BACKGROUND
plants respond to pathogens through constitutive and inducible mechanisms  <cit> .
structural barriers represent preformed constitutive defences, while the accumulation of pathogenesis-related proteins , phytoalexins and reactive oxygen species is part of an active mechanism stimulated by the pathogen  <cit> .
grapevine also responds to fungal infection via pr-protein synthesis and phytoalexin accumulation  <cit> .
plant phytoalexins are low-molecular-weight secondary metabolites with antimicrobial properties and they show wide chemical diversity among different plant species  <cit> .
in grapevine they mainly belong to the stilbene family and consist of trans-resveratrol  its oligomers, called viniferins  <cit>  and pterostilbene, a dimethylated derivative of resveratrol  <cit> .
stilbene synthesis in berries  <cit>  and leaves can be elicited by fungal infection  <cit> , but also by treatment with uv-irradiation  <cit> , ozone  <cit>  and heavy metals  <cit> .
plant cell cultures are a useful tool for studying plant cell defence response to biotic and abiotic elicitors  <cit> .
stilbene accumulation has been reported in grapevine cells treated with different elicitors: fungal cell wall fragments  <cit> , na-orthovanadate, jasmonic acid and methyljasmonate  <cit>  and laminarin, a β-glucan polysaccharide from brown algae  <cit> .
in addition, special attention has been given to the β-cyclodextrin molecular class.
these are cyclic oligosaccharides consisting of seven α-d-glucopyranose residues linked by α  <dig> →  <dig> glucosidic bonds forming a structure with a hydrophobic central cavity and a hydrophilic external surface  <cit> .
among β-cyclodextrins, heptakis-β-cyclodextrin , was reported to be the most effective resveratrol elicitor in different vitis vinifera cultivars  <cit> .
the ability of the modified β-cyclodextrins to act as elicitors probably resides in their chemical similarity to the alkyl-derivatized pectic oligosaccharides released from the cell walls during fungal infection  <cit> .
along with stilbene accumulation these experiments highlighted a more general response involving peroxidase activity as well as inhibition of botrytis cinerea growth  <cit> .
zamboni et al.
<cit>  further investigated dimeb activity on additional vitis genotypes and observed that its effect was more pronounced when tested on vitis riparia × vitis berlandieri cell cultures.
the kinetics of resveratrol synthesis showed that trans-resveratrol, the induced form, started to accumulate from  <dig> h after treatment and reached its maximum at  <dig> h. moreover, this metabolite was much more localized in the medium than within the cell.
with these results  <cit>  as our starting point, we report here the first large-scale transcriptional characterization of the early response of vitis riparia × vitis berlandieri cells to dimeb treatment.
after  <dig> h,  <dig> positively modulated genes were identified by suppression subtractive hybridization , whereas after  <dig> h,  <dig> genes turned out to be differentially expressed when control and treated cells on the vitis vinifera genechip® genome array  were compared.
these results showed that dimeb specifically modulates the expression of a small number of genes involved in resveratrol and lignin biosynthesis, pr synthesis, cell division and cell wall modification.
RESULTS
the ability of dimeb to elicit defence responses in grapevine cell culture was suggested by previous results showing stilbene accumulation, changes in peroxidase activity, as well as inhibition of botrytis cinerea growth  <cit> .
considerable stilbene accumulation in response to dimeb treatment was also observed by our group using non-vinifera  liquid cell cultures  <cit> .
in this study we analyzed the changes in gene expression of these cells elicited with dimeb after  <dig> h and  <dig> h using ssh and microarray experiments, respectively.
the rationale behind the two approaches was that after  <dig> h of treatment, a small number of genes are expected to be modulated, and only to a limited extent, whereas after  <dig> h an increase in the number of genes and in their expression level is envisaged.
the ssh technique appeared then the right choice for identifying the low abundance differential transcripts at  <dig> h, while the affymetrix genechip® microarray was used to measure the expression of a larger number of genes  after  <dig> h of treatment  <cit> .
