activity that are induced by stress are collectively named phytoalexins, and are an important part of the plant defense repertoire. Phytoalexins are a heterogeneous group of compounds [START_REF] Ahuja | Bones AM: Phytoalexins in defense against pathogens[END_REF] that show biological activity towards a variety of pathogens and are considered as molecular markers of disease resistance.

Phytoalexins from the Vitaceae family have been the subject of numerous studies during the past decade, because these compounds are thought to have implications in both phytopathology and human health [START_REF] Jeandet | Phytoalexins from the Vitaceae: Biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism[END_REF]. Although most phytoalexins are less phytotoxic than synthetic fungicides, they can accumulate in large quantities within plant tissues, far exceeding the concentrations necessary to inhibit fungal growth [START_REF] Jeandet | Phytoalexins from the Vitaceae: Biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism[END_REF].

The general phenylpropanoid metabolism generates an array of secondary metabolites, which are based on the few intermediates of the shikimate pathway as the core unit [START_REF] Slaughter | Beta-aminobutyric acid-induced resistance in grapevine against downy mildew: involvement of pterostilbene[END_REF]. The more relevant Vitaceae phytoalexins comprise a group of molecules belonging to the stilbene family [START_REF] Jeandet | Phytoalexins from the Vitaceae: Biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism[END_REF]. Stilbenes are synthesized via the phenylpropanoid/malonate pathway from phenylalanine that, in turn, is converted into cinnamic acid by phenylalanine ammonialyase (PAL). The consecutive action of cinnamate 4-hydroxylase (C4H) and 4coumarate CoA ligase (4CL) transform cinnamic acid into p-coumaryl-CoA. Compounds derived from this pathway, Figure 1 Phenylpropanoid biosynthetic pathway modified according to Jeandet et al. [START_REF] Jeandet | Phytoalexins from the Vitaceae: Biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism[END_REF] and Lijavetzky et al. [START_REF] Lijavetzky | Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures[END_REF]. Genes encoding phenylpropanoid pathway enzymes are shaded in grey.

collectively referred to as polyphenols, are originated from this branching point through the action of the enzymes chalcone synthase (CHS), stilbene synthase (STS) and cinnamoyl-CoA reductase (CCR) for flavonoids, stilbenes, and lignin, respectively (Figure 1) [START_REF] Jeandet | Phytoalexins from the Vitaceae: Biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism[END_REF][START_REF] Lijavetzky | Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures[END_REF].

Members of the stilbene family are derivatives of the trans-resveratrol structure (3,5,4 0 -trihydroxystilbene). Piceide (5,4 0 -dihydroxystilbene-3-O-β-glucopyranoside) is a glycosylated resveratrol derivative and pterostilbene (3,5-dimethoxy-4 0 -hydroxystilbene) is a dimethylated resveratrol derivative [START_REF] Jeandet | Phytoalexins from the Vitaceae: Biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism[END_REF]. Recently, it was shown that an isomer of ε-viniferin, δ-viniferin, is one of the major stilbenes produced from resveratrol oxidation in grapevine leaves infected by P. viticola [START_REF] Pezet | δ-viniferin, a resveratrol dehydrodimer: One of the major stilbenes synthesized by grapevine leaves[END_REF]. Pezet et al. [START_REF] Pezet | Effects of resveratrol, viniferins and pterostilbene on Plasmopara viticola zoospore mobility and disease development[END_REF] tested the toxicity of these stilbenes against zoospores of P. viticola (in-vitro) and found that δ-viniferin and pterostilbene were the most toxic stilbenes.

Currently, there is tremendous scientific and commercial interest in identifying chemicals whose exogenous application would activate plant defenses and afford protection from pathogen infection.

Elicitors induce production of phytoalexins by mimicking a pathogen attack or other stress [START_REF] Sticher | Systemic acquired resistance[END_REF], and can be substances of pathogenic origin (exogenous) or compounds released by the plants in response to the action of a pathogen (endogenous).

Inducers have potential uses in sustainable crop production. As critical components of many physiological processes, vitamins may influence the outcome of plantpathogen interactions. In recent years, the importance of vitamins as nutrients and as control agents for different diseases has been demonstrated [START_REF] Ahn | Vitamin B1 functions as an activator of plant disease resistance[END_REF][START_REF] Ahn | Vitamin B1-induced priming is dependent on hydrogen peroxide and the NPR1 gene in Arabidopsis[END_REF]. Ahn et al. [START_REF] Ahn | Vitamin B1 functions as an activator of plant disease resistance[END_REF] have reported that thiamine (vitamin B1) functions as an activator of plant disease resistance. Thiamine-treated rice, Arabidopsis thaliana, and vegetable crops showed resistance to fungal, bacterial, and viral infections. Thiamine-IR in rice and Arabidopsis plants was accompanied with a transient expression of PR genes through the salicylic acid-and Ca 2+ -related signaling pathways. In a previous study, we have reported the ability of thiamine to induce resistance against P. viticola in a susceptible grapevine cultivar "Chardonnay" by a dual mode of action involving direct antifungal activity and elicitation of host-defense responses including H 2 O 2 generation, upregulation of PR genes, and hypersensitive cell death [START_REF] Boubakri | Thiamine induced resistance to Plasmopara viticola in grapevine and elicited host-defense responses including HR-like cell death[END_REF]. However, the mechanisms underlying vitamin-IR, and especially thiamine-IR, in grapevine are poorly known. In this study, we investigated the role of phenylpropanoid pathway metabolism in thiamine-IR to P. viticola in grapevine. Our experiments using real-time quantitative polymerase chain reaction (Real-Time q-PCR) demonstrated that phenylpropanoid pathway genes were upregulated by thiamine treatment. In addition, qualitative and quantitative analysis using high-performance liquid chromatography-diode array detection (HPLC-DAD), ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS), chromatographic online antioxidant detection system (COADS), and histochemical analyses revealed that phenylpropanoidderived phytoalexins such as flavonoids, phenols, lignin, and stilbenes were efficiently induced following treatment of grapevine plants with thiamine. Furthermore, epifluorescence microscopy observations suggested the possible involvement of stilbenes in the restriction of P. viticola mycelial growth in the leaf mesophyll of thiamine-treated grapevine plants.

