BACKGROUND
grapevine  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  <cit> .
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  <cit> , environmental pollution, and toxic residues on food.
in the last decade, a new technology for disease control, which involves the induction of host-defense mechanisms, was developed as an alternative to chemical fungicides.
this activation of the plant’s own defense system, known as induced resistance , could be achieved by the application of inducers that mimic pathogen invasion  <cit> .
ir in grapevine against p. viticola is characterized by the generation of h2o <dig>  <cit> , enhanced expression of pathogenesis-related  proteins with antimicrobial activity, such as chitinases and glucanases  <cit> , phytoalexin production  <cit> , and callose deposition  <cit> .
recently, the involvement of the phenylpropanoid pathway in ir mechanisms in grapevine was shown using a pharmacological approach  <cit> .
phenylpropanoid pathway-derived defense responses such as the synthesis of flavonoids, lignin, stilbenes and phenols have been shown to be associated with β-aminobutyric acid -ir to p. viticola in grapevine  <cit> .
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  <cit> .
in addition, β- <dig> -glucan-ir to p. viticola in grapevine was accompanied by a substantial accumulation of phenolic compounds  <cit> , which are secondary metabolites that encompass several structurally diverse classes of natural products biogenetically arising from the phenylpropanoid pathway  <cit> .
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  <cit> .
phenolics seem to inhibit disease development via different mechanisms involving the inhibition of extracellular fungal enzymes , inhibition of fungal oxidative phosphorylation, nutrient deprivation , and antioxidant activity in plant tissues  <cit> .
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  <cit>  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  <cit> .
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  <cit> .
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  <cit> .
the more relevant vitaceae phytoalexins comprise a group of molecules belonging to the stilbene family  <cit> .
stilbenes are synthesized via the phenylpropanoid/malonate pathway from phenylalanine that, in turn, is converted into cinnamic acid by phenylalanine ammonialyase .
the consecutive action of cinnamate 4-hydroxylase  and 4-coumarate coa ligase  transform cinnamic acid into p-coumaryl-coa.
compounds derived from this pathway, collectively referred to as polyphenols, are originated from this branching point through the action of the enzymes chalcone synthase , stilbene synthase  and cinnamoyl-coa reductase  for flavonoids, stilbenes, and lignin, respectively   <cit> .
members of the stilbene family are derivatives of the trans-resveratrol structure .
piceide  is a glycosylated resveratrol derivative and pterostilbene  is a dimethylated resveratrol derivative  <cit> .
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  <cit> .
pezet et al.
<cit>  tested the toxicity of these stilbenes against zoospores of p. viticola  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  <cit> , and can be substances of pathogenic origin  or compounds released by the plants in response to the action of a pathogen .
inducers have potential uses in sustainable crop production.
as critical components of many physiological processes, vitamins may influence the outcome of plant-pathogen interactions.
in recent years, the importance of vitamins as nutrients and as control agents for different diseases has been demonstrated  <cit> .
ahn et al.
<cit>  have reported that thiamine  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 ca2+-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 h2o <dig> generation, upregulation of pr genes, and hypersensitive cell death  <cit> .
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  demonstrated that phenylpropanoid pathway genes were upregulated by thiamine treatment.
in addition, qualitative and quantitative analysis using high-performance liquid chromatography-diode array detection , ultra-performance liquid chromatography coupled with mass spectrometry , chromatographic online antioxidant detection system , and histochemical analyses revealed that phenylpropanoid-derived 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  <dig> correspond to disease incidence on grapevine plants as mean % of plants with visible symptoms according to unger et al.
<cit> .
this data indicated that in control conditions, the average of plants showing pathogen sporulation was  <dig> %, the average of plants showing pathogen oil spots was  <dig> %, 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  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  <dig> time points.
the first peak was detected within 12 hours post-treatment  with a 370-fold expression, whereas the second peak was observed at  <dig> hpt  before decreasing progressively till  <dig> hpt .
c4h <dig>  and 4cl <dig>  mrna transcripts were also induced by thiamine treatment showing similar kinetics.
in fact, both c4h <dig> and 4cl <dig> inductions peaked first time within  <dig> hpt  and next time within  <dig> hpt  before declining progressively till  <dig> hpt .
thiamine treatment also upregulated the expression of ccr <dig> gene  in grapevine plants.
the ccr <dig> induction peaked the first time within  <dig> hpt  and the second time within  <dig> hpt  before being decreased at  <dig> hpt .
both sts <dig>  and chs <dig>  genes were induced by thiamine treatment following a similar kinetic profile as that of the pal gene.
indeed, sts <dig> and chs <dig> mrna transcripts peaked the first time at  <dig> hpt  and a second time within  <dig> hpt .
subsequently, sts <dig> and chs <dig> gene expressions decreased progressively until  <dig> hpt .