starting with  <dig> clones from the constructed cdna subtractive library and then performing a hybridization screening to eliminate clones which were not really differentially expressed , we obtained  <dig> high-quality sequences which clustered in  <dig> tentative consensuses .
the microarray experiments instead identified  <dig>  significantly modulated probe sets in the treated cells compared with the control ones .
sequence annotation and classification according to gene ontology categories  <cit> , revealed that at both time points primary  and secondary metabolisms, together with response to the stimulus, were the most affected categories .
at  <dig> h, the analysis also highlighted downregulation of the cellular component organization and the biogenesis category .
in general, the two experiments showed modulation of specific mechanisms had already occurred at  <dig> h and continued more extensively at  <dig> h after dimeb treatment.
the data summarized in table  <dig> suggest that the grapevine cell responds to the elicitor by the activation of a signal transduction cascade which leads to the induction of specific classes of transcription factors.
the downstream effect of this process is, on the one hand, the induction of some branches of the secondary metabolism and defence response, and, on the other hand, the blockage of cell duplication .
acluster or affy id of transcripts modulated at  <dig> or  <dig> h.  and  refer to up- and down-regulation in the treated sample with respect to the control.
buniprotid  <cit> of the first hit obtained by “blast” analysis.
ctc: corresponding grapevine tentative consensus sequence obtained by a search  against the grape gene index database  <cit> .
at  <dig> h the treatment caused positive transcriptional regulation of a grapevine gene  encoding a protein with homology to an arabidopsis kinase-associated protein phosphatase  .
kapp protein may function as a signalling component in the pathway involving the serine-threonine receptor-like kinase, rlk <dig> of arabidopsis  <cit> .
in rice the rlk xa <dig> confers resistance to bacterial blight disease  <cit> .
other genes possibly involved in signal transduction showed overexpression at  <dig> h: a gene  with homology to a putative receptor-like protein kinase ark <dig> of oryza sativa and a gene  homologous to a glycine max salt overly sensitive gene encoding a sos2-like protein kinase .
in arabidopsis thaliana ark genes seem to be involved in plant defence response to wounding and to bacterial infections  <cit> , while sos <dig> is a signalling kinase involved in salt tolerance response  <cit> .
phospholipid-derived molecules are emerging as novel second messengers in plant defence signalling and phospholipases are key enzymes for their synthesis  <cit> .
in the array experiment we observed the overexpression of a putative phospholipase gene , which may generate lipid messengers for the signalling response .
the activation of a signal cascade generally induces the expression of genes encoding for specific transcription factors, which in turn regulate downstream effector genes.
two genes, upregulated at  <dig> h, showed homology to a hot pepper wrky-b  and arabidopsis wrky <dig>  respectively .
wrky proteins are plant-specific transcription factors whose expression is modulated in response to wounding, pathogen infection and abiotic stress  <cit> .
other classes of transcription factors appeared to take part in regulation of the response of grapevine cells to dimeb treatment.
the grape homologue  of a tomato pathogenesis-related gene transcriptional activator pti <dig> was upregulated at  <dig> h .
this transcription factor binds to the gcc-box cis element present in the promoter region of many plant pr genes  <cit>  and its upregulation could explain the observed induction of many pr proteins in this experiment.
another sequence , induced at  <dig> h, which might modulate the expression of pr genes is the homologue of the tobacco bzip tga <dig> factor .
it has been reported that this protein can bind to the regulatory activation sequence- <dig>   <cit>  identified in the promoter of arabidopsis pr- <dig> gene  <cit> .
our results indicated that one of the final grapevine cell responses to the dimeb-elicited signal consists in the modulation of phenolic metabolism, especially stilbene and monolignol biosynthesis .
genes encoding enzymes involved in phenylalanine biosynthesis such as 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase , 3-dehydroquinate synthase , prephenate dehydratase  and chorismate mutase  were positively modulated both at  <dig> and  <dig> h after dimeb treatment .