Results

Effect of thiamine treatment on downy mildew incidence

Results shown in Figure 2 correspond to disease incidence on grapevine plants as mean % of plants with visible symptoms according to Unger et al. [START_REF] Unger | The course of colonization of two different Vitis genotypes by Plasmopara viticola indicates compatible and incompatible host-pathogen interactions[END_REF]. This data indicated that in control conditions, the average of plants showing pathogen sporulation was 63.4%, the average of plants showing pathogen oil spots was 70.3%, and the average of plants showing necrosis was 0%. In contrast, in the case of thiamine treatment, the average of plants showing pathogen sporulation symptoms was 0%, the average of plants showing pathogen oil spots was 0%, and the average of plants showing necrosis was 70%. Collectively, this data confirms the efficiency of thiamine in downy mildew control in grapevine plants.

Thiamine upregulated phenylpropanoid pathway gene expression

The data obtained (Figure 3A) showed that thiamine treatment induced a rapid and strong accumulation of PAL mRNA transcripts in grapevine plant leaves. The PAL induction by thiamine peaked at 2 time points. The first peak was detected within 12 hours post-treatment (hpt) with a 370-fold expression, whereas the second peak was observed at 36 hpt (459.11-fold expression) before decreasing progressively till 72 hpt (32.62-fold expression). C4H1 (Figure 3B) and 4CL1 (Figure 3C) mRNA transcripts were also induced by thiamine treatment showing similar kinetics. In fact, both C4H1 and 4CL1 inductions peaked first time within 12 hpt (3.27-and 10.11-fold expressions, respectively) and next time within 48 hpt (4.64 and 5.56-fold expression, respectively) before declining progressively till 72 hpt (1.77-and 2.15-fold expression).

Thiamine treatment also upregulated the expression of CCR2 gene (Figure 3D) in grapevine plants. The CCR2 induction peaked the first time within 24 hpt (26.67-fold expression) and the second time within 48 hpt (41.65fold expression) before being decreased at 72 hpt (2.01fold expression).

Both STS1 (Figure 3E) and CHS1 (Figure 3F) genes were induced by thiamine treatment following a similar kinetic profile as that of the PAL gene. Indeed, STS1 and CHS1 mRNA transcripts peaked the first time at 12 hpt (1548 and 3.72-fold expression, respectively) and a second time within 36 hpt (1641.5-and 4.715-fold expressions, respectively). Subsequently, STS1 and CHS1 gene expressions decreased progressively until 72 hpt (188.14-and 1.06-fold expressions, respectively). Collectively, these results clearly demonstrate the ability of thiamine to elicit grapevine plants for an enhanced expression of phenylpropanoid pathway genes.

Thiamine induced production of stilbenes

To look for a correlation between STS1 gene induction by thiamine treatment and stilbenes production, the amounts of different stilbenic compounds (stilbenoids) were determined using HPLC-DAD at different time intervals post-treatment (Figure 4). The identity of the different stilbenoids in samples used for HPLC-DAD analysis was also confirmed via ULPC-DAD-MS (Figure 5). Our results showed that the contents of both trans-piceide (Figure 4A) and cis-piceide (Figure 4B) were markedly elevated in thiamine-treated plants as compared to those of water-treated samples for the 4 investigated time points. The trans-piceide content increased with the increase in the time interval after thiamine application and reached its maximum level of 191.52 μg/g dry weight (DW) within 4 days posttreatment (dpt). However, the highest concentration of cis-piceide in thiamine-treated plants occurred at 2 dpt (196.52 μg/g DW). In contrast, lesser amounts of both trans-and cis-piceide were detected in water-treated samples at all the sampling times. Trans-resveratrol was quantitatively the most abundant stilbenoid produced after thiamine treatment (Figure 4C). Trans-resveratrol induction was detected starting from 1 dpt (197.6 μg/g DW), increased progressively with time and reached its maximum level at 3 dpt (326.23 μg/g DW) before decreasing at 4 dpt (148.76 μg/g DW). Only very meager amounts of trans-resveratrol were detected in watertreated plants at the different time points of sampling.

Trans-ε-viniferin (Figure 4D) and trans-δ-viniferin (Figure 4E) were detected only in thiamine-treated plants. Both trans-ε-and trans-δ-viniferins were produced starting from 1 dpt in thiamine-treated plants; thereafter, their content increased progressively reaching maximal levels within 3 dpt (81.34 and 36.56 μg/g DW, respectively) before decreasing at 4 dpt (42.03 and 19.78 μg/g DW, respectively). Trans-pterostilbene, like both trans-ε-and trans-δ-viniferins was not detected in water-treated plants at any of the time points of sampling. In contrast, it was significantly produced in thiamine-treated plants (Figure 4F) starting from 1 dpt. Then its content increased gradually with time, reaching maximum level at 3 dpt (6.34 μg/g DW) before decreasing within 4 dpt (2.35 μg/g DW).

Thiamine treatment elicited the accumulation of phenols and lignin

The effect of thiamine on lignin and phenol synthesis was investigated using a histochemical approach. Grapevine plants were treated with water or thiamine and leaves were harvested at 1 dpt. For lignin detection, leaf discs were punched from water-and thiamine-treated plant leaves, stained with phloroglucinol, and examined under a light microscope. Red coloration of the leaf discs indicates the presence of lignin. A positive reaction, (red coloration) was observed in leaf discs of thiaminetreated plants (Figure 5A,C), indicating a marked induction of lignin synthesis in parenchymal tissues, whereas in the leaf discs of water-treated plants (Figure 5B), we did not observe any lignin accumulation.

For detection of phenols, leaf sections (10 μm in thickness) were obtained using a cryostat microtome and examined under an epifluorescence microscope. Production of phenols was observed as a blue coloration under UV-light. The leaf sections from thiamine-treated plants showed intense blue coloration, indicating accumulation of phenols (Figure 5D,F), unlike the leaf discs from water-treated plants (Figure 5E).