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 sts <dig> gene induction by thiamine treatment and stilbenes production, the amounts of different stilbenic compounds  were determined using hplc-dad at different time intervals post-treatment .
the identity of the different stilbenoids in samples used for hplc-dad analysis was also confirmed via ulpc-dad-ms .
our results showed that the contents of both trans-piceide  and cis-piceide  were markedly elevated in thiamine-treated plants as compared to those of water-treated samples for the  <dig> investigated time points.
the trans-piceide content increased with the increase in the time interval after thiamine application and reached its maximum level of  <dig>  μg/g dry weight  within 4 days post-treatment .
however, the highest concentration of cis-piceide in thiamine-treated plants occurred at  <dig> dpt .
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 .
trans-resveratrol induction was detected starting from  <dig> dpt , increased progressively with time and reached its maximum level at  <dig> dpt  before decreasing at  <dig> dpt .
only very meager amounts of trans-resveratrol were detected in water-treated plants at the different time points of sampling.
trans-ε-viniferin  and trans-δ-viniferin  were detected only in thiamine-treated plants.
both trans-ε- and trans-δ-viniferins were produced starting from  <dig> dpt in thiamine-treated plants; thereafter, their content increased progressively reaching maximal levels within  <dig> dpt  before decreasing at  <dig> dpt .
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  starting from  <dig> dpt.
then its content increased gradually with time, reaching maximum level at  <dig> dpt  before decreasing within  <dig> dpt .
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  <dig> 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,  was observed in leaf discs of thiamine-treated plants , indicating a marked induction of lignin synthesis in parenchymal tissues, whereas in the leaf discs of water-treated plants , we did not observe any lignin accumulation.
for detection of phenols, leaf sections  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 , unlike the leaf discs from water-treated plants .
thiamine induced a prominent induction of flavonoids
results of our molecular investigations have demonstrated that thiamine upregulated the expression of chs <dig> 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  at different time points by hplc-dad.
our results  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 .
the amount of quercetin 3-o-glucoside flavonol was not significantly affected by thiamine treatment of grapevine plants .
considering the large diversity of basal flavonoid structures in plants , 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
figure  <dig> shows 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 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  <dig> determinations .
the total antioxidant activity results were expressed as trolox equivalent .
the total antioxidant potential in thiamine-treated plants was 43 ±  <dig>  μg/ml trolox equivalent, whereas in water treated-plants, it was 12 ±  <dig>  μg/ml, indicating that the total antioxidant potential of thiamine-treated plants was  <dig> -fold that of control plants .
the largest antioxidant contribution came from only  <dig> compounds, which were responsible for 97% of the total antioxidant potential of thiamine-treated 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 .
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 <cit> .
here, we assessed their effect on the mycelia growth of the pathogen in grapevine plants, elicited by thiamine .
accumulation of both stilbenes and pathogen mycelia was detected in foliar tissues by epifluorescence microscopy .
the results , clearly showed the presence of altered , 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 .
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 , we observed a heavy infection of p. viticola in the leaves of water-treated plants, evidenced by the development of white sporulation symptoms , whereas in thiamine-treated samples, we observed only a development of necrotic zones corresponding to hypersensitive reaction related-cell death .
discussion
the major phytoalexins produced by v. vinifera cultivars during pathogen infections are resveratrol and its derivatives, piceide , δ-viniferin and ε-viniferin , and pterostilbene   <cit> .
in our experiment, thiamine treatment led to an induction of trans- and cis-piceide forms with maximal levels obtained within  <dig> and  <dig> dpt, respectively for each compound.
thiamine treatment also induced the accumulation of trans-resveratrol with a maximal level observed within  <dig> 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  <dig> times as toxic as ε-viniferin  <cit> .