these enzymes participate in the synthesis of aromatic amino acids, particularly of phenylalanine, which is the link between primary and secondary metabolism, being a precursor of general phenylpropanoid metabolism.
a recent report showed that cyclodextrins stimulates the expression of the structural genes of the general phenylpropanoids metabolism which sustains the synthesis of p-cumaroyl coa, one of the two precursors of stilbenes  <cit> .
although we focused on the earlier cell response time, at both time points we also observed upregulation of this pathway's genes, namely phenylalanine ammonia lyase , cinnamic acid 4-hydroxylase  and 4-coumarate-coa ligase  .
similarly, several stilbene synthase genes were induced at  <dig> h and  <dig> h .
according to the classification proposed by richter et al.
<cit> , they correspond to  <dig> different stilbene synthase genes plus one pseudogene .
in particular, the probeset 1616575_at, encoding a stilbene synthase  <dig>  appeared to be the most induced one, being  <dig> times higher in the dimeb treated sample with respect to the control.
in agreement, the chemical analysis proved stilbene accumulation in the medium already at  <dig> h and at higher levels after  <dig> h, as previously reported  <cit> .
the accumulation of stilbenes in the growth medium requires, besides stilbene biosynthesis, the presence of export machinery.
in fact, induction of genes encoding putative secondary metabolite transporters, such as those belonging to the atp-binding cassette  transporter family, was found.
genes encoding for pleiotropic drug resistance -like abc transporters , abc transporter-like proteins  and a cjmdr transporter  were indeed induced .
the abc transporters play an important role in some host-pathogen interactions  <cit> .
in some pathogenic fungi they are involved in resistance to plant phytolexins and antifungal compounds, while in plants they seem to take part in plant defence response  <cit> .
the induction of genes encoding glutathione s-transferase  at  <dig> h correlates well with the abc-mediated transport .
a glutathione moiety seems to function as a "recognition tag" for the transport of phenols  <cit> .
resveratrol translocation outside the cells has two main objectives: to mediate the defence response against pathogens and to avoid intracellular accumulation of this compound at cytotoxic levels.
phenylpropanoid metabolism also produces the precursors  for the synthesis of monolignols, which are used to reinforce the cell wall during defence response  <cit> .
dimeb treatment caused a general induction of genes involved in their synthesis at  <dig> h: the genes for caffeic acid o-methyltransferase , caffeoyl-coa o-methyltransferase , cinnamyl alcohol dehydrogenase , ferulate 5-hydroxylase  and cinnamoyl-coa reductase  were overexpressed .
genes coding for enzymes such as polyphenol oxidase and diphenol oxidase, probably responsible for the lignin polymerization process  <cit> , were induced as well  .
the other branches of phenolic metabolism seemed not to be affected by dimeb.
only two genes of the anthocyanin pathway  and a flavonol-3-o-glucosyltransferase ) were induced at  <dig> h but not at  <dig> h .
interestingly, selective induction of the early steps of phenylpropanoid metabolism and of the late steps leading to monolignol biosynthesis was also described in arabidopsis in the early response to oligogalacturonide treatment  <cit> .
the results strongly suggest that dimeb acts as an elicitor modifying cell metabolism to promote the accumulation of phytoalexins and cell wall lignification.
these two defence responses have been described as typical biochemical responses occurring in vegetal cells after elicitor exposure  <cit> .
the transcriptional profiling results, however, show that the response to dimeb seems to include other defence mechanisms.
overexpression of sequences for pathogenesis-related proteins such as chitinase , pr- <dig>  and pr- <dig> , but also for a prolin-rich protein  and a protease inhibitor  was observed in both experiments, while upregulation of two genes encoding the s-adenosyl-l-methyonine:salicylic acid carboxyl methyltransferase  was recorded at  <dig> h .