Thiamine induced a prominent induction of flavonoids

Results of our molecular investigations have demonstrated that thiamine upregulated the expression of CHS1 gene, which is responsible of flavonoid biosynthesis in grapevine. Therefore, we investigated whether the activation of this gene by thiamine correlated with an accumulation of flavonoids. The subcellular detection of flavonoids was achieved after staining with Wilson's Reagent. In addition, we have assessed the amount of quercetin 3Oglucoside (a flavonol) at different time points by HPLC-DAD. Our results (Figure 6H, I) showed that thiamine treatment induced a prominent accumulation of flavonoids evidenced by the presence of an intense yellow coloration after Wilson's Reagent staining, whereas the intensity of this yellow coloration was markedly less evident in water-treated plants (Figure 6F). The amount of quercetin 3-O-glucoside flavonol was not significantly affected by thiamine treatment of grapevine plants (Figure 7). Considering the large diversity of basal flavonoid structures in plants (flavones, dihydroflavonols, flavanones, isoflavonoids, isoflavans, and pterocarpans), other specific metabolites would be induced by thiamine.

Thiamine enhanced the anti-oxidant potential of grapevine plants through induction of specific phenolic compounds, including a caftaric acid detection at 254 nm, whereas that at the lower part was obtained using visible detection at 412 nm after post-column reaction. For each treatment, COADS chromatograms were determined and peak areas were summed and used to represent the total antioxidant activity, which were expressed as average ± SD from triplicates of 3 determinations (Figure 8A). The total antioxidant activity results were expressed as Trolox equivalent (μg/ml). The total antioxidant potential in thiamine-treated plants was 43 ± 6.8 μg/ml Trolox equivalent, whereas in water treated-plants, it was 12 ± 0.9 μg/ml, indicating that the total antioxidant potential of thiamine-treated plants was 3.58-fold that of control plants (Figure 9A). The largest antioxidant contribution came from only 4 compounds, which were responsible for 97% of the total antioxidant potential of thiaminetreated plants. Among them, we identified a caftaric acid belonging to the family of hydroxyl-cinnamic acids, which by itself contributes about 20% in the total antioxidant potential (Figure 9B). Work is under way to identify the other unknown phenolic compounds.

A possible role for stilbenes in P. viticola restriction by thiamine Stilbenes have been reported to possess a biocidal effect against P. viticola zoospores in vitro [START_REF] Pezet | Effects of resveratrol, viniferins and pterostilbene on Plasmopara viticola zoospore mobility and disease development[END_REF]. Here, we assessed their effect on the mycelia growth of the pathogen in grapevine plants, elicited by thiamine (in vivo). Accumulation of both stilbenes and pathogen mycelia was detected in foliar tissues by epifluorescence microscopy (under UV light). The results (Figure 10), clearly showed the presence of altered (Figure 10A), unbranched pathogen mycelia inside foliar zones that showed intense blue colorations corresponding to the accumulation of stilbenes in thiamine-treated plant leaves. In contrast, in water-treated plants, we observed normal and extensive hyphae development in the leaf mesophyll, and we did not observe any stilbene accumulation (Figure 10B). Thus, the concomitant presence of altered, unbranched mycelia and stilbene production in thiamine-treated plant leaves suggests a possible contribution of this phytoalexin family to P. viticola restriction by inhibition of mycelia spreading in the leaf mesophyll. In addition, direct effects of thiamine might also play a role in the alteration of pathogen mycelia. At 5 days post-inoculation (dpi), we observed a heavy infection of P. viticola in the leaves of water-treated plants, evidenced by the development of white sporulation symptoms (Figure 10D), whereas in thiamine-treated samples, we observed only a development of necrotic zones corresponding to hypersensitive reaction relatedcell death (Figure 10C).

Discussion

The major phytoalexins produced by V. vinifera cultivars during pathogen infections are resveratrol and its derivatives, piceide (the glycosylated form), δ-viniferin and ε-viniferin (oxidised dimers), and pterostilbene (the methylated derivative) [START_REF] Pezet | Glycosylation and oxidative dimerization of resveratrol are respectively associated to sensitivity and resistance of grapevine cultivars to downy mildew[END_REF]. In our experiment, thiamine treatment led to an induction of trans-and cis-piceide forms with maximal levels obtained within 4 and 2 dpt, respectively for each compound. Thiamine treatment also induced the accumulation of trans-resveratrol with a maximal level observed within 3 dpt. In susceptible grapevine cultivars, resveratrol is synthesized in large amounts, but it is rapidly glycosylated into the non-toxic compound piceide. This could explain the high concentrations of both cis-and trans-piceide in thiamine-treated plants, which is due to the high concentration of trans-resveratrol. Further, thiamine elicited the accumulation of important stilbenic forms, the viniferins, which are active against P. viticola; δ-viniferin is 5 times as toxic as ε-viniferin [START_REF] Pezet | Effects of resveratrol, viniferins and pterostilbene on Plasmopara viticola zoospore mobility and disease development[END_REF]. The maximal levels of both εand δ-viniferin were observed at 3 dpt. Both εand δ-viniferin were not present in water-treated plants. The increase in resveratrol synthesis at 3 dpt for thiamine-treated plants provided an important pool for the synthesis of viniferins that also occurred at 3 dpt. In addition, thiamine treatment induced pterostilbene synthesis with a maximal induction level observed also at 3 dpt.

