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
]. Considerable stilbene accumulation in response to DIMEB treatment was also observed by our group using non-vinifera
× 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
). 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 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 ).
List of transcripts modulated by DIMEB and reported in the Discussion
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 ). 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 ). 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
]. 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 ).
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 ). 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 ). 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 ). It has been reported that this protein can bind to the regulatory activation sequence-1 (as-1
] identified in the promoter of Arabidopsis PR-1
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 ).
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 (more ...)
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 ). 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 ). 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 ). 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 ). 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 , Figure ). 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 ).
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 , Figure ). 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 ). 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 ). 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
]. It appears from the finding that a gene (1616358_at) homologous to an MLO-like 11 of Arabidopsis was downregulated at 6 h (Table ), 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
], 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 ). 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 ). 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 ). 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.