Abstract
γ-glutamyl transferases/transpeptidases (E.C. 2.3.2.2, GGTs) are involved in the catabolism of many compounds that are conjugated to glutathione (GSH), which have a variety of roles. GSH can act as storage and transport vehicle for reduced sulfur; it is involved in the detoxification of xenobiotics and also acts as a redox buffer by utilizing its thiol residue to protect against reactive oxygen species, which accumulate in response to biotic and abiotic stress. Furthermore, many distinctive flavor and aroma compounds in Sauvignon blanc wines originate from odorless C5- and C6-GSH conjugates or their GGT catabolized derivatives. These precursors are then processed into their volatile forms by yeast during fermentation. In many plant species, two or more isoforms of GGTs exist that target GSH-conjugates to either the apoplast or the vacuole. A bioinformatics approach identified multiple GGT candidates in grapevine (Vitis vinifera). However, only a single candidate, VvGGT3, has all the conserved residues needed for GGT activity. This is intriguing given the variety of roles of GSH and GGTs in plant cells. Characterization of VvGGT3 from cv. Sauvignon blanc was then undertaken. The VvGGT3 transcript is present in roots, leaves, inflorescences, and tendril and at equal abundance in the skin, pulp, and seed of mature berries and shows steady accumulation over the course of whole berry development. In addition, the VvGGT3 transcript in whole berries is upregulated upon Botrytis cinerea infection as well as mechanical damage to leaf tissue. VvGGT3-GFP fusion proteins transiently over-expressed in onion cells were used to study subcellular localization. To confirm VvGGT3 activity and localization in vivo, the fluorescent γ-glutamyl-7-amido-4-methylcoumarin substrate was added to Nicotiana benthamiana leaves transiently over-expressing VvGGT3. In combination, these results suggest that the functional VvGGT3 is associated with membrane-like structures. This is not consistent with its closely related functionally characterized GGTs from Arabidopsis, radish and garlic.
Highlights
In plants, glutathione (GSH, γ-Glu-Cys-Gly) is involved in a multitude of cellular processes
We explored subcellular localization as well as VvGGT3 transcript abundance in a range of tissues, including whole grape berry development and whole berries infected with Botrytis
Pulp and seed fractions were separated from mature whole Sauvignon blanc grape berries at 21 °Brix and B. cinerea-infected berries were collected from the Lincoln University research vineyard in the 2012 and 2014 seasons, respectively
Summary
Glutathione (GSH, γ-Glu-Cys-Gly) is involved in a multitude of cellular processes It is often conjugated by glutathione S-transferases (GSTs) to a wide range of chemicals, both derived from cellular metabolism and externally occurring xenobiotics (Rouhier et al, 2008). Mammalian as well as Escherichia coli GGTs undergo an autoproteolytic cleavage that is dependent on a conserved threonine residue (Hashimoto et al, 1995; Okada et al, 2006; Taniguchi and Ikeda, 2006) into a large and a small subunit These subunits self-assemble into functional heterodimers (Tate and Meister, 1978; Masi et al, 2007; Castellano and Merlino, 2013). GGTs play a role in maintaining cellular redox homeostasis by regulating the cellular levels of GSH (Castellano and Merlino, 2012)
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