Pterostilbene was found to be very effective as a lipid/ lipoprotein-lowering agent in hypercholesterolemic hamsters, probably because, like fibrate drugs, pterostilbene was shown to act as a peroxisome proliferator-activated receptor agonist [START_REF] Rimando | Pterostilbene, a new agonist for the peroxisome proliferator-activated receptor alphaisoform, lowers plasma lipoproteins and cholesterol in hypercholesterolemic hamsters[END_REF]. Moreover, pterostilbene exhibits antioxidant and anticancer properties similar to those of resveratrol [START_REF] Jang | Cancer chemopreventive activity of resveratrol, a natural product derived from grapes[END_REF]. Pterostilbene was found to be very effective in preventing carcinogen-induced preneoplastic lesions in a mouse mammary organ culture model [START_REF] Rimando | Resveratrol, pterostilbene, and piceatannol in vaccinium berries[END_REF], and it showed preventive activity against colon carcinogenesis in rats [START_REF] Suh | Pterostilbene, an active constituent of blueberries, suppresses aberrant crypt foci formation in the azoxymethane-induced colon carcinogenesis model in rats[END_REF].

Flavonols are phenylpropanoid metabolites, most of which are synthesized from p-coumaroyl-CoA and malonyl-CoA and share their precursors with the biosynthetic pathway for lignin biosynthesis [START_REF] Stafford | Flavonoid Evolution: An Enzymic Approach[END_REF]. However, some rare flavonoids are synthesized from CoA esters of substrates such as cinnamic acid or dihydrocoumaric acid. Flavonoids also contribute to resistance against pathogens. In our experiment, thiamine induced the accumulation of flavonoids. In contrast, such metabolic response is absent in all susceptible grapevine cultivars after pathogen inoculation [START_REF] Dai | Histochemical studies on the interaction between three species of grapevine, Vitis vinifera, V. rupestris and V. rotundifolia and the downy fungus Plasmopara viticola[END_REF]. Many specific flavonoids belonging to flavone and flavanone classes have been shown to be active against fungal pathogens commonly found during the storage of fruits and vegetables, i.e. Aspergillus sp., Botrytis cinerea and Fusarium oxysporum [START_REF] Lattanzio | Role of phenolics in the resistance mechanisms of plants against fungal pathogens and insects[END_REF]. Flavonoids have also been reported to be involved in the restriction of P. viticola growth in intermediate resistant species Vitis rotundifolia and Vitis rupestris [START_REF] Dai | Polyphenols and resistance of grapevines (Vitis. spp) to downy mildew (Plasmopara viticola)[END_REF].

A part of phytoalexins functions as antioxidant and thereby contribute to plant resistance against pathogens [START_REF] Treutter | Managing phenol contents in crop plants by phytochemical farming and breeding. Visions and constraints[END_REF]. In our study, thiamine application induced a prominent accumulation of specific phenolic compounds with antioxidant properties, which led to an increase in the total antioxidant potential of grapevine plants as demonstrated by COADS analysis. Previous studies have demonstrated that, in resistant plants, cells usually respond by increasing the level of pre-existing antifungal phenols at the infection site, after an elicited increased activity of the key enzymes (PAL and CHS) of the phenylpropanoid pathway [START_REF] Hammerschmidt | Phytoalexin accumulation: response or defense[END_REF]. Phenolic esterification in cell wall materials is usually considered as an increase in resistance to fungal hydrolytic enzymes as well as a physical barrier against fungal penetration [START_REF] Stadnik | Inhibition of phenylalanine ammonia-lyase suppresses the resistance induced by benzothiadiazole in wheat to Blumeria graminis F.SP. tritici[END_REF]. The first demonstrated example of phenolics providing disease resistance was the case of onion scales accumulating sufficient quantities of catechol (I) and protocatechuic acid (II) to prevent onion smudge disease, Colletotrichum circinans. The colored outer onion scales of resistant onion varieties contain enough of these 2 phenols to reduce spore germination of C. circinans to below 2%, whereas in susceptible varieties, which lack these compounds, the germination rate is over 90% [START_REF] Hammerschmidt | Phytoalexin accumulation: response or defense[END_REF]. Adequate levels of chlorogenic acid (III) account for the resistance of potato tubers against Phytophthora infestans [START_REF] Hammerschmidt | Phytoalexin accumulation: response or defense[END_REF]. Phenolics have also been reported to be involved in the restriction of P. viticola growth in intermediate resistant species V. rotundifolia and V. rupestris [START_REF] Dai | Polyphenols and resistance of grapevines (Vitis. spp) to downy mildew (Plasmopara viticola)[END_REF]. In contrast, such metabolic response is absent in susceptible grapevine cultivars after pathogen inoculation [START_REF] Dai | Histochemical studies on the interaction between three species of grapevine, Vitis vinifera, V. rupestris and V. rotundifolia and the downy fungus Plasmopara viticola[END_REF]. In addition, accumulation of phenolics has been reported to be associated with IR mechanisms in grapevine, such as β-1,3-glucanand chitosan-IR to P. viticola [START_REF] Trouvelot | A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death[END_REF][START_REF] Aziz | Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew[END_REF].

On the other hand, the increased level of phenolics may provide an adequate substrate for oxidative reactions catalyzed by polyphenol oxidase (PPO) and/or peroxidase (POD), which, by consuming oxygen and producing fungitoxic quinones, make the medium unfavorable to the further development of pathogens [START_REF] Lattanzio | Role of phenolics in the resistance mechanisms of plants against fungal pathogens and insects[END_REF]. Vanci et al. [START_REF] Vance | Lignification as a mechanism of disease resistance[END_REF] have reported that peroxidase activity using specific phenolic compounds as substrate lead to lignin formation.

Our study revealed enhanced accumulation of phenolics and lignin in thiamine-treated grapevine plants. These compounds have been reported to play a key role in limiting fungal extension [START_REF] Robertsen | Endo-polygalacturonase from Cladosporium cucumerinum elicits lignification in cucumber hypocotyls[END_REF]. In addition, Cohen et al. [START_REF] Cohen | Ultrasctrcuture of Pseudoperenospora cubensis in muskmelon genotypes susceptible and resistant to downy mildew[END_REF] assumed that lignin encasing cells of muskmelon invaded with haustoria of Pseudoperenospora cubensis prevent fungal growth and induced host cell death by interrupting the nutrient flow into and out of the cells. Taken together, these findings suggest that both phenolics and lignin might play a possible role in thiamine-IR to P. viticola in grapevine.

