Open Access

Grapevine cell early activation of specific responses to DIMEB, a resveratrol elicitor

  • Anita Zamboni1, 2,
  • Pamela Gatto1, 3, 2,
  • Alessandro Cestaro1,
  • Stefania Pilati1,
  • Roberto Viola1,
  • Fulvio Mattivi1,
  • Claudio Moser1Email author and
  • Riccardo Velasco1
BMC Genomics200910:363

https://doi.org/10.1186/1471-2164-10-363

Received: 25 March 2009

Accepted: 6 August 2009

Published: 6 August 2009

Abstract

Background

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.

Results

The transcriptome changes of Vitis riparia × Vitis berlandieri grapevine cells in response to the modified β-cyclodextrin, DIMEB, were analyzed 2 and 6 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 6 h we also observed a down-regulation of the genes involved in cell division and cell-wall loosening.

Conclusions

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.

Background

Plants respond to pathogens through constitutive and inducible mechanisms [1]. Structural barriers represent preformed constitutive defences, while the accumulation of pathogenesis-related proteins (PR), phytoalexins and reactive oxygen species is part of an active mechanism stimulated by the pathogen [2]. Grapevine also responds to fungal infection via PR-protein synthesis and phytoalexin accumulation [3]. Plant phytoalexins are low-molecular-weight secondary metabolites with antimicrobial properties and they show wide chemical diversity among different plant species [4]. In grapevine they mainly belong to the stilbene family and consist of trans-resveratrol (3,5,4'-trihydroxystilbene) its oligomers, called viniferins [57] and pterostilbene, a dimethylated derivative of resveratrol [8]. Stilbene synthesis in berries [9] and leaves can be elicited by fungal infection [5, 10], but also by treatment with UV-irradiation [11], ozone [12] and heavy metals [13].

Plant cell cultures are a useful tool for studying plant cell defence response to biotic and abiotic elicitors [14]. Stilbene accumulation has been reported in grapevine cells treated with different elicitors: fungal cell wall fragments [15], Na-orthovanadate, jasmonic acid and methyljasmonate [16, 17] and laminarin, a β-glucan polysaccharide from brown algae [18]. 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 1 → 4 glucosidic bonds forming a structure with a hydrophobic central cavity and a hydrophilic external surface [19]. Among β-cyclodextrins, heptakis(2,6-di-O-methyl)-β-cyclodextrin (DIMEB), was reported to be the most effective resveratrol elicitor in different Vitis vinifera cultivars [19, 20]. 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 [20]. Along with stilbene accumulation these experiments highlighted a more general response involving peroxidase activity as well as inhibition of Botrytis cinerea growth [19, 20].

Zamboni et al. [21] 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 6 h after treatment and reached its maximum at 24 h. Moreover, this metabolite was much more localized in the medium than within the cell.

With these results [21] 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 2 h, 127 positively modulated genes were identified by suppression subtractive hybridization (SSH), whereas after 6 h, 371 genes turned out to be differentially expressed when control and treated cells on the Vitis vinifera GeneChip® Genome Array (Affymetrix) 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 and discussion

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 [19, 20]. Considerable stilbene accumulation in response to DIMEB treatment was also observed by our group using non-vinifera (Vitis riparia × Vitis berlandieri) liquid cell cultures [21]. In this study we analyzed the changes in gene expression of these cells elicited with DIMEB after 2 h and 6 h using SSH and microarray experiments, respectively.

The rationale behind the two approaches was that after 2 h of treatment, a small number of genes are expected to be modulated, and only to a limited extent, whereas after 6 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 2 h, while the Affymetrix GeneChip® microarray was used to measure the expression of a larger number of genes (~14,500 unigenes) after 6 h of treatment [22].

Starting with 384 clones from the constructed cDNA subtractive library and then performing a hybridization screening to eliminate clones which were not really differentially expressed (false positives), we obtained 168 high-quality sequences which clustered in 127 tentative consensuses (Additional File 1). The microarray experiments instead identified 371 (223 upregulated and 148 downregulated) significantly modulated probe sets in the treated cells compared with the control ones (Additional File 2). Sequence annotation and classification according to Gene Ontology categories [23], revealed that at both time points primary (mainly signal transduction related genes) and secondary metabolisms, together with response to the stimulus, were the most affected categories (Additional Files 3 and 4). At 6 h, the analysis also highlighted downregulation of the cellular component organization and the biogenesis category (Additional file 4).

In general, the two experiments showed modulation of specific mechanisms had already occurred at 2 h and continued more extensively at 6 h after DIMEB treatment. The data summarized in Table 1 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 (Figure 1).
Table 1

List of transcripts modulated by DIMEB and reported in the Discussion

IDa

Description

Uniprot IDb

TC-IDc

2 h

6 h

    

+

+

-

Signal trasduction

CLU090

Kinase associated protein phosphatase

P46014

EC987592

x

  

1608981_at

Putative phospholipase

Q8RXN7

TC69626

 

x

 

1620080_at

Putative receptor-like protein kinase ARK1

Q5ZAK8

CB922377

 

x

 

1611172_at

SOS2-like protein kinase

Q8LK24

TC52484

 

x

 

Transcription factors

1619311_at

Pathogenesis-related genes transcriptional activator PTI5

O04681

TC55556

 

x

 

1611285_s_at

Probable WRKY transcription factor 11

Q9SV15

TC65678

 

x

 

CLU059

TGA10 transcription factor

Q52MZ2

TC99087

x

  

1610775_s_at

WRKY transcription factor-b

Q5DJU0

TC55553

 

x

 

Effector genes

Phe biosynthesis

CLU083

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase precursor

O24051

TC74975

x

  

1611211_at

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase precursor

O24046

TC57386

 

x

 

1614440_at

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase

Q6YH16

TC54321

 

x

 

1619357_at

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase

O24046

TC57642

 

x

 

1621405_at

Plastidic 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 2

O22407

TC51974

 

x

 

1609646_at

3-Dehydroquinate synthase-like protein

Q9FKX0

TC56854

 

x

 

1609932_at

Prephenate dehydratase

Q6JJ29

TC53641

 

x

 

1621307_at

Prephenate dehydratase

Q6JJ29

TC53641

 

x

 

1611895_at

Putative chorismate mutase

Q5JN19

TC62307

 

x

 

General phenylpropanoid metabolism

1613113_at

Phenylalanine ammonia lyase

Q6UD65

TC60180

   

CLU024

Trans-cinnamate 4-monooxygenase

Q43240

TC71512

x

  

1610821_at

Cinnamic acid 4-hydroxylase

Q948S8

TC70715

 

x

 

1616191_s_at

Cinnamic acid 4-hydroxylase

Q948S8

TC70715

 

x

 

1615801_at

4-Coumarate:CoA ligase

Q5S017

TC60943

 

x

 

1619320_at

4-Coumarate--CoA ligase 2

P31687

TC66743

 

x

 

Stilbene biosynthesis

CLU009

Stilbene synthase

Q9SPW2

TC89701

x

  

CLU022

Stilbene synthase

Q6BAU9

TC89632

x

  

CLU023

Stilbene synthase

P28343

TC84974

x

  

CLU049

Stilbene synthase

Q8LPP4

TC78210

x

  

CLU097

Stilbene synthase

Q9S982

TC84974

x

  

