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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.

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(A) Two-dimensional gel electropherogram of glutathione affinity chromatography fractions possessing GST activity. (B) Enlarged image of the boxed region showing grouping based on mass spectrometry fingerprints (refer to Table 1). IEF strip pI3-10NL was rehydrated in the protein precipitate from GST active fractions of the GSTrap column. This was focused for 48 kVh and then separated on a 10% polyacrylamide gel and stained with SYPRO Ruby.
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fig2: (A) Two-dimensional gel electropherogram of glutathione affinity chromatography fractions possessing GST activity. (B) Enlarged image of the boxed region showing grouping based on mass spectrometry fingerprints (refer to Table 1). IEF strip pI3-10NL was rehydrated in the protein precipitate from GST active fractions of the GSTrap column. This was focused for 48 kVh and then separated on a 10% polyacrylamide gel and stained with SYPRO Ruby.

Mentions: Fractions from the glutathione affinity chromatography with GST activity were pooled and separated by 2D-GE (Fig. 2). Proteins were visualized using the MS-compatible SYPRO Ruby stain, following verification that overexposed silver staining (data not shown) did not indicate the presence of otherwise undetected low abundance proteins.


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)

(A) Two-dimensional gel electropherogram of glutathione affinity chromatography fractions possessing GST activity. (B) Enlarged image of the boxed region showing grouping based on mass spectrometry fingerprints (refer to Table 1). IEF strip pI3-10NL was rehydrated in the protein precipitate from GST active fractions of the GSTrap column. This was focused for 48 kVh and then separated on a 10% polyacrylamide gel and stained with SYPRO Ruby.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: (A) Two-dimensional gel electropherogram of glutathione affinity chromatography fractions possessing GST activity. (B) Enlarged image of the boxed region showing grouping based on mass spectrometry fingerprints (refer to Table 1). IEF strip pI3-10NL was rehydrated in the protein precipitate from GST active fractions of the GSTrap column. This was focused for 48 kVh and then separated on a 10% polyacrylamide gel and stained with SYPRO Ruby.
Mentions: Fractions from the glutathione affinity chromatography with GST activity were pooled and separated by 2D-GE (Fig. 2). Proteins were visualized using the MS-compatible SYPRO Ruby stain, following verification that overexposed silver staining (data not shown) did not indicate the presence of otherwise undetected low abundance proteins.

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