Previously [START_REF] Boubakri | Thiamine induced resistance to Plasmopara viticola in grapevine and elicited host-defense responses including HR-like cell death[END_REF], we have reported that a time interval of 3 days between thiamine treatment and pathogen inoculation is the optimal time for a maximal resistance to P. viticola. In the present work, we found that the maximal induction of specific stilbenes (both εand δ-viniferin and pterostilbene) by thiamine treatment also occurred at 3 dpt. Thus, these results might indicate a close correlation between stilbene accumulation and IR to P. viticola by thiamine treatment. Furthermore, our microscopic observations clearly demonstrated a concomitant presence of altered, unbranched P. viticola mycelia and stilbene accumulation in thiamine-treated plants. Collectively, these results indicate a possible contribution of stilbenes in thiamine-IR to P. viticola in grapevine.

Conclusion

As a conclusion, the findings denoted in this paper emphasized the role of phenylpropanoid pathway metabolism in thiamine-IR to P. viticola in grapevine. In fact, thiamine application upregulated phenylpropanoid pathway gene expression and elicited the plants to accumulate specific stilbenes like both εand δ-viniferins and pterostilbene, that are otherwise not present in the plants. Moreover, thiamine induced the accumulation of phenolics, lignin, and flavonoids, which are markers of resistance to P. viticola in intermediate resistant grapevine species. As a result of these molecular and cellular changes, the antioxidant potential of grapevine plants was increased, which could provide additional mechanisms contributing in downy mildew restriction by thiamine. Furthermore, we found a concomitant presence of phytoalexins-derived stilbenes and degenerated pathogen mycelial structures in the leaf mesophyll of thiamine-treated plants, suggesting a possible involvement of stilbenes in thiamine-IR to P. viticola in grapevine. Therefore, we could suggest that the induction of phenylpropanoid pathway-derived phytoalexins by thiamine might be valuable in developing alternative strategies to chemical fungicides for controlling downy mildew in vineyards. Moreover, this finding would open the door to a large community of vitro culturists, who extensively studied the possible use of plant cultures for the production of secondary compounds of industrial interest (mainly pharmaceutics and dyes).

Methods

Plant material and growth conditions

Vitis vinifera "Chardonnay cv" plants were obtained from herbaceous cuttings. They were cultivated in pots containing a mixture of peat and perlite (4:1, v/v). Plants were grown under controlled glasshouse conditions (24°C, 16 h light and 8 h dark photoperiod and 70% RH) until they developed 11 leaves. Plants were watered every 2 days with a nutritive solution (1 g/l) (Plant Prod 15-10-30, France).

Pathogen

Plasmopara viticola sporangia [START_REF] Diez-Navajas | Two simplified fluorescent staining techniques to observe infection structures of the oomycete Plasmopara viticola in grapevine leaf tissues[END_REF], were propagated on V. vinifera cv. Chardonnay detached leaves maintained in sealed Petri dishes on humid Whatman 3MM paper. Abaxial leaf surfaces were sprayed with freshly collected sporangia re-suspended in water at 5 × 10 4 sporangia/ml. Inoculated leaves were placed in a growth chamber at 20°C and 100% RH for 24 h in the dark, then under a 16 h light and 8 h dark photoperiod and 70% RH for 6days. Inoculum of P. viticola sporangia was prepared by washing the lower side of grapevine leaves that carried freshly sporulating lesions with distilled water. The sporangia suspension was then adjusted to a concentration of 10 4 sporangia/ml after counting with a haemocytometer under a light microscope.

Treatments

Grapevine plants were treated with 30 mM thiamine or water (control) on both upper and lower leaf surfaces until the point of run-off using a compressed air handsprayer device. Treated plants were kept in a growth chamber at 25°C, a 16 h light and 8 h dark photoperiod, and 70% RH.

Pathogen inoculation procedure and determination of disease incidence

Grapevine plants grown under glasshouse, controlled conditions were treated with water (control) or 30 mM thiamine and the abaxial leaf surfaces were inoculated with a sporangial suspension at 5 × 10 4 sporangia/ml using a compressed-air hand sprayer device. Inoculated plants were incubated overnight in dark at 80% RH and 20°C and then kept in a growth chamber under controlled conditions at 24°C, 16 h light and 8 h dark photoperiod, and 70% RH. At 5 dpi, plants were incubated overnight in darkness at 80% RH and 20°C to allow downy mildew sporulation and disease incidence was assessed as proportion of plants showing necrosis, oil spots, and sporulation symptoms [START_REF] Unger | The course of colonization of two different Vitis genotypes by Plasmopara viticola indicates compatible and incompatible host-pathogen interactions[END_REF]. Six plants were used per treatment and the experiment was repeated 3 times.

RNA extraction and reverse transcription (RT)

Total RNA was isolated from grapevine plant leaves by using the RNeasy plant mini kit (Qiagen, Germany) according to the manufacturer's instructions and quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA). Residual genomic DNA was removed by performing on-column DNase I digestion by using the RNase-Free DNase set (Qiagen). Total RNA (500 ng) was used as the template for RT by using the SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) and oligodT-18 as recommended by the supplier.

Real-Time Quantitative Polymerase Chain Reaction (RT q-PCR)

The expression pattern of phenylpropanoid pathway genes (Table 1) was determined in grapevine cultivar Chardonnay plants after thiamine treatment. RT q-PCR was performed using the absolute qPCR SYBR Green Mix (Eurogentec, Belgium) in 96-well plates in a volume of 25 μl in a buffer containing 1× SYBR Green Mix (including Taq polymerase, dNTP, and SYBR Green dye), 100 nM primers and 5-fold diluted reverse-transcribed RNA. PCR conditions were 5 min at 95°C followed by 40 cycles, each consisting of a step of denaturation (20 s at 95°C), a step of hybridization (30 s at 55°C), and a step of annealing (1 min at 60°C). To check the specificity of the PCR product, melting curves were analyzed for each data point. The absence of primer dimer formation was checked in controls without template. The kinetic of transcript levels were determined with ubiquitin gene (Ubq) as internal control. Sequences of the primers for Ubq (accession No.TC32075) are 5 0 GTGGTATT ATTGAGCCATCCTT3 0 for forward reaction and 5 0 -AACCTCCAATCCAGTCATCTAC3 0 for reverse. Each time point was determined as an average of 3 independent experiments. Relative gene expression was determined using the formula, fold induction = 2 -ΔΔCt , where ΔΔC T = (C T GI [unknown sample] -C T GI [reference sample]) -(C T Ubq [unknown sample] -C T Ubq [reference sample]) as previously reported by Trouvelot et al. [START_REF] Trouvelot | A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death[END_REF]. GI is the gene of interest. The reference sample is the water-treated samples chosen to represent 1× expression of the gene of interest.