CLU103

Stilbene synthase

P28343

TC88894

x

  

1606750_at

Stilbene synthase

Q6BAL2

TC67020

 

x

 

1608009_s_at

Stilbene synthase

P51070

  

x

 

1609696_x_at

Stilbene synthase

P28343

TC67020

 

x

 

1609697_at

Stilbene synthase

Q944W7

TC60946

 

x

 

1610824_s_at

Stilbene synthase

Q93YX5

TC52746

 

x

 

1610850_at

Stilbene synthase

P28343

  

x

 

1611190_s_at

Resveratrol synthase

Q94G58

TC67020

 

x

 

1612804_at

Stilbene synthase

Q9SPW2

TC52746

 

x

 

1614621_at

Stilbene synthase

P28343

TC67020

 

x

 

1616575_at

Stilbene synthase

Q944W7

TC52746

 

x

 

1620964_s_at

Stilbene synthase

P28343

  

x

 

1622638_x_at

Stilbene synthase

Q9SPW2

TC52746

 

x

 

Secondary metabolite transport

CLU106

PDR-like ABC transporter

Q8GU88

TC76318

x

  

CLU119

Pleiotropic drug resistance protein 12

Q5Z9S8

TC81892

x

  

1613763_at

ABC transporter-like protein

Q9LYS2

TC60768

 

x

 

1618493_s_at

ABC transporter-like protein

Q9LYS2

TC64210

 

x

 

1610363_at

CjMDR1

Q94IH6

TC69843

 

x

 

1609330_at

Glutathione S-transferase

Q6YEY5

NP864091

 

x

 

1611890_at

Glutathione S-transferase GST 14

Q9FQE4

TC61062

 

x

 

1619682_x_at

Caffeic acid O-methyltransferase

Q9M560

TC52364

 

x

 

1620342_at

Caffeic acid 3-O-methyltransferase 1

Q00763

TC64352

 

x

 

Lignin biosynthesis

1611897_s_at

Caffeoyl-CoA O-methyltransferase

Q8H9B6

TC63685

 

x

 

1614643_at

Caffeoyl-CoA O-methyltransferase

Q43237

TC51729

 

x

 

1613900_at

Cinnamyl alcohol dehydrogenase

Q9ATW1

TC52904

 

x

 

1614045_at

Ferulate 5-hydroxylase

Q6IV45

TC64493

 

x

 

1614502_at

Ferulate 5-hydroxylase

Q6IV45

TC63764

 

x

 

1619065_at

Putative cinnamoyl-CoA reductase

Q8W3H0

TC53437

 

x

 

1622651_at

Polyphenol oxidase

Q68NI4

TC58764

 

x

 

1610806_at

Putative diphenol oxidase

Q6Z8L2

CD007812

 

x

 

CLU122

Chalcone-flavonone isomerase

P51117

TC78712

x

  

CLU048

Flavonol 3-O-glucosyltransferase 6

Q40288

TC85607

x

  

1621051_at

Flavonol 3-O-glucosyltransferase 2

Q40285

CN006197

  

x

Defence response

CLU088

Chitinase (Class II)

Q43322

TC95665

x

  

1613871_at

Class IV chitinase

Q9M2U5

TC57889

 

x

 

1617192_at

Class IV chitinase

Q7XB39

TC63731

 

x

 

1617430_s_at

Basic endochitinase precursor

P51613

TC51704

 

x

 

CLU001

Pathogenesis-related protein10

Q9FS42

TC72098

x

  

1610011_s_at

Pathogenesis-related protein10

Q9FS42

  

x

 

1618568_s_at

Pathogenesis-related protein10

Q9FS42

  

x

 

CLU021

Pathogenesis-related protein PR-4A precursor

P29062

TC91296

x

  

CLU036

Merlot proline-rich protein 2

Q6QGY1

TC85591

x

  

1609875_at

Protease inhibitor

Q6YEY6

  

x

 

1611666_s_at

Protease inhibitor

Q6YEY6

TC70006

 

x

 

1612552_at

Putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase

Q9C9W8

TC57170

 

x

 

1620309_at

Putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase

Q9C9W8

TC63451

 

x

 

1622147_at

1-Aminocyclopropane-1-carboxylate oxidase 3

Q08507

TC60326

 

x

 

1616358_at

MLO-like protein 11

Q9FI00

BQ798612

  

x

Cell wall metabolism

1608074_s_at

Expansin

Q84UT0

TC62965

  

x

1620840_at

Alpha-expansin

Q8LKJ8

TC53122

  

x

1615995_at

Xyloglucan endotransglycosylase XET2

Q9LLC2

CF212592

  

x

1620003_at

Xyloglucan endotransglycosylase 1

Q9ZRV1

TC63269

  

x

1608799_at

Pectin methylesterase

Q96497

TC58800

  

x

1619468_at

Pectin methylesterase PME1

Q94B16

TC53043

  

x

1619522_at

Putative beta-galactosidase BG1

Q94B17

TC56838

  

x

1608756_at

Polygalacturonase-like protein

Q84LI7

TC59719

  

x

1606763_at

Putative beta-1,3-glucanase

Q8L868

TC67051

  

x

1609506_at

Putative cellulase CEL2

Q94B13

NP596365

  

x

1610263_at

Putative beta-1,3-glucanase

Q8L868

TC67051

  

x

Cell duplication

1612320_a_at

Tubulin alpha chain

P33629

TC57547

  

x

1616815_at

Tubulin beta-8 chain

Q41785

TC55048

  

x

1618413_at

Tubulin alpha chain

P33629

TC63601

  

x

1619167_at

Tubulin beta-8 chain

Q41785

TC62643

  

x

1621015_at

Alpha-tubulin 1

Q8H6M1

TC65238

  

x

1622466_at

Tubulin beta-8 chain

Q41785

TC62809

  

x

1608927_at

Putative histone H2A

Q6L500

TC53574

  

x

1612573_at

Histone H3

A2Y533

TC56731

  

x

1613041_at

Histone H4

Q76H85

TC61904

  

x

1613076_at

Histone H4

Q76H85

TC62637

  

x

1620332_at

Histone H3

A2Y533

TC59489

  

x

1622440_at

Histone H3

A2Y533

TC64779

  

x

1622737_at

Histone H2B

O22582

TC64405

  

x

1610854_at

Proliferating cell nuclear antigen

P22177

TC54817

  

x

1610422_at

Patellin-6

Q9SCU1

TC61622

  

x

1610607_at

Gip1-like protein

Q93WR4

TC66111

  

x

1613373_at

Formin-like protein 1

Q8S0F0

TC55249

  

x

1607792_at

Putative DNA polymerase alpha catalytic subunit

O48653

TC59012

  

x

aCluster or Affy ID of transcripts modulated at 2 or 6 h. (+) and (-) refer to up- and down-regulation in the treated sample with respect to the control.

bUniprotID [73]of the first hit obtained by “Blast” analysis.

cTC: corresponding grapevine Tentative Consensus sequence obtained by a search (BlastN) against the Grape Gene Index database [75].

Figure 1

Molecular events triggered by DIMEB as deduced by transcriptional profiling.

