DIGE analysis of proteome changes accompanying large resveratrol production by grapevine (Vitis vinifera cv. Gamay) cell cultures in response to methyl-ß-cyclodextrin and methyl jasmonate elicitors.

We had previously shown that Vitis vinifera cv. Gamay cell suspension accumulates extracellularly large amounts of the phytoalexin trans-resveratrol (tR) in response to elicitation with methylated cyclodextrins (MBCD), which can be triplicated when the elicitor is combined with methyl jasmonate (MeJA). In parallel, new pathogenesis-related proteins accumulated in the apoplast-like extracellular space. The aim of this study was to investigate changes in the grapevine cell proteome potentially related to tR accumulation in response to the above elicitors. The DIGE technique was used to detect statistically significant changes in the cell's proteome. A total number of 1031 unique spots were detected, 67 of which were de-regulated upon elicitation. Sixty-four spots were successfully identified by nLC-MS/MS database search analysis. The tR biosynthetic pathway enzymes were up-regulated by MBCD alone or combined with MeJA, but not by treatment with MeJA alone, in agreement with tR accumulation pattern. Seven spots contained stilbene synthase encoded by four different isogenes. Likewise, four glutathione-S-transferases, potentially involved in tR trafficking within the cell and across membranes, were up-regulated in the same fashion as stilbene synthases. The relation of other de-regulated proteins with other effects caused by elicitors on grapevine cells, namely defense response and cell growth inhibition, is discussed.

Changes of defense proteins in the extracellular proteome of grapevine (Vitis vinifera cv. Gamay) cell cultures in response to elicitors.

In plant cells, elicitors induce defense responses that resemble those triggered by pathogen attack, such as the synthesis of phytoalexins and pathogen-related proteins which accumulate in the extracellular space. In the search for the particular proteins involved in defense responses, we investigated the changes in the extracellular proteome of a grapevine (Vitis vinifera cv. Gamay) cell suspension in response to elicitation with methylated cyclodextrins (MBCD) and methyl jasmonate (MeJA). Twenty-five of the 39 spots differentially expressed in 2-D gels were identified and found to be encoded by 10 different genes: three secretory peroxidases, chitinase-III, beta-1,3-glucanase, thaumatin-like, SGNH plant lipase-like, NtPR27-like, xyloglucan endotransglycosylase and subtilisin-like protease. Most of them belong to the pathogenesis-related type proteins. A new class III secretory basic peroxidase and chitinase III were strongly induced in cultures treated with MBCD alone or combined with MeJA, while cultures treated with MeJA alone displayed a general repression of most of the extracellular proteins. Some of the proteins induced in grapevine cell cultures by MBCD are induced in other species by activators of systemic acquired resistance (SAR), a form of plant immunity. Collectively, the results suggest that treatment with MBCD resembles the effect of SAR induction agents in cell cultures.