Chemical solvents used for HPLC-DAD and UPLC-MS analysis and their references HPLC-MS grade acetonitrile and formic acid were supplied by Thermo Fisher Scientific; water was provided by a Millipore water purification system (Millipore, Bedford, MA). Reference compounds including transresveratrol, trans-piceide, trans-pterostilbene, and εviniferins were purchased or kindly provided by R. Pezet, Changins, Switzerland. The peak identity related to trans-resveratrol, trans-piceide, and trans-pterostilbene was verified by comparing the retention time, the UV, and the exact mass spectra of authentic standard.

HPLC-DAD quantification of stilbenes

Grapevine cuttings grown under glasshouse conditions were treated with water (control) or thiamine (30 mM) and leaves on the fourth and fifth positions from the apex were harvested at various time intervals posttreatment, weighed, and used for methanolic extraction. Stilbene extractions and HPLC-DAD analysis were performed according to Poutaraud et al. [START_REF] Poutaraud | Fast and local assessment of stilbene content in grapevine leaf by in vivo fluorometry[END_REF]. Briefly, 800 μl of methanol was added to 160 mg (FW) of leaf tissues followed by incubation at 60°C for 45 min using a heating block, then the mixture was centrifuged twice at 12000 rpm for 10 min. Twenty microliter of the recuperated supernatant were injected in the HPLC-DAD system. The HPLC system consisted of a 1100 quaternary pump (Hewlett-Packard, Agilent Technologies, Massy, France) equipped with a 1100 photodiode array multiwavelength detector (Hewlett-Packard), a 1100 vacuum degasser (Hewlett-Packard), and a 234 automatic injection module (Gilson, Villiers-le-Bel, France). The analyses were carried out at 20°C on a Lichrospher end-capped RP-18 column (5 μm, 250mm × 4.6 mm, Merck, Lyon, France). The absorption was measured between 200 and 400 nm, and chromatograms were recorded at 307 nm. Stilbenes were identified by comparing their retention times and their UV absorption spectra with those of the reference standards.

UHPLC-MS conditions

The identity of each peak of stilbenes on leaf methanolic extracts analyzed by HPLC-DAD was confirmed using an Ultra High Performance Liquid Chromatography system (UHPLC, Ultimate 3000, Dionex, Thermofisher Scientific, San Jose, CA) equipped with a binary pump, an online degasser, a thermostated autosampler, a thermostatically controlled column compartment, and a diode array detector. The chromatographic separation was performed on an Agilent RRHD C18 SB column (150 × 2.1 mm, 2.7 μm particle size) maintained at 20°C. The mobile phase was composed of 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in water (solvent B) at a flow rate of 0.25 ml/min. The gradient elution program was: 0-1 min, 80% B; 1-11 min, 80-0% B; and 11-12 min, 0% B. The sample volume injected for each extract was 1 μl. The absorption was measured Table 1 Primers used for the expression analysis of phenylpropanoid pathway genes by using RT q-PCR [START_REF] Lijavetzky | Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures[END_REF] Gene abbreviation

Gene definition Gen Bank accession

Unigene ID Primer pair 5 0 -Forward-3 0 /5 0 -Reverse-3 0 between 190 and 400 nm, and chromatograms were recorded at 280 and 307 nm. The liquid chromatography system was coupled to an Exactive Orbitrap mass spectrometer (Thermo Fischer Scientific). The mass spectrometer consisted of an electrospray ionization (ESI) source operating in negative mode. Parameters were set at 300°C for ion-transfer capillary temperature and -2500 V needle voltage. Nebulization with nitrogen sheath gas and auxiliary gas were maintained at 40 and 6 arbitrary units, respectively. The instrument was operated at 50 000 resolution, and the spectra were acquiring within the m/z mass range of 100-1000 a.m.u. The instrument was operated using the program ExactiveTune, and data were processed using the XcaliburQual software.

STS1

The system was calibrated externally using the calibration mixture of Thermo Fischer Scientific in the scan range of m/z 100-2000 a.m.u. The calibration was performed every 7 days with MSCAL6-1EA. Such a calibration gave at least 2 ppm accuracy. The identification of stilbenes was confirmed by comparing their retention times and their mass spectra with those of the reference standards and by obtaining their pseudo molecular ion peak (M-H), which permitted the establishment of their precise molecular formula.

Determination of the total antioxidant potential and phenolic compounds by using COADS

The grapevine plant extracts were screened for radical scavenging capacity of individual phenolic compounds, using an on-line HPLC post-column reaction assay [START_REF] Leitao | Effects of processing steps on the phenolic content and antioxidant activity of beer[END_REF]. The assay is based on the ability of radical scavenging compounds to reduce the green-blue-colored 2,2 0azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS[*] [+]) radical cation into non-colored form. This results in a decrease of the absorbance at 412 nm. COADS chromatograms were determined for each treatment and peak areas were summed and used to represent the total antioxidant activity. The HPLC system (Ultimate 3000, Dionex, ThermoScientific, Gometz le Châtel, France) consists of a DGP 3600 Pump, a WPS-3000T Autosampler, a 3000 Diode Array Detector, and a 3100 Variable Wavelength Detector. A 3-m long and 0.25-m i.d. PEEK reaction coil was used. Data were acquired and processed using Dionex Chromeleon 6.8 data system.