At 2 h the treatment caused positive transcriptional regulation of a grapevine gene (CLU090) encoding a protein with homology to an Arabidopsis kinase-associated protein phosphatase (KAPP) (Table 1). KAPP protein may function as a signalling component in the pathway involving the serine-threonine receptor-like kinase, RLK5 of Arabidopsis [24]. In rice the RLK XA21 confers resistance to bacterial blight disease [25]. Other genes possibly involved in signal transduction showed overexpression at 6 h: a gene (1620080_at) with homology to a putative receptor-like protein kinase ARK1 of Oryza sativa and a gene (1611172_at) homologous to a Glycine max Salt Overly Sensitive gene encoding a SOS2-like protein kinase (Table 1). In Arabidopsis thaliana ARK genes seem to be involved in plant defence response to wounding and to bacterial infections [26], while SOS2 is a signalling kinase involved in salt tolerance response [27]. Phospholipid-derived molecules are emerging as novel second messengers in plant defence signalling and phospholipases are key enzymes for their synthesis [14, 28]. In the array experiment we observed the overexpression of a putative phospholipase gene (1608981_at), which may generate lipid messengers for the signalling response (Table 1).

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 6 h, showed homology to a hot pepper WRKY-b (1610775_s_at) and Arabidopsis WRKY11 (1611285_s_at) respectively (Table 1). WRKY proteins are plant-specific transcription factors whose expression is modulated in response to wounding, pathogen infection and abiotic stress [29]. Other classes of transcription factors appeared to take part in regulation of the response of grapevine cells to DIMEB treatment. The grape homologue (1619311_at) of a tomato pathogenesis-related gene transcriptional activator PTI5 was upregulated at 6 h (Table 1). This transcription factor binds to the GCC-box cis element present in the promoter region of many plant PR genes [30] and its upregulation could explain the observed induction of many PR proteins in this experiment. Another sequence (CLU059), induced at 2 h, which might modulate the expression of PR genes is the homologue of the tobacco bZIP TGA10 factor (Table 1). It has been reported that this protein can bind to the regulatory activation sequence-1 (as-1) [31] identified in the promoter of Arabidopsis PR-1 gene [32].

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 (Figure 2).
Figure 2

Modulation of secondary metabolism at 2 and 6 h after DIMEB treatment. Modulation (+ or -) of genes encoding enzymes of phenylalanine biosynthesis, general phenylpropanoid metabolism, monolignol, stilbene and anthocyanin pathways are reported within a simplified secondary metabolism scheme. Abbreviations: DHAP synthase, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase; DHQ synthase, 3-dehydroquinate synthase; CM, chorismate mutase; PDT, prephenate dehydratase; PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CAD, cinnamyl alchol dehydrogenase; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; COMT, caffeic acid O-methyltransferase; CCR, cinnamoyl-CoA reductase; F5H, ferulate-5-hydroxylase; STS, stilbene synthase; CHI, chalcone isomerase; UFGT, flavonoid-3-O-glucosyltransferase.

Genes encoding enzymes involved in phenylalanine biosynthesis such as 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (CLU083; 1611211_at; 1614440_at; 1619357_at; 1621405_at), 3-dehydroquinate synthase (1609646_at), prephenate dehydratase (1609932_at; 1621307_at) and chorismate mutase (1611895_at) were positively modulated both at 2 and 6 h after DIMEB treatment (Table 1). 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 [17].

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 (1613113_at), cinnamic acid 4-hydroxylase (CLU024; 1610821_at; 1616191_s_at) and 4-coumarate-CoA ligase (1615801_at; 1619320_at) (Table 1). Similarly, several stilbene synthase genes were induced at 2 h and 6 h (CLU009, CLU022, CLU023, CLU049, CLU097, CLU103, 1606750_at, 1608009_s_at, 1609696_x_at, 1609697_at, 1610824_s_at, 1610850_at, 1611190_s_at, 1612804_at, 1614621_at, 1616575_at, 1620964_s_at, 1622638_x_at). According to the classification proposed by Richter et al. [33], they correspond to 7 different stilbene synthase genes plus one pseudogene (1606750_at). In particular, the probeset 1616575_at, encoding a stilbene synthase 2, appeared to be the most induced one, being 23 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 2 h and at higher levels after 6 h, as previously reported [21].

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 (ABC) transporter family, was found. Genes encoding for pleiotropic drug resistance (PDR)-like ABC transporters (CLU106; CLU119), ABC transporter-like proteins (1613763_at; 1618493_s_at) and a CjMDR transporter (1610363_at) were indeed induced (Table 1). The ABC transporters play an important role in some host-pathogen interactions [34]. 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 [34]. The induction of genes encoding glutathione S-transferase (1609330_at; 1611890_at) at 6 h correlates well with the ABC-mediated transport (Table 1). A glutathione moiety seems to function as a "recognition tag" for the transport of phenols [35]. 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 (p-coumarate and p-coumaroyl-CoA) for the synthesis of monolignols, which are used to reinforce the cell wall during defence response [36]. DIMEB treatment caused a general induction of genes involved in their synthesis at 6 h: the genes for caffeic acid O-methyltransferase (1607475_s_at, 1619682_x_at, 1620342_at), caffeoyl-CoA O-methyltransferase (1611897_s_at; 1614643_at), cinnamyl alcohol dehydrogenase (1613900_at), ferulate 5-hydroxylase (1614045_at; 1614502_at) and cinnamoyl-CoA reductase (1619065_at) were overexpressed (Table 1, Figure 2). Genes coding for enzymes such as polyphenol oxidase and diphenol oxidase, probably responsible for the lignin polymerization process [36], were induced as well (1622651_at; 1610806_at) (Table 1).

The other branches of phenolic metabolism seemed not to be affected by DIMEB. Only two genes of the anthocyanin pathway (a chalcone-flavonone isomerase (CLU122) and a flavonol-3-O-glucosyltransferase (CLU048)) were induced at 2 h but not at 6 h (Table 1, Figure 2). 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 [37].

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 [14].

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 (CLU088; 1613871_at; 1617192_at; 1617430_s_at), PR-10 (CLU001; 1610011_s_at; 1618568_s_at) and PR-4 (CLU021), but also for a prolin-rich protein (CLU036) and a protease inhibitor (1609875_at; 1611666_s_at) was observed in both experiments, while upregulation of two genes encoding the S-adenosyl-L-methyonine:salicylic acid carboxyl methyltransferase (1612552_at; 1620309_at) was recorded at 6 h (Table 1). Interestingly, this enzyme mediates the synthesis of gaseous methyl salicylate which was recently demonstrated to be a key mediator in plant systemic acquired resistance [38] in tobacco, as well as an inducer of the expression of PR-1 gene and TMV resistance [39]. 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 (1622147_at), would suggest the involvement of ethylene as well (Table 1). This hormone is a major regulator of the plant's reaction to pathogen attack [40] 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 [41, 42]. It appears from the finding that a gene (1616358_at) homologous to an MLO-like 11 of Arabidopsis was downregulated at 6 h (Table 1), 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[43], and in the dicot Arabidopsis thaliana, resistance to powdery mildews also depends on loss-of-function mlo alleles [44].

