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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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Inference of functional residues in anthocyanin transport by protein modelling. Three-dimensional model of E. coli GST chain A, interacting with its substrate (glutathione sulphonic acid). The highlighted regions are within 3 Å of the substrate and expanded to show the respective coding sequences for the aligned GSTs. Alignment of VvGSTs with known anthocyanin-transporting GSTs complementing the bz2 maize model. Amino acids shaded in black are present in ≥50% of the proteins at that residue, while alternative conserved residues at that site are shaded in grey, with other unique residues left unshaded.
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fig8: Inference of functional residues in anthocyanin transport by protein modelling. Three-dimensional model of E. coli GST chain A, interacting with its substrate (glutathione sulphonic acid). The highlighted regions are within 3 Å of the substrate and expanded to show the respective coding sequences for the aligned GSTs. Alignment of VvGSTs with known anthocyanin-transporting GSTs complementing the bz2 maize model. Amino acids shaded in black are present in ≥50% of the proteins at that residue, while alternative conserved residues at that site are shaded in grey, with other unique residues left unshaded.

Mentions: Bestfit alignments were made between the amino acid sequence of all V. vinifera GSTs and those plant GSTs previously demonstrated to complement the bz2 mutant; An9, TT19, and Bz2 (Table 2; Marrs et al., 1995; Alfenito et al., 1998; Mueller et al., 2000). It was found that VvGST1 possessed the second highest similarity to Bz2 (48.2% similarity), while the most similar sequence, VvGST5 (48.3% similarity), did not complement bz2 in this assay. VvGST4 was most similar to An9 and TT19, 75.8% and 68.4% similarity, respectively. VvGST4 also gave a similar level of complementation in the maize bombardment assay to these GSTs (Alfenito et al., 1998), inferring that the protein sequence was influential in governing anthocyanin transport in the bz2 complementation model. All VvGST sequences were aligned with Bz2, TT19, ZmGSTIII, and An9 to identify conserved residues (Fig. 8). Few key regions were identified as being unique to anthocyanin-transporting GSTs, with other single or double amino acid residues differing between GSTs with low and high activity in the bz2 complementation assay.


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)

Inference of functional residues in anthocyanin transport by protein modelling. Three-dimensional model of E. coli GST chain A, interacting with its substrate (glutathione sulphonic acid). The highlighted regions are within 3 Å of the substrate and expanded to show the respective coding sequences for the aligned GSTs. Alignment of VvGSTs with known anthocyanin-transporting GSTs complementing the bz2 maize model. Amino acids shaded in black are present in ≥50% of the proteins at that residue, while alternative conserved residues at that site are shaded in grey, with other unique residues left unshaded.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: Inference of functional residues in anthocyanin transport by protein modelling. Three-dimensional model of E. coli GST chain A, interacting with its substrate (glutathione sulphonic acid). The highlighted regions are within 3 Å of the substrate and expanded to show the respective coding sequences for the aligned GSTs. Alignment of VvGSTs with known anthocyanin-transporting GSTs complementing the bz2 maize model. Amino acids shaded in black are present in ≥50% of the proteins at that residue, while alternative conserved residues at that site are shaded in grey, with other unique residues left unshaded.
Mentions: Bestfit alignments were made between the amino acid sequence of all V. vinifera GSTs and those plant GSTs previously demonstrated to complement the bz2 mutant; An9, TT19, and Bz2 (Table 2; Marrs et al., 1995; Alfenito et al., 1998; Mueller et al., 2000). It was found that VvGST1 possessed the second highest similarity to Bz2 (48.2% similarity), while the most similar sequence, VvGST5 (48.3% similarity), did not complement bz2 in this assay. VvGST4 was most similar to An9 and TT19, 75.8% and 68.4% similarity, respectively. VvGST4 also gave a similar level of complementation in the maize bombardment assay to these GSTs (Alfenito et al., 1998), inferring that the protein sequence was influential in governing anthocyanin transport in the bz2 complementation model. All VvGST sequences were aligned with Bz2, TT19, ZmGSTIII, and An9 to identify conserved residues (Fig. 8). Few key regions were identified as being unique to anthocyanin-transporting GSTs, with other single or double amino acid residues differing between GSTs with low and high activity in the bz2 complementation assay.

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