interestingly, this enzyme mediates the synthesis of gaseous methyl salicylate which was recently demonstrated to be a key mediator in plant systemic acquired resistance  <cit>  in tobacco, as well as an inducer of the expression of pr- <dig> gene and tmv resistance  <cit> .
this result strengthens the hypothesis that dimeb acts as a true elicitor.
the increase in the expression of a gene encoding for a 1-aminocyclopropane-1-carboxylate oxidase , would suggest the involvement of ethylene as well .
this hormone is a major regulator of the plant's reaction to pathogen attack  <cit>  and via the action of a group of ethylene responsive factors it modulates the expression of plant defence-related genes such as, for example, phenylalanine ammonia-lyase, hydroxylproline-rich glycoprotein and acid class ii chitinase  <cit> .
it appears from the finding that a gene  homologous to an mlo-like  <dig> of arabidopsis was downregulated at  <dig> h , that the similarities between the cell's responses upon dimeb treatment and upon pathogen attack are even greater.
in barley, downregulation of the mlo gene is involved in response to powdery mildew caused by the fungus blumeria graminis f.sp.hordei  <cit> , and in the dicot arabidopsis thaliana, resistance to powdery mildews also depends on loss-of-function mlo alleles  <cit> .
our data support another effect of dimeb on grapevine cells: blockage of the cell-division process.
upon treatment, we measured a lower expression of the genes involved in modification of the cell wall structure, cell division and microtubule organization.
at  <dig> h, downregulation of genes related to cell wall modification  <cit> , such as those encoding expansins , xyloglucan endotransglycosylase , pectin methylesterases , a β-galactosidase , a polygalacturonase  and endoglucanases , was observed .
the sequence 1609506_at corresponds to the vvcel <dig> transcript which encodes a grapevine cellulase.
since in arabidopsis the expression of the cel <dig> gene was related to growing tissues  <cit> , downregulation of vvcel <dig> could be related to repression of the cell growth.
microtubules play an essential role in cell division and cell elongation too.
they set the cellular division planes and axes of elongation and influence the deposition and orientation of cellulose microfibrils  <cit> .
the downregulation of genes coding for α- and β-tubulin  is indication of a stop in cell expansion and cell division .
mrna degradation of a β-tubulin isoform was observed in soybean cells elicited by phytophthora sojae-derived glucan fragments suggesting re-routing of the cellular resources towards the defence-related metabolism and repression of the cellular growth  <cit> .
further indication of cell division reduction were the lower transcription of genes coding for histones h2a, h <dig>  h <dig> and h2b , a cyclin , a pattelin protein , a ga-induced-like protein  , a putative formin homology  protein  and a dna polymerase alpha catalytic subunit gene  .
all these proteins are either related to dna organization and synthesis or to the cytokinesis process.
the down-regulated grapevine gip gene is homologous to gip- <dig> of petunia hybrida, which is expressed during the cell division phase in stems and corollas  <cit> .
in arabidopsis patellin <dig> plays a role in membrane-trafficking when the cell-plate is formed during cytokinesis  <cit> , and formins are plant cytoskeleton-organizing proteins which take part in cytokinesis and in the establishment and maintenance of cell polarity  <cit> .
very similar effects on cell growth have been reported upon elicitation of parsley cell cultures with an oligopeptide elicitor.
pep  <dig> provoked the repression of genes regulating the cell cycle, such as cdc <dig>  cyclin and histones  <cit> .
a likely explanation for the repression of cell division would be the need of the cell to use, almost exclusively, the transcription system as well as the available resources to establish a defence-related metabolism.
CONCLUSIONS
the transcriptional profiles measured at  <dig> h and  <dig> h after dimeb treatment highlight the fact that this compound is able to induce an early and specific defence response in grapevine liquid cell cultures, supporting the hypothesis of its role as a true elicitor.
the classes of genes modulated by the treatment reveal that dimeb triggers a signal transduction cascade which activates different families of transcription factors, in turn modulating the effector genes of specific metabolisms.
these results thus suggest that in grapevine cells dimeb induces a stop in cell division, reinforcement of the cell wall and the production of resveratrol and defence proteins .
this response largely resembles that occurring upon pathogen attack.