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 6 h, downregulation of genes related to cell wall modification [45], such as those encoding expansins (1608074_s_at; 1620840_at), xyloglucan endotransglycosylase (1615995_at; 1620003_at), pectin methylesterases (1608799_at; 1619468_at), a β-galactosidase (1619522_at), a polygalacturonase (1608756_at) and endoglucanases (1606763_at; 1609506_at; 1610263_at), was observed (Table 1). The sequence 1609506_at corresponds to the VvCEL2 transcript which encodes a grapevine cellulase. Since in Arabidopsis the expression of the cel1 gene was related to growing tissues [46], downregulation of VvCEL2 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 [47]. The downregulation of genes coding for α- and β-tubulin (1612320_a_at; 1616815_at; 1618413_at; 1619167_at; 1621015_at; 1622466_at) is indication of a stop in cell expansion and cell division (Table 1). 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 [48].

Further indication of cell division reduction were the lower transcription of genes coding for histones H2A, H3, H4 and H2B (1608927_at; 1612573_at; 1613041_at; 1613076_at; 1620332_at; 1622440_at; 1622737_at), a cyclin (1610854_at), a pattelin protein (1610422_at), a GA-induced-like protein (GIP-like) (1610607_at), a putative formin homology (FH) protein (1613373_at) and a DNA polymerase alpha catalytic subunit gene (1607792_at) (Table 1). 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-5 of Petunia hybrida, which is expressed during the cell division phase in stems and corollas [49]. In Arabidopsis patellin1 plays a role in membrane-trafficking when the cell-plate is formed during cytokinesis [50], and formins are plant cytoskeleton-organizing proteins which take part in cytokinesis and in the establishment and maintenance of cell polarity [51]. Very similar effects on cell growth have been reported upon elicitation of parsley cell cultures with an oligopeptide elicitor. Pep 25 provoked the repression of genes regulating the cell cycle, such as cdc2, cyclin and histones [52].

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.

Conclusion

The transcriptional profiles measured at 2 h and 6 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 (Figure 3). This response largely resembles that occurring upon pathogen attack.
Figure 3

Cellular processes triggered by DIMEB as deduced by transcriptional profiling. Grapevine cell model showing the major genes involved in the cellular processes modulated by DIMEB treatment. Abbreviations: CAD, cinnamyl alchol dehydrogenase; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; COMT, caffeic acid O-methyltransferase; CCR, cinnamoyl-CoA reductase; F5H, ferulate-5-hydroxylase; PME, pectin methylesterase; PPO, polyphenol oxidase, PR protein, pathogenesis-related protein; STS, stilbene synthase; XET, xyloglucan endotransglycosylase.

Methods

Plant material

Liquid cell cultures of a cross between Vitis riparia and Vitis berlandieri were used to carry out the treatment experiments with DIMEB (50 mM) [21]. Cell cultures were collected 2 h and 6 h after DIMEB treatment from control and treated samples. Cells and medium were separated by centrifugation at 12.000 ×g for 10 min at room temperature.

Total RNA extraction

Total RNA was extracted from control and treated samples using a modified hot-borate method, as described by Moser et al. [53]. DNA traces were removed by DNase I treatment (Sigma-Aldrich, St.Louis, MO, USA) according to the manufacturer's procedure. RNA was isolated from one replicate for the SSH experiment (2 h) and from 3 biological replicates for the microarray experiment (6 h).

cDNA synthesis and SSH library construction

Double-stranded cDNA was synthesized from 0.6 μg of total RNA of the control and treated samples (2 h) using the SMART™ PCR cDNA synthesis kit (Clontech Laboratories, Mountain View, CA) as recommended by the manufacturer.

Suppression subtractive hybridization (SSH) was carried out using the PCR-Select cDNA subtraction Kit (Clontech Laboratories) according to the manufacturer's procedure. The cDNA from the treated sample was used as the "tester" while the cDNA from the control sample was used as the "driver". Following hybridization, the subtracted cDNA molecules were inserted into a pCR® 2.1-TOPO® Vector (Invitrogen, Carlsbad, CA) and then used to transform One Shot® TOP10 Chemically Competent Escherichia coli cells (Invitrogen). Positive transformants, based on blue/white screening, were picked and arrayed in a 384-well plate containing LB medium (Sigma-Aldrich) supplemented with ampicillin (50 μg mL-1) and glycerol (10% v/v). The SSH cDNA library was stored at -80°C.

Amplification of cDNA inserts and spotting on filters

The SSH library clones were cultured overnight at 37°C in a 384-well plate with LB medium and ampicillin (50 μg mL-1). A small aliquot (1 μl) of each liquid culture was then transferred into four 96-well plates containing PCR mix and used as template to amplify the corresponding cDNA inserts. PCR reactions (95°C for 15 min, 94°C for 45 sec, 68°C for 45 sec, 72°C for 2 min for 35 cycles, 72°C for 7 min) contained 300 nM Nested Primer PCR 1 and 300 nM Nested Primer PCR 2R (Clontech Laboratories), 0.5 U HotStartTaq DNA polymerase (Qiagen, Shanghai, China), 200 μM dNTPs, 1.5 M betain (Sigma-Aldrich) and 80 μM Cresol Red (Sigma-Aldrich). The 40 μl PCR reactions were then concentrated by overnight incubation at 37°C. The human nebulin cDNA (NM_004543) was PCR amplified in the same way to serve as a positive control. One microliter of each concentrated cDNA insert together with one microliter of a 2 ng/μl solution of amplified nebulin were transferred onto 8 × 12 cm Hybond+ nylon membranes (Amersham, GE Healthcare Bio-Sciences AB, Little Chalfont, UK) using a manual 96-pin tool. The samples were arrayed in duplicate according to a 4 × 4 grid pattern. Before and after spotting, membranes were denatured on Whatmann 3 MM paper saturated with denaturation buffer (0.5 M NaOH, 1.5 M NaCl) for 15 min. Membranes were then neutralised on Whatmann 3 MM paper saturated with neutralization buffer (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.2) for 15 min, rinsed in 2× SSC, air dried and crosslinked at 80°C for 2 h.

Target labelling

To assess whether the isolated clones were truly positive, they were hybridized with the same total RNAs used for SSH library construction. The RNAs were DIG-labelled by reverse transcription according to Vernon et al. [54] with the following modifications: 7.5 μl of PCR DNA Labelling MIX 10× (Roche, Basel, Switzerland) and 1.5 μl of 50 μM of Oligo(dT)20 were added to 5 μg of total RNA of each sample (tester and driver). After incubation of the two samples at 65°C for 10 min and then on ice for 2 min, a mix of 6 μl of RT Buffer 5× (Invitrogen), 3 μl of 0.1 M DTT (Invitrogen), 1.5 μl of RNase OUT (40 U/μl) (Invitrogen) and 1.5 μl of Superscript II (200 U/μl) (Invitrogen) was added to each sample. Reverse transcription was performed at 42°C for 1 h and then continued for a further hour after addition of another 1.5 μl of Superscript II (200 U/μl) (Invitrogen). The reaction was stopped by incubation at 70°C for 15 min and was followed by treatment with 1.5 μl of RNase H (2 U/μl) (Invitrogen) at 37°C for 20 min. The digoxigenin-labelled probe of the control target was synthesized by PCR amplification of a portion of human nebulin cDNA cloned in pBluescript II SK/KS (-) (Stratagene) in the presence of PCR DNA Labelling MIX 10×. PCR reaction was carried out in 50 μl using 7 ng/μl of pBluescript II SK (-) containing human nebulin cDNA as template and the primers nebulin-for 5'-CAGGAGACTATTACAGGTTT-3' and nebulin-rev 5'-ACCCATAGGCAGCTTGAGAA-3', according to the manufacturer's procedure. PCR conditions were 95°C for 15 min, 35 cycles of 94°C for 45 sec, 52°C for 45 sec, 72°C for 1 min, followed by 72°C for 7 min.

