Abstract
Vitis vinifera cell cultures respond to pathogens and elicitors by synthesizing and extracellularly accumulating stilbenoid phytoalexins. Large amounts of trans-resveratrol (t-R) are produced when a cell culture is elicited with methylated cyclodextrins (MBCD), either alone or combined with methyl jasmonate (MeJA). t-R transport to the extracellular medium, which represents the apoplastic space, would place this antifungal defense right in the battlefield to efficiently fight against pathogen attack. Yet despite their physiological relevance, these transport pathways are mostly unknown. A broad hypothesis-free DIGE-based proteomic experiment of a temporal series of elicited grapevine cell cultures was performed to explore the expression profiles of t-R biosynthetic proteins and other co-expressing proteins potentially involved in such a cell response. A correlation between two tau class glutathione-S-transferases (GSTs) with several stilbene synthase and phenylalanine ammonia-lyase isoforms, and with the t-R metabolite itself, was found and further assessed by a qRT-PCR gene expression analysis. The best candidate, GSTU-2, was cloned from the cDNA of the MBCD + MeJA-elicited grapevine cells and used for Agrobacterium-mediated grapevine cell transformation. The non-elicited lines that overexpressed GSTU-2 displayed an extracellular t-R accumulating phenotype, but stabilization of t-R required the addition to culture medium of adsorbent compounds, e.g., PVP or β-cyclodextrin. The wild-type cell cultures accumulated no t-R, not even in the presence of adsorbents. The transient expression of the GSTU-2-GFP fusion proteins in grapevine cells showed localisation in the plasma membrane, and the immunoprecipitation of HA-tagged GSTU-2 revealed its interaction with HIR, a plasma membrane-bound protein. These findings are consistent with a functional role in transport. This is the first report providing several pieces of experimental evidence for the involvement of a specific tau class GST in t-R transport to the extracellular medium.
Highlights
Vitis species defend themselves from fungal infection by accumulating phytoalexins and PR proteins (Derckel et al, 1999)
From the four intracellular compounds, only t-R followed the trend observed in the extracellular medium, and increased in a time-dependent manner in treatments MBCD and MBCD + methyl jasmonate (MeJA), while the piceid and c-R content did not respond to treatments
In a previous proteomic study that used DIGE at a fixed 96-h time point, we found that t-R accumulation in the MBCD- and MBCD + MeJA-elicited cultures correlated positively with the spots that contained stilbene/resveratrol synthase (STS) isoforms, and with the spots that contained the tau class glutathione-S-transferase (GST) isoforms (MartínezEsteso et al, 2011b)
Summary
Vitis species defend themselves from fungal infection by accumulating phytoalexins and PR proteins (Derckel et al, 1999). A phylogenetic analysis performed with other GSTs identified in proteomic studies of grapevine berry skin (Martínez-Esteso et al, 2011a) and flesh (Martínez-Esteso et al, 2013), and in elicited cell cultures (Martínez-Esteso et al, 2011b), and those co-expressed with STS clustered apart, indicates a role of specific GSTs in t-R accumulation, which is potentially related with the transport of the metabolite to the extracellular medium. To explore this possibility, we carried out a broader DIGEbased proteomic co-expression analysis to better select the potential GST candidates involved in t-R transport. The physiological and biotechnological relevance of the results is discussed
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