Separation was carried out at room temperature on a Hypersil BDS C18 HPLC column (5 μm, 250 × 4.6 mm i.d., ThermoScientific, Gometz-le-Châtel, France). The mobile phase, delivered at 1 ml/min, consisted of a gradient mixture of water containing 0.1% formic acid (eluent A) and acetonitrile (eluent B). The following gradient was used: 0-25 min, 3-25% B; 25-29 min, 25% B; 29-37 min, 25-30% B; 37-47 min, 30% B; 47-57 min, 30-65% B; 57-67 min, 65% B. The (ABTS[*][+]) solution was delivered at 0.5 ml/min. Detection was carried out at 280 nm for phenolic compounds and at 412 nm for their respective antioxidant activities after postcolumn reaction with (ABTS[*][+]). The results are expressed in μg/ml Trolox equivalent, a water-soluble synthetic vitamin E derivative 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid, which was used as standard anti-oxidant.

Histochemical detection of flavonoids, phenols, and lignin

Leaves from plants treated with water (control) or thiamine (30 mM) were used for preparing leaf sections (10 μm thickness) using a cryostat microtome (Leica 2800) operating at -20°C. For detection of flavonoids, sections were immersed for 15 min in Wilson's reagent consisting of citric acid: boric acid (5/5, w/w) in 100 ml absolute ethanol, mounted in glycerol 75% (v/v) and examined using an epifluorescence microscope (NIKON, F 1200) under UV (BP 340-380 nm, LP 425 nm). Yellow fluorescence indicated the presence of flavonoids [START_REF] Dai | Histochemical studies on the interaction between three species of grapevine, Vitis vinifera, V. rupestris and V. rotundifolia and the downy fungus Plasmopara viticola[END_REF].

For detecting phenolics, leaf sections were placed on a glass slide, covered with a glass cover slip, and examined with the aid of an epifluorescence microscope (NIKON, F 1200) (excitation filter of 390-420 nm and an emission filter of 425-450 nm). Phenolics emitted blue fluorescence under UV light [START_REF] Dai | Polyphenols and resistance of grapevines (Vitis. spp) to downy mildew (Plasmopara viticola)[END_REF]. For lignin observation, leaf disks were punched from control and thiamine-treated plant leaves, boiled for 10 min in 95% ethanol and then treated with phloroglucinol (10 g in 95 ml of absolute ethanol) for 3 min. The disks were washed in 25% HCl, mounted in glycerol (75%), and examined under a light microscope [START_REF] Dai | Thiamine modulates metabolism of the phenylpropanoid pathway leading to enhanced resistance to Plasmopara viticola in grapevine[END_REF].

Microscopic assessment of P. viticola structures

For the observation of P. viticola mycelia growing in grapevine foliar tissues, leaf discs (1.4 cm diameter) from thiamine-or water-treated and inoculated plants were incubated at 100°C in 1 M KOH for 15 min, washed 3 times in water for 15 min, stained with 0.05% (w/v) aniline blue in 0.067 M K 2 HPO4 (pH 9-9.5) for 15 min and observed with an epifluorescence microscope (NIKON, F 1200) under UV light (excitation 340 nm, emission 380 nm, stop filter LP 430 nm) according to the method of Diez-Navajaz et al. [START_REF] Diez-Navajas | Two simplified fluorescent staining techniques to observe infection structures of the oomycete Plasmopara viticola in grapevine leaf tissues[END_REF]. The reaction of aniline blue with β-1,3-glucans, makes P. viticola mycelia visible under these conditions as a blue fluorescence. For each treatment, 20 leaf discs from 6 plants were randomly isolated for microscopic observations and the whole experiment was repeated 3 times.

Statistical analysis

Data were analyzed by ANOVA using the Statistica 7.1 Software program (StatSoft, Tulsa, OK, USA). Almost, significance of the differences between mean values was determined with Tukey's HSD test (P ≤ 0.05).

Figure 2

 2 Figure 2 Disease incidence (mean % of plants with visible symptoms) on grapevine cuttings treated with thiamine (solid bars) or water (white bars). Grapevine plants grown under glasshouse, controlled conditions were treated with water (control) or 30 mM thiamine and the abaxial leaf surfaces were inoculated with a sporangial suspension at 5 × 10 4 sporangia/ml. Disease incidence was assessed 7 dpi as proportion of plants showing necrosis, oil spots, and sporulation symptoms [20]. Six plants were used per treatment and the experiment was repeated 3 times. All figures in thiamine treatments are significantly different at 5% level from the controls.

Figure 3

 3 Figure 3 Time course of transcript accumulation of genes of the phenylpropanoid pathway after thiamine treatment. Grapevine plants (cv. Chardonnay) were treated with 30 mM thiamine (solid bars) or water (white bars) and leaves were collected at different time intervals posttreatment[START_REF] Ahuja | Bones AM: Phytoalexins in defense against pathogens[END_REF][START_REF] Rimando | Resveratrol, pterostilbene, and piceatannol in vaccinium berries[END_REF][START_REF] Leitao | Effects of processing steps on the phenolic content and antioxidant activity of beer[END_REF] 48, and 72 hpt). Gene expression was analyzed by real-time q-PCR. Genes of interest are: (A) PAL (phenylalanine ammonialyase), (B) C4H1 (cinnamate 4-hydroxylase), (C) 4CL1 (4-coumarate:coenzyme A ligase), (D) CCR2 (cinnamoyl:coenzyme A reductase), (E) CHS1 (chalcone synthase) and (F) STS1 (stilbene synthase1). Results are presented as normalized fold-expression of defense genes in thiaminetreated samples compared to expression in water-treated controls at the same time point, which is set to 1. Results are mean ± standard deviations of 3 independent experiments.

Figure 4

 4 Figure 4 Quantitative estimation of stilbenes in grapevine plants by HPLC-DAD. Grapevine plants (cv. Chardonnay) were treated with 30 mM thiamine (solid bars) or water (white bars) and leaves were collected at different time intervals post-treatment (1, 2, 3, and 4 dpt) for methanolic extraction of stilbenes. Data are the mean of 3 independent experiments. (A) Trans-piceide, (B) Cis-piceide, (C) Trans-resveratrol, (D) Trans-ε-viniferin, (E)Trans-δ-viniferin and (F) Trans-pterostilbene.