Hybridization, washing and detection

Two filters were incubated with 20 ml of pre-hybridization solution (5× SSC, 0.1% (w/v) N-lauroylsarcosine, 0.02% (w/v) SDS, 1% (v/v) blocking solution in 1× acid maleic buffer) at 72°C for 30 min. Two different probes were prepared: the first was obtained by mixing the DIG-labelled "tester" DNA (30 μl) with the DIG-labelled human nebulin (2 μl), the second by mixing the DIG-labelled "driver" DNA (30 μl) with the DIG-labelled human nebulin (2 μl). After a short denaturation step (95°C for 3 min) the two probes were incubated separately with one filter each overnight at 68°C in hybridization solution (20 ml, 5× SSC, 0.1% (w/v) N-lauroylsarcosine, 0.02% (w/v) SDS, 1% (v/v) blocking solution in 1× acid maleic buffer). After hybridization, four high-stringency washings at 68°C for 20 min (2× SSC, 0.5% (w/v) SDS) followed by two low-stringency washings (0.2× SSC, 0.5% (w/v) SDS) at 68°C for 20 min, were carried out. Chemiluminescence was detected by 30-min exposure to Kodak® BioMax Light Film (Kodak, Rochester, NY) after incubation with anti-DIG antibodies and CDP-Star, according to the manufacturer's procedure (Roche).

Sequencing of transcripts identified by SSH

Following the screening procedure, the 289 positive clones were amplified, as described above for filter production, but without betain and Cresol Red in the PCR reaction mix. Five microliters of each PCR reaction were purified from primers and nucleotides using 1.5 μl of ExoSAP-IT™ (Amersham) at 37°C for 1 h. The reaction was stopped at 75°C for 15 min. Three nanograms for every 100 bp of amplified fragment were used for the sequencing reaction with Nested PCR Primer 1. Sequencing of 243 positively amplified clones was outsourced to the BMR Sequencing Service of C.R.I.B.I. (University of Padua, Padua, Italy) [55]. Electropherograms were analyzed with Phred [56, 57] to assign a quality score and with a perl script using the UniVec Database [58] to identify any vector and adaptors sequences. Interspersed repeats and low complexity DNA sequences were identified through analysis with RepeatMasker [59]. The sequences were then organized in transcript consensus sequences (clusters) using the CAP3 DNA sequence program [60].

Affymetrix GeneChip experiments

Total RNA of the control and treated cells after 6 h of DIMEB treatment (3 biological replicates for each type of sample) were used to hybridize 6 different GeneChip®Vitis vinifera Genome Arrays (Affymetrix, Santa Clara, CA). Ten micrograms of total RNA for each replicate were purified as described above (Total RNA extraction), subjected to further purification using "RNeasy" columns (Qiagen) and sent to an external service (IFOM-IEO Campus for ONCOGENOMICS, Milan, Italy) for labelling and hybridization. RNA samples passed the quality check as determined by electrophoresis run on a Agilent BioAnalyzer (Agilent, Palo Alto, CA, USA). Biotin-labelling, hybridization, washing, staining and scanning procedures were performed according to the Affymetrix technical manual. Analysis of raw data was performed using the open source software of the Bioconductor project [61, 62] with the statistical R programming language [63, 64]. The quality of the hybridization reactions was checked using the affyPLM package. Intensity distribution of PM for each chip and the quality of the 3 biological replicates of both control and treated conditions were analyzed with the functions and plots (histogram and MA plots) of the affy package [6567]. Background adjustment, normalization and summarization were performed using gcrma and the affy package. Data, before and after application of the gcrma algorithm [68], were compared through the graphical representation of box-plots and MA plots. Probe sets which were not expressed or were non-differentially expressed between the two conditions considered were eliminated in a filtering step based on the inter-quantile range method (IQR = 0.25) using the genefilter package. A two-class paired SAM analysis (Δ = 0.9; FDR = 13.3%) [69] was performed using the probe sets resulting from the filtering procedure in order to identify differentially expressed probe sets between the control and treated conditions. A fold-change of two was then applied.

Functional annotation of the SSH transcripts and Affymetrix probesets

Protein sequences encoded by the SSH transcripts or by the representative sequence of each probeset as provided by the NetAffx Analysis Center [70] were predicted using a consensus generated by three different CDS predictors [71]. Blastp analyses [72] of the polypeptides obtained from the predicted CDSs were performed by searching against the UniProt database [73]. GO terms (molecular function, biological process and cellular component) [23] were linked at every consensus sequence on the basis of the results of the Blastp analysis (Additional files 1 and 2). The sequences were organized in main functional categories according to the GO term biological process (Additional files 3 and 4). In cases of non significant Blastp results (Evalue <1e-8; sequence alignment length <75% of the query polypeptide length), these were classified as "No hits found".

The SSH transcripts were deposited at the NCBI database [74] under the sequence IDs reported in the Additional file 1. Both SSH transcripts and probesets were also referred to corresponding Tentative Consensus sequences obtained by a search (BlastN) against the Grape Gene Index database [75] and to the corresponding genomic locus on Pinot Noir clone ENTAV 115 [76] (Additional files 1 and 2).

Real-time reverse transcription (RT)-PCR

To validate the SSH and microarray data, 12 genes and 5 genes identified by SSH and GeneChip array respectively, were also analyzed by quantitative RT-PCR experiments (Additional file 5). Specific primers were designed to generate 100–200 bp PCR products (Additional file 5). The actin gene (TC45156) was used to normalize the data (actin forward: 5'-TCCTTGCCTTGCGTCATCTAT-3'; actin reverse: 5'-CACCAATCACTCTCCTGCTACAA-3') since in preliminary trials it appeared to be constantly expressed in the RNA samples subjected to gene expression analyses. For RT-PCR, total RNA from control and treated samples of the SSH experiment and from 3 biological replicates of control and treated samples of the GeneChip experiments were used. DNA traces were removed with DNase I treatment (Sigma-Aldrich) according to the manufacturer's procedure. Reverse transcription reactions and real-time RT-PCR reactions were performed using the SuperScript™ III Platinum® Two-Step qRT-PCR Kit with SYBR® Green (Invitrogen) according to the manufacturer's protocols with minor modification (300 nM of each primer in a final volume of 12.5 μl). PCR reactions contained 20 ng of cDNA and were replicated 3 times (technical replicates). Amplification reactions were performed with an ABI PRISM® 7000 Sequence Detection System (Applied Biosystems). The following thermal profile was used: 50°C for 2 min; 95°C for 10 min; 40 cycle of 95°C for 15 sec and 55°C for 1 min. Data were analysed with the ABI PRISM® 7000 SDS Software (Applied Biosystems). PCR reaction efficiencies were calculated with the LinRegPCR program [77]. For all the consensus sequences, the differential expression between treated and control samples was expressed as a ratio calculated with the Pfaffl equation [78]. The overall standard error of the mean normalized expression was obtained by applying the error calculation based on Taylor's series as developed for REST© software [79].

