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

Conn S, Curtin C, Bézier A, Franco C, Zhang W - J. Exp. Bot. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biotechnology, Flinders University, Adelaide, Australia.

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.

Show MeSH

Related in: MedlinePlus

Anthocyanin accumulation and relative mRNA transcript levels for key anthocyanin biosynthetic genes in cultures following induction after 4 d with sucrose, jasmonic acid, and continuous light irradiation. (A) Anthocyanin content in V. vinifera cell suspensions following induction, compared with control cultures. Data are presented as the mean ±SD of triplicate analyses. The fold increase in steady-state transcript was calculated using the ΔΔ-Ct equation (Pfaffl, 2001), with β-tubulin as internal control, for (B) phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and UDP-glucose:flavonoid glucosyltransferase (UFGT), and (C) glutathione S-transferases 1–5 (VvGST1–5). All data are expressed relative to untreated cultures at each time point and are presented as the mean of duplicate analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2561157&req=5

fig5: Anthocyanin accumulation and relative mRNA transcript levels for key anthocyanin biosynthetic genes in cultures following induction after 4 d with sucrose, jasmonic acid, and continuous light irradiation. (A) Anthocyanin content in V. vinifera cell suspensions following induction, compared with control cultures. Data are presented as the mean ±SD of triplicate analyses. The fold increase in steady-state transcript was calculated using the ΔΔ-Ct equation (Pfaffl, 2001), with β-tubulin as internal control, for (B) phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and UDP-glucose:flavonoid glucosyltransferase (UFGT), and (C) glutathione S-transferases 1–5 (VvGST1–5). All data are expressed relative to untreated cultures at each time point and are presented as the mean of duplicate analyses.

Mentions: GST activity of anthocyanin-accumulating V. vinifera L. Gamay Freaux suspension cells fluctuated over time, reflecting the combined activities of individual GSTs during the culture period (Fig. 1a). Both GST activity and anthocyanin accumulation were enhanced by treatment with sucrose, jasmonic acid, and light (Figs. 1a and 5a, respectively). Glutathione-binding proteins were purified using glutathione affinity chromatography on protein from 5-day-old cells, corresponding to high GST activity (Fig. 1a) and the highest rate of anthocyanin accumulation in both treated and control cultures (data not shown). Control cultures, rather than treated cultures, were used for purification of GSTs in order to identify those proteins necessary and sufficient for anthocyanin transport. Multiple proteins were identified in the fractions displaying GST activity by one-dimensional gel electrophoresis (1D-GE; Fig. 1b). The GST activity of the fractions was proportional to the prevalence of proteins with a molecular weight of 21–26 kDa (Fig. 1b, c). The 45 kDa band, present in most fractions, may represent another glutathione-binding protein, with no apparent correlation with GST activity. GSTs were estimated to comprise >95% of protein in the bound fractions from the 1D-GE (equivalent to 2.3% of total protein from V. vinifera cell cultures).


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

Conn S, Curtin C, Bézier A, Franco C, Zhang W - J. Exp. Bot. (2008)

Anthocyanin accumulation and relative mRNA transcript levels for key anthocyanin biosynthetic genes in cultures following induction after 4 d with sucrose, jasmonic acid, and continuous light irradiation. (A) Anthocyanin content in V. vinifera cell suspensions following induction, compared with control cultures. Data are presented as the mean ±SD of triplicate analyses. The fold increase in steady-state transcript was calculated using the ΔΔ-Ct equation (Pfaffl, 2001), with β-tubulin as internal control, for (B) phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and UDP-glucose:flavonoid glucosyltransferase (UFGT), and (C) glutathione S-transferases 1–5 (VvGST1–5). All data are expressed relative to untreated cultures at each time point and are presented as the mean of duplicate analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2561157&req=5

fig5: Anthocyanin accumulation and relative mRNA transcript levels for key anthocyanin biosynthetic genes in cultures following induction after 4 d with sucrose, jasmonic acid, and continuous light irradiation. (A) Anthocyanin content in V. vinifera cell suspensions following induction, compared with control cultures. Data are presented as the mean ±SD of triplicate analyses. The fold increase in steady-state transcript was calculated using the ΔΔ-Ct equation (Pfaffl, 2001), with β-tubulin as internal control, for (B) phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and UDP-glucose:flavonoid glucosyltransferase (UFGT), and (C) glutathione S-transferases 1–5 (VvGST1–5). All data are expressed relative to untreated cultures at each time point and are presented as the mean of duplicate analyses.
Mentions: GST activity of anthocyanin-accumulating V. vinifera L. Gamay Freaux suspension cells fluctuated over time, reflecting the combined activities of individual GSTs during the culture period (Fig. 1a). Both GST activity and anthocyanin accumulation were enhanced by treatment with sucrose, jasmonic acid, and light (Figs. 1a and 5a, respectively). Glutathione-binding proteins were purified using glutathione affinity chromatography on protein from 5-day-old cells, corresponding to high GST activity (Fig. 1a) and the highest rate of anthocyanin accumulation in both treated and control cultures (data not shown). Control cultures, rather than treated cultures, were used for purification of GSTs in order to identify those proteins necessary and sufficient for anthocyanin transport. Multiple proteins were identified in the fractions displaying GST activity by one-dimensional gel electrophoresis (1D-GE; Fig. 1b). The GST activity of the fractions was proportional to the prevalence of proteins with a molecular weight of 21–26 kDa (Fig. 1b, c). The 45 kDa band, present in most fractions, may represent another glutathione-binding protein, with no apparent correlation with GST activity. GSTs were estimated to comprise >95% of protein in the bound fractions from the 1D-GE (equivalent to 2.3% of total protein from V. vinifera cell cultures).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biotechnology, Flinders University, Adelaide, Australia.

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.

Show MeSH
Related in: MedlinePlus