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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) Genomic structure of V. vinifera GSTs cloned in this study. Exons are indicated by black arrows, and introns are indicated by double lines. (B) Phylogenetic tree of GSTs in the plant kingdom, showing classification into type I, II, and III GSTs as per Droog (1997). Sequences aligned are listed on the right-hand side of the tree and were obtained by retrieval of protein sequences from GenBank. Multiple sequence alignments were created by ClustalW (Thompson et al., 1994) and the phylogenetic tree was generated using PHYLIP under default settings and viewed with Treeview version 1.6.6. Origins of the proteins are indicated by a two-letter prefix to the protein name: At, Arabidopsis, Dc, carnation; Nt, tobacco; Sc, cucumber; St, potato; Ta, wheat; Vv, grape (highlighted); Zm, maize. An9, TT19, and Bz2 are shown without prefixes for consistency.
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fig3: (A) Genomic structure of V. vinifera GSTs cloned in this study. Exons are indicated by black arrows, and introns are indicated by double lines. (B) Phylogenetic tree of GSTs in the plant kingdom, showing classification into type I, II, and III GSTs as per Droog (1997). Sequences aligned are listed on the right-hand side of the tree and were obtained by retrieval of protein sequences from GenBank. Multiple sequence alignments were created by ClustalW (Thompson et al., 1994) and the phylogenetic tree was generated using PHYLIP under default settings and viewed with Treeview version 1.6.6. Origins of the proteins are indicated by a two-letter prefix to the protein name: At, Arabidopsis, Dc, carnation; Nt, tobacco; Sc, cucumber; St, potato; Ta, wheat; Vv, grape (highlighted); Zm, maize. An9, TT19, and Bz2 are shown without prefixes for consistency.

Mentions: Amplification of GST gene sequences from genomic DNA revealed the presence of a single intron in both VvGST1 and VVGST5, two introns in VvGST2 and VvGST3, while VvGST4 did not contain an intron (Fig. 3a). Alignments with other plant GST proteins indicate VvGST4 to be a type I GST (Fig. 3b) and support the classification of the other GSTs based on intron/exon structure alone.


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) Genomic structure of V. vinifera GSTs cloned in this study. Exons are indicated by black arrows, and introns are indicated by double lines. (B) Phylogenetic tree of GSTs in the plant kingdom, showing classification into type I, II, and III GSTs as per Droog (1997). Sequences aligned are listed on the right-hand side of the tree and were obtained by retrieval of protein sequences from GenBank. Multiple sequence alignments were created by ClustalW (Thompson et al., 1994) and the phylogenetic tree was generated using PHYLIP under default settings and viewed with Treeview version 1.6.6. Origins of the proteins are indicated by a two-letter prefix to the protein name: At, Arabidopsis, Dc, carnation; Nt, tobacco; Sc, cucumber; St, potato; Ta, wheat; Vv, grape (highlighted); Zm, maize. An9, TT19, and Bz2 are shown without prefixes for consistency.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: (A) Genomic structure of V. vinifera GSTs cloned in this study. Exons are indicated by black arrows, and introns are indicated by double lines. (B) Phylogenetic tree of GSTs in the plant kingdom, showing classification into type I, II, and III GSTs as per Droog (1997). Sequences aligned are listed on the right-hand side of the tree and were obtained by retrieval of protein sequences from GenBank. Multiple sequence alignments were created by ClustalW (Thompson et al., 1994) and the phylogenetic tree was generated using PHYLIP under default settings and viewed with Treeview version 1.6.6. Origins of the proteins are indicated by a two-letter prefix to the protein name: At, Arabidopsis, Dc, carnation; Nt, tobacco; Sc, cucumber; St, potato; Ta, wheat; Vv, grape (highlighted); Zm, maize. An9, TT19, and Bz2 are shown without prefixes for consistency.
Mentions: Amplification of GST gene sequences from genomic DNA revealed the presence of a single intron in both VvGST1 and VVGST5, two introns in VvGST2 and VvGST3, while VvGST4 did not contain an intron (Fig. 3a). Alignments with other plant GST proteins indicate VvGST4 to be a type I GST (Fig. 3b) and support the classification of the other GSTs based on intron/exon structure alone.

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