Data Availability

All microarray expression data are available at EBI ArrayExpress under the series entry E-MEXP-2114.

Abbreviations

DIMEB: 

(heptakis(2,6-di-O-methyl)-β-cyclodextrin)

SSH: 

Suppression subtractive hybridization

cDNA: 

Complementary DNA

CDS: 

Coding Sequence

EST: 

Expressed Sequence Tag

GO: 

Gene Ontology

NCBI: 

National Center for Biotechnology Information

SAM: 

Significance Analysis of Microarrays

RT-PCR: 

Real time polymerase chain reaction.

Declarations

Acknowledgements

We wish to thank Dr. H H Kassermeyer for cell supply and helpful discussion. This work was supported by the Fondo Unico of the Provincia Autonoma di Trento (Resveratrol Project).

Authors’ Affiliations

(1)
Fondazione Edmund Mach, IASMA Research and Innovation Center
(2)
Department for Sciences, Technologies and Markets of Grapevine and Wine
(3)
Centre for Integrative Biology (CIBIO), University of Trento

References

  1. Dixon RA, Harrison MJ: Activation, structure and organization of genes involved in microbial defence in plants. Adv Genet. 1990, 28: 165-234.View ArticlePubMedGoogle Scholar
  2. Jeandet P, Douillt-Breuil AC, Bessis R, Debord S, Sbaghi M, Adrian M: Phytoalexins from the Vitaceae: biosynthesis, phytoalexin gene expression in transgenic plants, antifungal activity, and metabolism. J Agr Food Chem. 2002, 50 (10): 2731-2741. 10.1021/jf011429s.View ArticleGoogle Scholar
  3. Derckel JP, Baillieul F, Manteau S, Audran JC, Haye B, Lambert B, Legendre L: Differential induction of grapevine defenses by two strains of Botrytis cinerea. Phytopathology. 1999, 89 (3): 197-203. 10.1094/PHYTO.1999.89.3.197.View ArticlePubMedGoogle Scholar
  4. Harborne JB: The comparative biochemistry of phytoalexin induction in plants. Biochem Syst Ecol. 1999, 27 (4): 335-367. 10.1016/S0305-1978(98)00095-7.View ArticleGoogle Scholar
  5. Langcake P, Pryce RJ: Production of resveratrol by Vitis Vinifera and other members of Vitaceae as a response to infection or injury. Physiol Plant Pathol. 1976, 9 (1): 77-86. 10.1016/0048-4059(76)90077-1.View ArticleGoogle Scholar
  6. Stein U, Hoos G: Induktions- und Nachweismethoden für Stilbene bei Vitaceen. Vitis. 1984, 23: 179-184.Google Scholar
  7. Dercks W, Creasy LL: The significance of stilbene phytoalexins in the Plasmopara viticola grapevine interaction. Physiol Mol Plant Pathol. 1989, 34 (3): 189-202. 10.1016/0885-5765(89)90043-X.View ArticleGoogle Scholar
  8. Langcake P, Cornford CA, Pryce RJ: Identification of pterostilbene as a phytoalexin from Vitis Vinifera leaves. Phytochemistry. 1979, 18: 1025-1027. 10.1016/S0031-9422(00)91470-5.View ArticleGoogle Scholar
  9. Gatto P, Vrhovsek U, Muth J, Segala C, Romualdi C, Fontana P, Pruefer D, Stefanini M, Moser C, Mattivi F, Velasco R: Ripening and genotype control stilbene accumulation in healthy grapes. J Agr Food Chem. 2008, 56 (24): 11773-11785. 10.1021/jf8017707.View ArticleGoogle Scholar
  10. Adrian M, Jeandet P, Veneau J, Weston LA, Bessis R: Biological activity of resveratrol, a stilbenic compound from grapevines, against Botrytis cinerea, the causal agent for gray mold. J Chem Ecol. 1997, 23 (7): 1689-1702. 10.1023/B:JOEC.0000006444.79951.75.View ArticleGoogle Scholar
  11. Langcake P, Pryce RJ: Production of resveratrol and viniferins by grapevines in response to UV irradiation. Phytochemistry. 1977, 6: 1193-1196. 10.1016/S0031-9422(00)94358-9.View ArticleGoogle Scholar
  12. Schubert R, Fischer R, Hain R, Schreier PH, Bahnweg G, Ernst D, Sandermann H: An ozone-responsive region of the grapevine resveratrol synthase promoter differs from the basal pathogen-responsive sequence. Plant Mol Biol. 1997, 34 (3): 417-426. 10.1023/A:1005830714852.View ArticlePubMedGoogle Scholar
  13. Adrian M, Jeandet P, Bessis R, Joubert JM: Induction of phytoalexin (resveratrol) synthesis in grapevine leaves treated with aluminum chloride (AlCl3). J Agr Food Chem. 1996, 44 (8): 1979-1981. 10.1021/jf950807o.View ArticleGoogle Scholar
  14. Radman R, Saez T, Bucke C, Keshavarz T: Elicitation of plants and microbial cell systems. Biotechnol Appl Bioc. 2003, 37 (Pt 1): 91-102. 10.1042/BA20020118.View ArticleGoogle Scholar
  15. Liswidowati , Melchior F, Hohmann F, Schwer B, Kindl H: Induction of stilbene synthase by Botrytis cinerea in cultured grapevine cells. Planta. 1991, 183 (2): 307-314. 10.1007/BF00197803.View ArticlePubMedGoogle Scholar
  16. Tassoni A, Fornale S, Franceschetti M, Musiani F, Michael AJ, Perry B, Bagni N: Jasmonates and Na-orthovanadate promote resveratrol production in Vitis vinifera cv. Barbera cell cultures. New Phytol. 2005, 166 (3): 895-905. 10.1111/j.1469-8137.2005.01383.x.View ArticlePubMedGoogle Scholar
  17. Lijavetzky D, Almagro L, Belchi-Navarro S, Martínez-Zapater J, Bru R, Pedreno MA: Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures. BMC Res Notes. 2008, 1: 132-10.1186/1756-0500-1-132.PubMed CentralView ArticlePubMedGoogle Scholar
  18. Aziz A, Poinssot B, Daire X, Adrian M, Bezier A, Lambert B, Joubert JM, Pugin A: Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola. Mol Plant Microbe Interact. 2003, 16 (12): 1118-1128. 10.1094/MPMI.2003.16.12.1118.View ArticlePubMedGoogle Scholar
  19. Morales M, Bru R, Garcia-Carmona F, Barcelo AR, Pedreno MA: Effect of dimethyl-beta-cyclodextrins on resveratrol metabolism in Gamay grapevine cell cultures before and after inoculation with Xylophilus ampelinus. Plant Cell Tiss Org. 1998, 53 (3): 179-187. 10.1023/A:1006027410575.View ArticleGoogle Scholar
  20. Bru R, Selles S, Casado-Vela J, Belchi-Navarro S, Pedreno MA: Modified cyclodextrins are chemically defined glucan inducers of defense responses in grapevine cell cultures. J Agr Food Chem. 2006, 54 (1): 65-71. 10.1021/jf051485j.View ArticleGoogle Scholar
  21. Zamboni A, Vrhovsek U, Kassemeyer HH, Mattivi F, Velasco R: Elicitor-induced resveratrol production in cell cultures of different grape genotypes (Vitis spp.). Vitis. 2006, 45 (2): 63-68.Google Scholar