 plants respond to pathogens through constitutive and inducible mechanisms. structural barriers represent preformed constitutive defences, while the accumulation of pathogenesis-related proteins, phytoalexins and reactive oxygen species is part of an active mechanism stimulated by the pathogen. grapevine also responds to fungal infection via pr-protein synthesis and phytoalexin accumulation. plant phytoalexins are low-molecular-weight secondary metabolites with antimicrobial properties and they show wide chemical diversity among different plant species. in grapevine they mainly belong to the stilbene family and consist of trans-resveratrol its oligomers, called viniferins and pterostilbene, a dimethylated derivative of resveratrol. stilbene synthesis in berries and leaves can be elicited by fungal infection, but also by treatment with uv-irradiation, ozone and heavy metals. plant cell cultures are a useful tool for studying plant cell defence response to biotic and abiotic elicitors. stilbene accumulation has been reported in grapevine cells treated with different elicitors: fungal cell wall fragments, na-orthovanadate, jasmonic acid and methyljasmonate and laminarin, a β-glucan polysaccharide from brown algae. in addition, special attention has been given to the β-cyclodextrin molecular class. these are cyclic oligosaccharides consisting of seven α-d-glucopyranose residues linked by α → glucosidic bonds forming a structure with a hydrophobic central cavity and a hydrophilic external surface. among β-cyclodextrins, heptakis-β-cyclodextrin, was reported to be the most effective resveratrol elicitor in different vitis vinifera cultivars. the ability of the modified β-cyclodextrins to act as elicitors probably resides in their chemical similarity to the alkyl-derivatized pectic oligosaccharides released from the cell walls during fungal infection. along with stilbene accumulation these experiments highlighted a more general response involving peroxidase activity as well as inhibition of botrytis cinerea growth. zamboni et al. further investigated dimeb activity on additional vitis genotypes and observed that its effect was more pronounced when tested on vitis riparia × vitis berlandieri cell cultures. the kinetics of resveratrol synthesis showed that trans-resveratrol, the induced form, started to accumulate from h after treatment and reached its maximum at h. moreover, this metabolite was much more localized in the medium than within the cell. with these results as our starting point, we report here the first large-scale transcriptional characterization of the early response of vitis riparia × vitis berlandieri cells to dimeb treatment. after h, positively modulated genes were identified by suppression subtractive hybridization, whereas after h, genes turned out to be differentially expressed when control and treated cells on the vitis vinifera genechip® genome array were compared. these results showed that dimeb specifically modulates the expression of a small number of genes involved in resveratrol and lignin biosynthesis, pr synthesis, cell division and cell wall modification.  the ability of dimeb to elicit defence responses in grapevine cell culture was suggested by previous results showing stilbene accumulation, changes in peroxidase activity, as well as inhibition of botrytis cinerea growth. considerable stilbene accumulation in response to dimeb treatment was also observed by our group using non-vinifera liquid cell cultures. in this study we analyzed the changes in gene expression of these cells elicited with dimeb after h and h using ssh and microarray experiments, respectively. the rationale behind the two approaches was that after h of treatment, a small number of genes are expected to be modulated, and only to a limited extent, whereas after h an increase in the number of genes and in their expression level is envisaged. the ssh technique appeared then the right choice for identifying the low abundance differential transcripts at h, while the affymetrix genechip® microarray was used to measure the expression of a larger number of genes after h of treatment. starting with clones from the constructed cdna subtractive library and then performing a hybridization screening to eliminate clones which were not really differentially expressed, we obtained high-quality sequences which clustered in tentative consensuses. the microarray experiments instead identified significantly modulated probe sets in the treated cells compared with the control ones. sequence annotation and classification according to gene ontology categories, revealed that at both time points primary and secondary metabolisms, together with response to the stimulus, were