the maximal levels of both ε- and δ-viniferin were observed at  <dig> dpt.
both ε- and δ-viniferin were not present in water-treated plants.
the increase in resveratrol synthesis at  <dig> dpt for thiamine-treated plants provided an important pool for the synthesis of viniferins that also occurred at  <dig> dpt.
in addition, thiamine treatment induced pterostilbene synthesis with a maximal induction level observed also at  <dig> 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  <cit> .
moreover, pterostilbene exhibits antioxidant and anticancer properties similar to those of resveratrol  <cit> .
pterostilbene was found to be very effective in preventing carcinogen-induced preneoplastic lesions in a mouse mammary organ culture model  <cit> , and it showed preventive activity against colon carcinogenesis in rats  <cit> .
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  <cit> .
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  <cit> .
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  <cit> .
flavonoids have also been reported to be involved in the restriction of p. viticola growth in intermediate resistant species vitis rotundifolia and vitis rupestris <cit> .
a part of phytoalexins functions as antioxidant and thereby contribute to plant resistance against pathogens  <cit> .
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  of the phenylpropanoid pathway  <cit> .
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  <cit> .
the first demonstrated example of phenolics providing disease resistance was the case of onion scales accumulating sufficient quantities of catechol  and protocatechuic acid  to prevent onion smudge disease, colletotrichum circinans.
the colored outer onion scales of resistant onion varieties contain enough of these  <dig> 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%  <cit> .
adequate levels of chlorogenic acid  account for the resistance of potato tubers against phytophthora infestans  <cit> .
phenolics have also been reported to be involved in the restriction of p. viticola growth in intermediate resistant species v. rotundifolia and v. rupestris <cit> .
in contrast, such metabolic response is absent in susceptible grapevine cultivars after pathogen inoculation  <cit> .
in addition, accumulation of phenolics has been reported to be associated with ir mechanisms in grapevine, such as β- <dig> -glucan- and chitosan-ir to p. viticola <cit> .
on the other hand, the increased level of phenolics may provide an adequate substrate for oxidative reactions catalyzed by polyphenol oxidase  and/or peroxidase , which, by consuming oxygen and producing fungitoxic quinones, make the medium unfavorable to the further development of pathogens  <cit> .
vanci et al.
<cit>  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  <cit> .
in addition, cohen et al.
<cit>  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  <cit> , 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  by thiamine treatment also occurred at  <dig> 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.
CONCLUSIONS
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 ε- and 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 .

 grapevine 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. 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, environmental pollution, and toxic residues on food. in the last decade, a new technology for disease control, which involves the induction of host-defense mechanisms, was developed as an alternative to chemical fungicides. this activation of the plant’s own defense system, known as induced resistance, could be achieved by the application of inducers that mimic pathogen invasion. ir in grapevine against p. viticola is characterized by the generation of h2o, enhanced expression of pathogenesis-related proteins with antimicrobial activity, such as chitinases and glucanases, phytoalexin production, and callose deposition. recently, the involvement of the phenylpropanoid pathway in ir mechanisms in grapevine was shown using a pharmacological approach. phenylpropanoid pathway-derived defense responses such as the synthesis of flavonoids, lignin, stilbenes and phenols have been shown to be associated with β-aminobutyric acid -ir to p. viticola in grapevine. 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. in addition, β- -glucan-ir to p. viticola in grapevine was accompanied by a substantial accumulation of phenolic compounds, which are secondary metabolites that encompass several structurally diverse classes of natural products biogenetically arising from the phenylpropanoid pathway. 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. phenolics seem to inhibit disease development via different mechanisms involving the inhibition of extracellular fungal enzymes, inhibition of fungal oxidative phosphorylation, nutrient deprivation, and antioxidant activity in plant tissues. 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 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. 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. 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. the more relevant vitaceae phytoalexins comprise a group of molecules belonging to the stilbene family. stilbenes are synthesized via the phenylpropanoid/malonate pathway from phenylalanine that, in turn, is converted into cinnamic acid by phenylalanine ammonialyase. the consecutive action of cinnamate 4-hydroxylase and 4-coumarate coa ligase transform cinnamic acid into p-coumaryl-coa. compounds derived from this pathway, collectively referred to as polyphenols, are originated from this branching point through the action of the enzymes chalcone synthase, stilbene synthase and cinnamoyl-coa reductase for flavonoids, stilbenes, and lignin, respectively. members of the stilbene family are derivatives of the trans-resveratrol structure. piceide is a glycosylated resveratrol derivative and pterostilbene is a dimethylated resveratrol derivative. 