  22. Cao WX, Epstein C, Liu H, DeLoughery C, Ge NX, Lin JY, Diao R, Cao H, Long F, Zhang X, Chen YD, Wright PS, Busch S, Wenck M, Wong K, Saltzman AG, Tang ZH, Liu L, Zilberstein A: Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study. BMC Genomics. 2004, 5 (1): 26-10.1186/1471-2164-5-26.PubMed CentralView ArticlePubMedGoogle Scholar
  23. The Gene Ontology. [http://www.geneontology.org/]
  24. Stone JM, Collinge MA, Smith RD, Horn MA, Walker JC: Interaction of a protein phosphatase with an Arabidopsis serine-threonine receptor kinase. Science. 1994, 266 (5186): 793-795. 10.1126/science.7973632.View ArticlePubMedGoogle Scholar
  25. Song WY, Wang GL, Chen LL, Kim HS, Pi LY, Holsten T, Gardner J, Wang B, Zhai WX, Zhu LH, Fauquet C, Ronald P: A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science. 1995, 270 (5243): 1804-1806. 10.1126/science.270.5243.1804.View ArticlePubMedGoogle Scholar
  26. Pastuglia M, Swarup R, Rocher A, Saindrenan P, Roby D, Dumas C, Cock JM: Comparison of the expression patterns of two small gene families of S gene family receptor kinase genes during the defence response in Brassica oleracea and Arabidopsis thaliana. Gene. 2002, 282 (1–2): 215-225. 10.1016/S0378-1119(01)00821-6.View ArticlePubMedGoogle Scholar
  27. Halfter U, Ishitani M, Zhu JK: The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3. Proc Natl Acad Sci USA. 2000, 97 (7): 3735-3740. 10.1073/pnas.040577697.PubMed CentralView ArticlePubMedGoogle Scholar
  28. Laxalt AM, Munnik T: Phospholipid signalling in plant defence. Curr Opin Plant Biol. 2002, 5 (4): 332-338. 10.1016/S1369-5266(02)00268-6.View ArticlePubMedGoogle Scholar
  29. Ulker B, Somssich IE: WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol. 2004, 7 (5): 491-498. 10.1016/j.pbi.2004.07.012.View ArticlePubMedGoogle Scholar
  30. Gu YQ, Wildermuth MC, Chakravarthy S, Loh YT, Yang CM, He XH, Han Y, Martin GB: Tomato transcription factors Pti4, Pti5, and Pti6 activate defense responses when expressed in Arabidopsis. Plant Cell. 2002, 14 (4): 817-831. 10.1105/tpc.000794.PubMed CentralView ArticlePubMedGoogle Scholar
  31. Schiermeyer A, Thurow C, Gatz C: Tobacco bZIP factor TGA10 is a novel member of the TGA family of transcription factors. Plant Mol Biol. 2003, 51 (6): 817-829. 10.1023/A:1023093101976.View ArticlePubMedGoogle Scholar
  32. Zhang YL, Fan WH, Kinkema M, Li X, Dong XN: Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proc Natl Acad Sci USA. 1999, 96 (11): 6523-6528. 10.1073/pnas.96.11.6523.PubMed CentralView ArticlePubMedGoogle Scholar
  33. Richter H, Pezet R, Viret O, Gindro K: Characterization of 3 new partial stilbene synthase genes out of over 20 expressed in Vitis vinifera during the interaction with Plasmopara viticola. Physiol Mol Plant Pathol. 2005, 67 (3–5): 248-260.View ArticleGoogle Scholar
  34. Campbell EJ, Schenk PM, Kazan K, Penninckx IAMA, Anderson JP, Maclean DJ, Cammue BPA, Ebert PR, Manners JM: Pathogen-responsive expression of a putative ATP-binding cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by multiple defense signaling pathways in Arabidopsis. Plant Physiol. 2003, 133 (3): 1272-1284. 10.1104/pp.103.024182.PubMed CentralView ArticlePubMedGoogle Scholar
  35. Yazaki K: Transporters of secondary metabolites. Curr Opin Plant Biol. 2005, 8 (3): 301-307. 10.1016/j.pbi.2005.03.011.View ArticlePubMedGoogle Scholar
  36. Whetten R, Sederoff R: Lignin biosynthesis. Plant Cell. 1995, 7 (7): 1001-1013. 10.1105/tpc.7.7.1001.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Ferrari S, Galletti R, Denoux C, De Lorenzo G, Ausubel FM, Dewdney J: Resistance to Botrytis cinerea induced in Arabidopsis by elicitors is independent of salicylic acid, ethylene, or jasmonate signaling but requires PHYTOALEXIN DEFICIENT3. Plant Physiol. 2007, 144 (1): 367-379. 10.1104/pp.107.095596.PubMed CentralView ArticlePubMedGoogle Scholar
  38. Park SW, Kaimoyo E, Kumar D, Mosher S, Klessig DF: Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science. 2007, 318 (5847): 113-116. 10.1126/science.1147113.View ArticlePubMedGoogle Scholar
  39. Shulaev V, Silverman P, Raskin I: Airborne signalling by methyl salicylate in plant pathogen resistance. Nature. 1997, 386 (6626): 738-738.Google Scholar
  40. Broekaert WF, Delauré SL, De Bolle MF, Cammue BP: The role of ethylene in host-pathogen interactions. Annu Rev Phytopathol. 2006, 44: 393-416. 10.1146/annurev.phyto.44.070505.143440.View ArticlePubMedGoogle Scholar
  41. Ecker JR, Davis RW: Plant defense genes are regulated by ethylene. Proc Natl Acad Sci USA. 1987, 84 (15): 5202-5206. 10.1073/pnas.84.15.5202.PubMed CentralView ArticlePubMedGoogle Scholar
  42. Marcos JF, Gonzalez-Candelas L, Zacarias L: Involvement of ethylene biosynthesis and perception in the susceptibility of citrus fruits to Penicillium digitatum infection and the accumulation of defence-related mRNAs. J Exp Bot. 2005, 56 (418): 2183-2193. 10.1093/jxb/eri218.View ArticlePubMedGoogle Scholar
  43. Buschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, vanDaelen R, vanderLee T, Diergaarde P, Groenendijk J, Topsch S, Vos P, Salamini F, Schulze-Lefert P: The barley mlo gene: A novel control element of plant pathogen resistance. Cell. 1997, 88 (5): 695-705. 10.1016/S0092-8674(00)81912-1.View ArticlePubMedGoogle Scholar
  44. Consonni C, Humphry ME, Hartmann HA, Livaja M, Durner J, Westphal L, Vogel J, Lipka V, Kemmerling B, Schulze-Lefert P, Somerville SC, Panstruga R: Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nat Genet. 2006, 38 (6): 716-720. 10.1038/ng1806.View ArticlePubMedGoogle Scholar
  45. Cosgrove DJ: Enzymes and other agents that enhance cell wall extensibility. Annu Rev Plant Phys. 1999, 50: 391-417. 10.1146/annurev.arplant.50.1.391.View ArticleGoogle Scholar
  46. Shani Z, Dekel M, Roiz L, Horowitz M, Kolosovski N, Lapidot S, Alkan S, Koltai H, Tsabary G, Goren R, Shoseyov O: Expression of endo-1,4-beta-glucanase (cel1) in Arabidopsis thaliana is associated with plant growth, xylem development and cell wall thickening. Plant Cell Rep. 2006, 25 (10): 1067-1074. 10.1007/s00299-006-0167-9.View ArticlePubMedGoogle Scholar