the most affected categories. at h, the analysis also highlighted downregulation of the cellular component organization and the biogenesis category. in general, the two experiments showed modulation of specific mechanisms had already occurred at h and continued more extensively at h after dimeb treatment. the data summarized in table suggest that the grapevine cell responds to the elicitor by the activation of a signal transduction cascade which leads to the induction of specific classes of transcription factors. the downstream effect of this process is, on the one hand, the induction of some branches of the secondary metabolism and defence response, and, on the other hand, the blockage of cell duplication. acluster or affy id of transcripts modulated at or h. and refer to up- and down-regulation in the treated sample with respect to the control. buniprotid of the first hit obtained by “blast” analysis. ctc: corresponding grapevine tentative consensus sequence obtained by a search against the grape gene index database. at h the treatment caused positive transcriptional regulation of a grapevine gene encoding a protein with homology to an arabidopsis kinase-associated protein phosphatase. kapp protein may function as a signalling component in the pathway involving the serine-threonine receptor-like kinase, rlk of arabidopsis. in rice the rlk xa confers resistance to bacterial blight disease. other genes possibly involved in signal transduction showed overexpression at h: a gene with homology to a putative receptor-like protein kinase ark of oryza sativa and a gene homologous to a glycine max salt overly sensitive gene encoding a sos2-like protein kinase. in arabidopsis thaliana ark genes seem to be involved in plant defence response to wounding and to bacterial infections, while sos is a signalling kinase involved in salt tolerance response. phospholipid-derived molecules are emerging as novel second messengers in plant defence signalling and phospholipases are key enzymes for their synthesis. in the array experiment we observed the overexpression of a putative phospholipase gene, which may generate lipid messengers for the signalling response. the activation of a signal cascade generally induces the expression of genes encoding for specific transcription factors, which in turn regulate downstream effector genes. two genes, upregulated at h, showed homology to a hot pepper wrky-b and arabidopsis wrky respectively. wrky proteins are plant-specific transcription factors whose expression is modulated in response to wounding, pathogen infection and abiotic stress. other classes of transcription factors appeared to take part in regulation of the response of grapevine cells to dimeb treatment. the grape homologue of a tomato pathogenesis-related gene transcriptional activator pti was upregulated at h. this transcription factor binds to the gcc-box cis element present in the promoter region of many plant pr genes and its upregulation could explain the observed induction of many pr proteins in this experiment. another sequence, induced at h, which might modulate the expression of pr genes is the homologue of the tobacco bzip tga factor. it has been reported that this protein can bind to the regulatory activation sequence- identified in the promoter of arabidopsis pr- gene. our results indicated that one of the final grapevine cell responses to the dimeb-elicited signal consists in the modulation of phenolic metabolism, especially stilbene and monolignol biosynthesis. genes encoding enzymes involved in phenylalanine biosynthesis such as 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, 3-dehydroquinate synthase, prephenate dehydratase and chorismate mutase were positively modulated both at and h after dimeb treatment. these enzymes participate in the synthesis of aromatic amino acids, particularly of phenylalanine, which is the link between primary and secondary metabolism, being a precursor of general phenylpropanoid metabolism. a recent report showed that cyclodextrins stimulates the expression of the structural genes of the general phenylpropanoids metabolism which sustains the synthesis of p-cumaroyl coa, one of the two precursors of stilbenes. although we focused on the earlier cell response time, at both time points we also observed upregulation of this pathway's genes, namely phenylalanine ammonia lyase, cinnamic acid 4-hydroxylase and 4-coumarate-coa ligase. similarly, several stilbene synthase genes were induced at h and h. according to the classification proposed by richter et al., they correspond to different stilbene synthase genes plus one pseudogene. in particular, the probeset 1616575_at, encoding a stilbene synthase