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. pezet et al. tested the toxicity of these stilbenes against zoospores of p. viticola 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, and can be substances of pathogenic origin or compounds released by the plants in response to the action of a pathogen. inducers have potential uses in sustainable crop production. as critical components of many physiological processes, vitamins may influence the outcome of plant-pathogen interactions. in recent years, the importance of vitamins as nutrients and as control agents for different diseases has been demonstrated. ahn et al. have reported that thiamine 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 ca2+-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 h2o generation, upregulation of pr genes, and hypersensitive cell death. 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 demonstrated that phenylpropanoid pathway genes were upregulated by thiamine treatment. in addition, qualitative and quantitative analysis using high-performance liquid chromatography-diode array detection, ultra-performance liquid chromatography coupled with mass spectrometry, chromatographic online antioxidant detection system, and histochemical analyses revealed that phenylpropanoid-derived 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.  effect of thiamine treatment on downy mildew incidence results shown in figure  correspond to disease incidence on grapevine plants as mean % of plants with visible symptoms according to unger et al.. this data indicated that in control conditions, the average of plants showing pathogen sporulation was %, the average of plants showing pathogen oil spots was %, 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 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 time points. the first peak was detected within 12 hours post-treatment with a 370-fold expression, whereas the second peak was observed at hpt before decreasing progressively till hpt. c4h and 4cl mrna transcripts were also induced by thiamine treatment showing similar kinetics. in fact, both c4h and 4cl inductions peaked first time within hpt and next time within hpt before declining progressively till hpt. thiamine treatment also upregulated the expression of ccr gene in grapevine plants. the ccr induction peaked the first time within hpt and the second time within hpt before being decreased at hpt. both sts and chs genes were induced by thiamine treatment following a similar kinetic profile as that of the pal gene. indeed, sts and chs mrna transcripts peaked the first time at hpt and a second time within hpt. subsequently, sts and chs gene expressions decreased progressively until hpt. 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 sts gene induction by thiamine treatment and stilbenes production, the amounts of different stilbenic compounds were determined using hplc-dad at different time intervals post-treatment. the identity of the different stilbenoids in samples used for hplc-dad analysis was also confirmed via ulpc-dad-ms. our results showed that the contents of both trans-piceide and cis-piceide were markedly elevated in thiamine-treated plants as compared to those of water-treated samples for the investigated time points. the trans-piceide content increased with the increase in the time interval after thiamine application and reached its maximum level of  μg/g dry weight within 4 days post-treatment. however, the highest concentration of cis-piceide in thiamine-treated plants occurred at dpt. 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. trans-resveratrol induction was detected starting from dpt, increased progressively with time and reached its maximum level at dpt before decreasing at dpt. only very meager amounts of trans-resveratrol were detected in water-treated plants at the different time points of sampling. trans-ε-viniferin and trans-δ-viniferin were detected only in thiamine-treated plants. both trans-ε- and trans-δ-viniferins were produced starting from dpt in thiamine-treated plants; thereafter, their content increased progressively reaching maximal levels within dpt before decreasing at dpt. 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 starting from dpt. then its content increased gradually with time, reaching maximum level at dpt before decreasing within dpt. 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 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, was observed in leaf discs of thiamine-treated plants, indicating a marked induction of lignin synthesis in parenchymal tissues, whereas in the leaf discs of water-treated plants, we did not observe any lignin accumulation. for detection of phenols, leaf sections 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, unlike the leaf discs from water-treated plants. thiamine induced a prominent induction of flavonoids results of our molecular investigations have demonstrated that thiamine upregulated the expression of chs 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 at different time points by hplc-dad. our results 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. the amount of quercetin 3-o-glucoside flavonol was not significantly affected by thiamine treatment of grapevine plants. considering the large diversity of basal flavonoid structures in plants, 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 figure  shows 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 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 determinations. the total antioxidant activity results were expressed as trolox equivalent. the total antioxidant potential in thiamine-treated plants was 43 ±   μg/ml trolox equivalent, whereas in water treated-plants, it was 12 ±   μg/ml, indicating that the total antioxidant potential of thiamine-treated plants was -fold that of control plants. the largest antioxidant contribution came from only compounds, which were responsible for 97% of the total antioxidant potential of thiamine-treated 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. 