Figure 5

 5 Figure 5 Confirmation of the identity of stilbenoids by LC-UV-ESIMS(-) in methanolic extract of thiamine-treated plants (partial chromatogram). (A) UV chromatogram at 307 nm, which permits the detection of stilbene derivatives specifically. (B) Extracted Ion Chromatogram of m/z 227.0703 corresponding to resveratrol derivatives (mass formula: C 14 H 11 O 3 ). (C) Extracted Ion Chromatogram of m/z 389.1231 corresponding to piceide isomers (mass formula: C 20 H 21 O 8 ). (D) Extracted Ion Chromatogram of m/z 453.1333 corresponding to viniferins (mass formula: C 28 H 21 O 6 ).

Figure 8

 8 Figure8shows COADS chromatograms corresponding to the phenolic compounds separated in a methanolic extract of thiamine-treated grapevine plants and their corresponding antioxidant activities. The result in the upper part of the figure was obtained using direct UV

Figure 6

 6 Figure 6 Histochemical detection of lignin, phenolics, and flavonoids. Leaves from plants treated with water (B, E, G) or 30 mM thiamine (A, C, D, F, H, I) were collected at 1 dpt, and used for microscopic studies. (A, B, C) Leaves from plants were used for subcellular detection of lignin accumulation as a red coloration under light microscope following phloroglucinol-HCl staining. (D, E, F) Leaf segments were examined under auto-fluorescence (UV light Filter); white-blue coloration indicates the presence of phenolics. (G, H, I) Leaf sections (10 μm of thickness) were stained with Wilson's reagent and observed under UV light; yellow coloration indicates presence of flavonoids. Six leaves from different plants were assessed for each treatment and the whole experiment was repeated three times with similar results. Bars = 40 μM for (A, B, D, E, G, H) and 20 μm for (C, F, I).

Figure 7

 7 Figure 7 Quantitative determination of quercetin 3-O-glucoside flavone in grapevine plants by HPLC-DAD. Plants were treated with 30 mM thiamine (solid bars) or sterile distilled water (white bars) and leaves were harvested at different time intervals posttreatment (dpt). Data are the mean of 3 independents experiments.

Figure 8

 8 Figure 8 Chromatographic determination of phenolic compounds (A) and their corresponding anti-oxidant activities (B) in extracts from thiamine-treated grapevine plants. Detection was done at 280 nm for phenolic compounds (LC-UV) and at 412 nm for their respective antioxidant activities after post-column reaction with (ABTS[*][+]) (LC-AOx). The whole experiment was repeated 3 times with similar results.

Figure 9

 9 Figure 9 Increase of the antioxidant potential of grapevine plants after thiamine treatment. (A) COADS determination of the total anti-oxidant potential of leaf extracts from water (white bars) and thiamine-treated (solid bars) grapevine plants. (B) Individual contribution of phenolic compounds to the anti-oxidant potential of water (white bars) and thiamine-treated (solid bars) plants. Data are the mean of 3 independents experiments.

Figure 10 A

 10 Figure 10 A possible role for stilbenes in the restriction of P. viticola development by thiamine treatment. Vitis vinifera cv. Chardonnay plants were treated with water or thiamine for 24 h and inoculated by spraying P. viticola sporangia suspension (10 4 sporangia/ml). (A) Presence of fluorescent material (discontinued arrows) under autofluorescence corresponding to stilbenes [6], surrounding unbranched and degenerated hyphae (discontinued arrows) of P. viticola in the mesophyll of thiamine-treated plant leaves. (B) Absence of stilbenes accumulation and extensive branching of P. viticola hyphae in the intercellular space of the mesophyll of water-treated plant leaves. (C) Development of necrotic zones (HR) and suppression of P. viticola sporulation in thiamine-treated plants. (D) Development of P. viticola sporulation symptoms in water-treated plants. Bars = 40 μM for (A, B) and 1 cm for (C, D).

Acknowledgments

The authors are thankful to Dr. Julie Chong and Pr. Christophe Bertsch (Laboratoire Vigne, Biotechnologies et Environnement, Université de Haute-Alsace de Colmar, France) for providing the plant material. This work was carried out in the "Institut National de Recherche Agronomique-Colmar, France" and supported by grants from the Tunisian Ministry of Higher Education and Scientific Research.

Abbreviations ABTS[*][+]: 2,2 0 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid); COADS: Chromatographic online antioxidant detection system; DPT: Days post-treatment; DPI: Days post-inoculation; HPT: Hours post-treatment; IR: Induced resistance; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions HB conceived the study, carried out the culture, treatment and inoculation of grapevine plants, assessed the disease severity, analyzed the expression of defense related-genes by Real Time q-PCR, analyzed cellular defense responses by histochemistry, assessed P. viticola development by epifluorescence microscopy, helped to HPLC analysis of stilbenes and wrote the manuscript. AP realized HPLC-DAD analysis of stilbenoids and quercetin flavonol and helped to draft the manuscript. M-AW participated in the statistical analysis of the data and helped to draft the manuscript. CC, DS and CM realized the HPLC-AOx analysis of the total antioxidant potential of grapevine plants and identified the phenolic compounds implicated. RB realized the ULPC-MS analysis in order to confirm the identity of the stilbenoids and helped in writing the manuscript. AM and IS-G coordinated the research project and supervised the work. All authors read and approved the final manuscript.

Author details

1 Laboratoire de Physiologie Moléculaire des Plantes, Centre de Biotechnologie de Borj-Cédria, 2050, Hammam Lif, Tunisie. 2 Unité Mixte de Recherche 1131, Université de Strasbourg /INRA-Colmar, 28 Rue de Herrlisheim, F68021, Colmar, France. 3 Technopole de Borj-Cédria-CWR, 2050, Hammam Lif, Tunisie. 4 Twistaroma, 28 Rue de Herrlisheim, 68021, Colmar, France. 5