appeared to be the most induced one, being times higher in the dimeb treated sample with respect to the control. in agreement, the chemical analysis proved stilbene accumulation in the medium already at h and at higher levels after h, as previously reported. the accumulation of stilbenes in the growth medium requires, besides stilbene biosynthesis, the presence of export machinery. in fact, induction of genes encoding putative secondary metabolite transporters, such as those belonging to the atp-binding cassette transporter family, was found. genes encoding for pleiotropic drug resistance -like abc transporters, abc transporter-like proteins and a cjmdr transporter were indeed induced. the abc transporters play an important role in some host-pathogen interactions. in some pathogenic fungi they are involved in resistance to plant phytolexins and antifungal compounds, while in plants they seem to take part in plant defence response. the induction of genes encoding glutathione s-transferase at h correlates well with the abc-mediated transport. a glutathione moiety seems to function as a "recognition tag" for the transport of phenols. resveratrol translocation outside the cells has two main objectives: to mediate the defence response against pathogens and to avoid intracellular accumulation of this compound at cytotoxic levels. phenylpropanoid metabolism also produces the precursors for the synthesis of monolignols, which are used to reinforce the cell wall during defence response. dimeb treatment caused a general induction of genes involved in their synthesis at h: the genes for caffeic acid o-methyltransferase, caffeoyl-coa o-methyltransferase, cinnamyl alcohol dehydrogenase, ferulate 5-hydroxylase and cinnamoyl-coa reductase were overexpressed. genes coding for enzymes such as polyphenol oxidase and diphenol oxidase, probably responsible for the lignin polymerization process, were induced as well. the other branches of phenolic metabolism seemed not to be affected by dimeb. only two genes of the anthocyanin pathway and a flavonol-3-o-glucosyltransferase ) were induced at h but not at h. interestingly, selective induction of the early steps of phenylpropanoid metabolism and of the late steps leading to monolignol biosynthesis was also described in arabidopsis in the early response to oligogalacturonide treatment. the results strongly suggest that dimeb acts as an elicitor modifying cell metabolism to promote the accumulation of phytoalexins and cell wall lignification. these two defence responses have been described as typical biochemical responses occurring in vegetal cells after elicitor exposure. the transcriptional profiling results, however, show that the response to dimeb seems to include other defence mechanisms. overexpression of sequences for pathogenesis-related proteins such as chitinase, pr- and pr-, but also for a prolin-rich protein and a protease inhibitor was observed in both experiments, while upregulation of two genes encoding the s-adenosyl-l-methyonine:salicylic acid carboxyl methyltransferase was recorded at h. interestingly, this enzyme mediates the synthesis of gaseous methyl salicylate which was recently demonstrated to be a key mediator in plant systemic acquired resistance in tobacco, as well as an inducer of the expression of pr- gene and tmv resistance. this result strengthens the hypothesis that dimeb acts as a true elicitor. the increase in the expression of a gene encoding for a 1-aminocyclopropane-1-carboxylate oxidase, would suggest the involvement of ethylene as well. this hormone is a major regulator of the plant's reaction to pathogen attack and via the action of a group of ethylene responsive factors it modulates the expression of plant defence-related genes such as, for example, phenylalanine ammonia-lyase, hydroxylproline-rich glycoprotein and acid class ii chitinase. it appears from the finding that a gene homologous to an mlo-like of arabidopsis was downregulated at h, that the similarities between the cell's responses upon dimeb treatment and upon pathogen attack are even greater. in barley, downregulation of the mlo gene is involved in response to powdery mildew caused by the fungus blumeria graminis f.sp.hordei, and in the dicot arabidopsis thaliana, resistance to powdery mildews also depends on loss-of-function mlo alleles. our data support another effect of dimeb on grapevine cells: blockage of the cell-division process. upon treatment, we measured a lower expression of the genes involved in modification of the cell wall structure, cell division and microtubule organization. at h, downregulation of genes related to cell wall modification, such as those encoding expansins, xyloglucan