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. here, we assessed their effect on the mycelia growth of the pathogen in grapevine plants, elicited by thiamine. accumulation of both stilbenes and pathogen mycelia was detected in foliar tissues by epifluorescence microscopy. the results, clearly showed the presence of altered, 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. 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, we observed a heavy infection of p. viticola in the leaves of water-treated plants, evidenced by the development of white sporulation symptoms, whereas in thiamine-treated samples, we observed only a development of necrotic zones corresponding to hypersensitive reaction related-cell death. discussion the major phytoalexins produced by v. vinifera cultivars during pathogen infections are resveratrol and its derivatives, piceide, δ-viniferin and ε-viniferin, and pterostilbene. in our experiment, thiamine treatment led to an induction of trans- and cis-piceide forms with maximal levels obtained within and dpt, respectively for each compound. thiamine treatment also induced the accumulation of trans-resveratrol with a maximal level observed within 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 times as toxic as ε-viniferin. the maximal levels of both ε- and δ-viniferin were observed at dpt. both ε- and δ-viniferin were not present in water-treated plants. the increase in resveratrol synthesis at dpt for thiamine-treated plants provided an important pool for the synthesis of viniferins that also occurred at dpt. in addition, thiamine treatment induced pterostilbene synthesis with a maximal induction level observed also at 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. moreover, pterostilbene exhibits antioxidant and anticancer properties similar to those of resveratrol. pterostilbene was found to be very effective in preventing carcinogen-induced preneoplastic lesions in a mouse mammary organ culture model, and it showed preventive activity against colon carcinogenesis in rats. 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. 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. 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. flavonoids have also been reported to be involved in the restriction of p. viticola growth in intermediate resistant species vitis rotundifolia and vitis rupestris. a part of phytoalexins functions as antioxidant and thereby contribute to plant resistance against pathogens. 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 of the phenylpropanoid pathway. 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. the first demonstrated example of phenolics providing disease resistance was the case of onion scales accumulating sufficient quantities of catechol and protocatechuic acid to prevent onion smudge disease, colletotrichum circinans. the colored outer onion scales of resistant onion varieties contain enough of these 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%. adequate levels of chlorogenic acid account for the resistance of potato tubers against phytophthora infestans. phenolics have also been reported to be involved in the restriction of p. viticola growth in intermediate resistant species v. rotundifolia and v. rupestris. in contrast, such metabolic response is absent in susceptible grapevine cultivars after pathogen inoculation. in addition, accumulation of phenolics has been reported to be associated with ir mechanisms in grapevine, such as β- -glucan- and chitosan-ir to p. viticola. on the other hand, the increased level of phenolics may provide an adequate substrate for oxidative reactions catalyzed by polyphenol oxidase and/or peroxidase, which, by consuming oxygen and producing fungitoxic quinones, make the medium unfavorable to the further development of pathogens. vanci et al. 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. in addition, cohen et al. 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, 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 by thiamine treatment also occurred at 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.  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 ε- and 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. 
the expression of these genes correlated with an accumulation of stilbenes, phenolic compounds, flavonoids and lignin. four phenolic compounds are responsible of 97% of the total anti-oxidant potential of thiamine-treated plants. 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. our data show that thiamine treatment elicited the expression of phenylpropanoid pathway genes in grapevine plants. 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. 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. among these compounds, is the caftaric acid, belonging to the hydroxy-cinnamic acids family. thiamineplasmopara viticolastilbenesphenylpropanoid pathway genesligninflavonoidsgrapevineinduced resistancereal-time q-pcrhplcuplc-ms however, mechanisms underlying vitamins, especially, thiamine-induced disease resistance in grapevine are still largely unknown.
 previously, we have reported the ability of thiamine 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.  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 -fold 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.  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. thiamineplasmopara viticolastilbenesphenylpropanoid pathway genesligninflavonoidsgrapevineinduced resistancereal-time q-pcrhplcuplc-ms 