Characterisation of Anthocyanin Transport and Storage in Vitis vinifera L. cv. Gamay Freaux Cell Suspension Cultures

Author: Simon James Conn

Conn, Simon James, 2006 Characterisation of Anthocyanin Transport and Storage in Vitis vinifera L. cv. Gamay Freaux Cell Suspension Cultures, Flinders University, School of Medicine

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Anthocyanins are ubiquitous plant pigments with strong antioxidant activity, stimulating interest in the development of a plant cell-based bioprocess for their production to replace toxic synthetic food dyes and for application as pharmaceuticals, or nutraceuticals. Anthocyanin-producing plant cell suspension cultures are the currently favoured model production system facilitating rapid scale-up of production and circumventing the seasonal growth of crop plants. However, the level of anthocyanin production in these cells is commonly less than that seen in the intact plant, requiring anthocyanin enhancement strategies to improve the commercial feasability of this approach. Attempts to enhance anthocyanin production by augmenting anthocyanin biosynthesis alone, without considering the post-biosynthetic limitations (transport and storage) have been largely unsuccessful in the development of a commercial bioprocess. The aims of this study were to characterise the anthocyanin transport pathway and storage sites in Vitis vinifera L. suspension cells towards significantly improving anthocyanin production by rational enhancement strategies at the molecular level. Anthocyanins are thought to be transported from their site of biosynthesis in the cytosol via the non-covalent (ligandin) activity of glutathione S-transferases (GSTs) to the vacuole where they are concentrated in insoluble bodies, called anthocyanic vacuolar inclusions (AVIs). Five GSTs were affinity purified from pigmented grape suspension cells, characterised by nano-LC MS/MS and Edman sequencing, with the coding sequences identified and cloned. Bombardment of anthocyanin transport-deficient maize kernels with V. vinifera L. GST sequences indicated the potential involvement of two GSTs, GST1 and GST4, in anthocyanin transport. Gene expression analyses by QPCR indicated a strong correlation of these two GSTs with anthocyanin accumulation. GST4 was enhanced 60-fold with veraison in Shiraz berry skins, while GST1 and to a lesser extent GST4, was induced in V. vinifera L. cv. Gamay Freaux suspension cells under elicitation with sucrose, jasmonic acid and light irradiation (S/JA/L) to enhance anthocyanin synthesis. Purified GSTs quantified by reverse-phase HPLC from control and S/JA/L-treated suspension cells supported the gene expression data. Sequence alignments of these genes with known anthocyanin-transporting GSTs have shown conserved putative anthocyanin-binding regions. Furthermore, analysis of short upstream regions identified anthocyanin transcription factor- (R/C1) binding regions in the promoter of GST1. Increasing the expression of these GSTs provides an avenue to enhance anthocyanin production by more rapid removal of anthocyanins from biosynthetic complexes, potentially increasing biosynthetic flux. AVIs have been documented in 45 of the highest anthocyanin-accumulating suspension cell cultures, with few detailed studies on their composition, or anthocyanin profile. AVIs in grape cell cultures were found to be highly dense, membrane-delimited bodies containing a complex mix of anthocyanins, long-chain tannins and other unidentified organic compounds. Furthermore, while the proportion of individual anthocyanin species were maintained between whole-cell and AVI extracts, the AVIs were found to selectively bind a subset of highly stable acylated (p-coumaroylated) anthocyanins. Strategies to enhance anthocyanin accumulation in grape suspension cultures lead to a proportionate increase in the abundance of AVIs. Unlike AVIs in sweet potato and, to a lesser extent lisianthus, protein was not a major component of AVIs in V. vinifera L. It is likely from this evidence that AVIs represent a by-product of ER-derived vesicular transport of anthocyanins, and therefore not a target for rational enhancement of anthocyanin production.

Keywords: anthocyanin,Vitis vinifera,GSTs,anthocyanic vacuolar inclusions (AVIs)

Subject: Medical Biotechnology thesis

Thesis type: Doctor of Philosophy
Completed: 2006
School: School of Medicine
Supervisor: Prof. Chirs Franco