  47. Dixon DC, Seagull RW, Triplett BA: Changes in the accumulation of alpha-tubulin and beta-tubulin isotypes during cotton fiber development. Plant Physiol. 1994, 105 (4): 1347-1353.PubMed CentralPubMedGoogle Scholar
  48. Ebel C, Gomez LG, Schmit AC, Neuhaus-Url G, Boller T: Differential mRNA degradation of two beta-tubulin isoforms correlates with cytosolic Ca2+ changes in glucan-elicited soybean cells. Plant Physiol. 2001, 126 (1): 87-96. 10.1104/pp.126.1.87.PubMed CentralView ArticlePubMedGoogle Scholar
  49. Ben-Nissan G, Lee JY, Borohov A, Weiss D: GIP, a Petunia hybrida GA-induced cysteine-rich protein: a possible role in shoot elongation and transition to flowering. Plant J. 2004, 37 (2): 229-238.View ArticlePubMedGoogle Scholar
  50. Peterman TK, Ohol YM, McReynolds LJ, Luna EJ: Patellin1, a novel Sec14-like protein, localizes to the cell plate and binds phosphoinositides. Plant Physiol. 2004, 136 (2): 3080-3094. 10.1104/pp.104.045369.PubMed CentralView ArticlePubMedGoogle Scholar
  51. Favery B, Chelysheva LA, Lebris M, Jammes F, Marmagne A, de Almeida-Engler J, Lecomte P, Vaury C, Arkowitz RA, Abad P: Arabidopsis formin AtFH6 is a plasma membrane-associated protein upregulated in giant cells induced by parasitic nematodes. Plant Cell. 2004, 16 (9): 2529-2540. 10.1105/tpc.104.024372.PubMed CentralView ArticlePubMedGoogle Scholar
  52. Logemann E, Wu SC, Schroder J, Schmelzer E, Somssich IE, Hahlbrock K: Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle-related genes. Plant J. 1995, 8 (6): 865-876.View ArticlePubMedGoogle Scholar
  53. Moser C, Gatto P, Moser M, Pindo M, Velasco R: Isolation of functional RNA from small amounts of different grape and apple tissues. Mol Biotechnol. 2004, 26 (2): 95-99. 10.1385/MB:26:2:95.View ArticlePubMedGoogle Scholar
  54. Vernon SD, Unger ER, Rajeevan M, Dimulescu IM, Nisenbaum R, Campbell CE: Reproducibility of alternative probe synthesis approaches for gene expression profiling with arrays. J Mol Diagn. 2000, 2 (3): 124-127.PubMed CentralView ArticlePubMedGoogle Scholar
  55. BMR Genomics. [http://bmr.cribi.unipd.it/]
  56. Ewing B, Hillier L, Wendl MC, Green P: Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998, 8 (3): 175-185.View ArticlePubMedGoogle Scholar
  57. Ewing B, Green P: Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998, 8 (3): 186-194.View ArticlePubMedGoogle Scholar
  58. The UniVec Database. [http://www.ncbi.nlm.nih.gov/VecScreen/UniVec]
  59. RepeatMasker. [http://www.repeatmasker.org/]
  60. Xuang X, Madan A: CAP3: a DNA sequence assembly program. Genome Res. 1999, 9: 868-877. 10.1101/gr.9.9.868.View ArticleGoogle Scholar
  61. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004, 5 (10): R80-10.1186/gb-2004-5-10-r80.PubMed CentralView ArticlePubMedGoogle Scholar
  62. The Bioconductor project. [http://www.bioconductor.org/]
  63. Ihaka R, Gentleman RC: R: a language for data analysis and graphics. J Comput Graph Stat. 1996, 5 (3): 299-314. 10.2307/1390807.Google Scholar
  64. The R Project for Statistical Computing. [http://www.r-project.org/]
  65. Bolstad BM, Irizarry RA, Astrand M, Speed TP: A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003, 19 (2): 185-193. 10.1093/bioinformatics/19.2.185.View ArticlePubMedGoogle Scholar
  66. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003, 4 (2): 249-264. 10.1093/biostatistics/4.2.249.View ArticlePubMedGoogle Scholar
  67. Gautier L, Cope L, Bolstad BM, Irizarry RA: affy – analysis of Affymetrix GeneChip data at the probe level. Bioinformatics. 2004, 20 (3): 307-315. 10.1093/bioinformatics/btg405.View ArticlePubMedGoogle Scholar
  68. Wu ZJ, Irizarry RA: Preprocessing of oligonucleotide array data. Nat Biotechnol. 2004, 22 (6): 656-658. 10.1038/nbt0604-656b.View ArticlePubMedGoogle Scholar
  69. Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001, 98 (9): 5116-5121. 10.1073/pnas.091062498.PubMed CentralView ArticlePubMedGoogle Scholar
  70. Affymetrix. [http://www.affymetrix.com/index.affx]
  71. Wasmuth JD, Blaxter ML: prot4EST: translating expressed sequence tags from neglected genomes. BMC Bioinformatics. 2004, 5: 187-10.1186/1471-2105-5-187.PubMed CentralView ArticlePubMedGoogle Scholar
  72. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25 (17): 3389-3402. 10.1093/nar/25.17.3389.PubMed CentralView ArticlePubMedGoogle Scholar
  73. The UniProt Database. [http://www.uniprot.org/]
  74. The NCBI database. [http://www.ncbi.nlm.nih.gov/]
  75. The DFCI Grape Gene Index. [http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=grape]
  76. Velasco R, Zharkikh A, Troggio M, Cartwright DA, Cestaro A, Pruss D, Pindo M, Fitzgerald LM, Vezzulli S, Reid J, Malacarne G, Iliev D, Coppola G, Wardell B, Micheletti D, Macalma T, Facci M, Mitchell JT, Perazzolli M, Eldredge G, Gatto P, Oyzerski R, Moretto M, Gutin N, Stefanini M, Chen Y, Segala C, Davenport C, Dematte L, Mraz A, Battilana J, Stormo K, Costa F, Tao Q, Si-Ammour A, Harkins T, Lackey A, Perbost C, Taillon B, Stella A, Solovyev V, Fawcett JA, Sterck L, Vandepoele K, Grando SM, Toppo S, Moser C, Lanchbury J, Bogden R, Skolnick M, Sgaramella V, Bhatnagar SK, Fontana P, Gutin A, Peer Van de Y, Salamini F, Viola R: A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. PLoS ONE. 2007, 2 (12): e1326-10.1371/journal.pone.0001326.PubMed CentralView ArticlePubMedGoogle Scholar
  77. Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM: Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett. 2003, 339 (1): 62-66. 10.1016/S0304-3940(02)01423-4.View ArticlePubMedGoogle Scholar
  78. Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29 (9): e45-10.1093/nar/29.9.e45.PubMed CentralView ArticlePubMedGoogle Scholar
  79. Pfaffl MW, Horgan GW, Dempfle L: Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002, 30 (9): e36-10.1093/nar/30.9.e36.PubMed CentralView ArticlePubMedGoogle Scholar

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