© 2008 The Author(s).
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RESEARCH PAPER |
Purification, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins
1Department of Medical Biotechnology, Flinders University, Adelaide, Australia, 5042
2School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Urrbrae, Australia, 5064
3Australian Wine Research Institute, Waite Campus, Urrbrae, Australia, 5064
4Laboratoire de Biologie et Physiologie Végétales, Equipe de Biochimie et Biologie Moléculaire des Plantes, Université de Reims Champagne-Ardenne, 51687 Reims cedex 2, France
* To whom correspondence should be addressed. E-mail: wei.zhang{at}flinders.edu.au
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.
Key words: Anthocyanin transport, glutathione S-transferase, ligandin, plant cell culture, Vitis vinifera
Received 27 May 2008; Revised 1 July 2008 Accepted 24 July 2008
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