endotransglycosylase, pectin methylesterases, a β-galactosidase, a polygalacturonase and endoglucanases, was observed. the sequence 1609506_at corresponds to the vvcel transcript which encodes a grapevine cellulase. since in arabidopsis the expression of the cel gene was related to growing tissues, downregulation of vvcel could be related to repression of the cell growth. microtubules play an essential role in cell division and cell elongation too. they set the cellular division planes and axes of elongation and influence the deposition and orientation of cellulose microfibrils. the downregulation of genes coding for α- and β-tubulin is indication of a stop in cell expansion and cell division. mrna degradation of a β-tubulin isoform was observed in soybean cells elicited by phytophthora sojae-derived glucan fragments suggesting re-routing of the cellular resources towards the defence-related metabolism and repression of the cellular growth. further indication of cell division reduction were the lower transcription of genes coding for histones h2a, h h and h2b, a cyclin, a pattelin protein, a ga-induced-like protein, a putative formin homology protein and a dna polymerase alpha catalytic subunit gene. all these proteins are either related to dna organization and synthesis or to the cytokinesis process. the down-regulated grapevine gip gene is homologous to gip- of petunia hybrida, which is expressed during the cell division phase in stems and corollas. in arabidopsis patellin plays a role in membrane-trafficking when the cell-plate is formed during cytokinesis, and formins are plant cytoskeleton-organizing proteins which take part in cytokinesis and in the establishment and maintenance of cell polarity. very similar effects on cell growth have been reported upon elicitation of parsley cell cultures with an oligopeptide elicitor. pep provoked the repression of genes regulating the cell cycle, such as cdc cyclin and histones. a likely explanation for the repression of cell division would be the need of the cell to use, almost exclusively, the transcription system as well as the available resources to establish a defence-related metabolism.  the transcriptional profiles measured at h and h after dimeb treatment highlight the fact that this compound is able to induce an early and specific defence response in grapevine liquid cell cultures, supporting the hypothesis of its role as a true elicitor. the classes of genes modulated by the treatment reveal that dimeb triggers a signal transduction cascade which activates different families of transcription factors, in turn modulating the effector genes of specific metabolisms. these results thus suggest that in grapevine cells dimeb induces a stop in cell division, reinforcement of the cell wall and the production of resveratrol and defence proteins. this response largely resembles that occurring upon pathogen attack. 
at both time points, we identified a specific set of induced genes belonging to the general phenylpropanoid metabolism, including stilbenes and hydroxycinnamates, and to defence proteins such as pr proteins and chitinases. at h we also observed a down-regulation of the genes involved in cell division and cell-wall loosening. this molecule seems to mimic a defence elicitor which enhances the physical barriers of the cell, stops cell division and induces phytoalexin synthesis. in response to pathogen attack, grapevine synthesizes phytoalexins belonging to the family of stilbenes.
 in response to pathogen attack, grapevine synthesizes phytoalexins belonging to the family of stilbenes. grapevine cell cultures represent a good model system for studying the basic mechanisms of plant response to biotic and abiotic elicitors. among these, modified β-cyclodextrins seem to act as true elicitors inducing strong production of the stilbene resveratrol.  the transcriptome changes of vitis riparia × vitis berlandieri grapevine cells in response to the modified β-cyclodextrin, dimeb, were analyzed and h after treatment using a suppression subtractive hybridization experiment and a microarray analysis respectively. at both time points, we identified a specific set of induced genes belonging to the general phenylpropanoid metabolism, including stilbenes and hydroxycinnamates, and to defence proteins such as pr proteins and chitinases. at h we also observed a down-regulation of the genes involved in cell division and cell-wall loosening.  we report the first large-scale study of the molecular effects of dimeb, a resveratrol inducer, on grapevine cell cultures. this molecule seems to mimic a defence elicitor which enhances the physical barriers of the cell, stops cell division and induces phytoalexin synthesis. 