Purification, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins

Abstract

The ligandin activity of specific glutathione S-transferases (GSTs) is necessary for the transport of anthocyanins from the cytosol to the plant vacuole. Five GSTs were purified from Vitis vinifera L. cv. Gamay Fréaux cell suspension cultures by glutathione affinity chromatography. These proteins underwent Edman sequencing and mass spectrometry fingerprinting, with the resultant fragments aligned with predicted GSTs within public databases. The corresponding coding sequences were cloned, with heterologous expression in Escherichia coli used to confirm GST activity. Transcriptional profiling of these candidate GST genes and key anthocyanin biosynthetic pathway genes (PAL, CHS, DFR, and UFGT) in cell suspensions and grape berries against anthocyanin accumulation demonstrated strong positive correlation with two sequences, VvGST1 and VvGST4, respectively. The ability of VvGST1 and VvGST4 to transport anthocyanins was confirmed in the heterologous maize bronze-2 complementation model, providing further evidence for their function as anthocyanin transport proteins in grape cells. Furthermore, the differential induction of VvGST1 and VvGST4 in suspension cells and grape berries suggests functional differences between these two proteins. Further investigation of these candidate ligandins may identify a mechanism for manipulating anthocyanin accumulation in planta and in vitro suspension cells.

The Phenylpropanoid Pathway Is Controlled at Different Branches by a Set of R2R3-MYB C2 Repressors in Grapevine

“…In particular, genes coding for PHE AM-MONIA LYASE (PAL), C4H, 4CL, CAFFEOYL-CoA O-METHYLTRANSFERASE (CCoAOMT), and CAFFEIC ACID 3-O-METHYLTRANSFERASE (COMT) were selected as SWP-related genes. Additionally, the anthocyanin-related genes MYBA1, UDP GLC: FLA-VONOID-3-O-GLUCOSYLTRANSFERASE (UFGT), and GLUTATHIONE S-TRANSFERASE4 (GST4; Walker et al, 2007;Conn et al, 2008) and the PA-related genes MYBPA1, LEUCOANTHOCYANIDIN REDUCTASE1 (LAR1), LAR2, and ANTHOCYANIDIN REDUCTASE (ANR; Bogs et al, 2005Bogs et al, , 2007 were included together with the bHLH regulator of the flavonoid pathway MYC1 (Hichri et al, 2010). Figure S1.…”

Oxidative stress homeostasis in grapevine (Vitis vinifera L.)

“…GSTs also play a role in the cellular response to auxins and during the normal metabolism of plant secondary products like anthocyanins and cinnamic acid. In grapevine, 107 GST isoforms were found (Supplementary Table 1) and seven genes belonging to the phi class and 57 belonging to the tau class, the two most important classes of plant-specific GSTs (Edwards et al, 2000), are implicated in anthocyanin metabolism during berry development (Zenoni et al, 2010), GST1 and GST4 being implicated in anthocyanin transport to the vacuole (Conn et al, 2008). Also in grapevine, the expression of several GSTs genes was affected by defoliation (Pastore et al, 2013).…”

The multidrug and toxic compound extrusion (MATE) family in plants

“…Analysis of these hairy roots showed that anthocyanins accumulated not only in vacuoles, but in small vesicles that actively moved around the tonoplast, suggesting that VvAM1 and VvAM3 are involved in the vesiclemediated transport of anthocyanins into vacuoles ( Figure 4). Because grapevine GST is also a candidate protein for anthocyanin transport (Conn et al 2008), and because it was the most up-regulated gene when VvMYBA1 was overexpressed in hairy roots (Cutanda-Perez et al 2009), its involvement in vesicle trafficking was also investigated. Suppression of GST reduced anthocyanin accumulation in vacuoles, while having no effect on the colors of small vesicles.…”