A Tau Class Glutathione-S-Transferase is Involved in Trans-Resveratrol Transport Out of Grapevine Cells

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.

Glutathione S-Transferase Enzymes in Plant-Pathogen Interactions

“…Grapevine ( Vitis ) species may resist fungal infections by accumulating secondary metabolites like stilbenoid phytoalexins (trans-resveratrols). A tau class GST (GSTU-2) was identified in V. vinifera cell cultures and shown to be involved in extracellular transport of trans-resveratrols: grapevine cell cultures overexpressing GSTU-2 accumulated trans-resveratrols in the extracellular medium even without any elicitation of plant defenses or pathogen infection (Martínez-Márquez et al, 2017). However, in Vitis flexuosa different GTSs may play diverse roles in pathogen defense, since only one out of five characterized GST genes was induced, while expression of the other GSTs was down-regulated following infection by the necrotrophic fungi B. cinerea and Elsinoe ampelina (Ahn et al, 2016).…”

Physiological Roles of Plant Methionine Sulfoxide Reductases in Redox Homeostasis and Signaling

“…GSTs form a complex family of enzymes detoxifying a broad range of molecules such as secondary metabolites and exogenous substrates that are referred as xenobiotics, and include herbicides [ 124 , 125 , 126 ]. Depending on their catalytic residues, GSTs catalyze glutathione conjugation, perform deglutathionylation or bind non-substrate ligands.…”

Functional, Structural and Biochemical Features of Plant Serinyl-Glutathione Transferases

“…Other molecules were isolated using ligand fishing approaches (Table 2). GSTU2 from V. vinifera binds trans- resveratrol, a polyphenol transported from the cells into extracellular medium and conferring antimicrobial properties (Martínez-Márquez et al, 2017). GSTF2 and GSTF3 from A. thaliana were described in vitro to bind various ligands such as lumichrome, harmane, norharmane, indole-3-aldehyde, camalexin, and quercetin-3-O-rhamnoside (Dixon et al, 2011a) but the physiological significance remains unknown.…”