Characterisation of anthocyanin transport and storage in Vitis vinifera L. cv. Gamay Fréaux cell suspension cultures

Abstract

Anthocyanins are ubiquitous plant pigments with strong antioxnidant activity, stimulating interest in the development of a plant cell-based bioprocess for their production to replace toxic synnthetic 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 enhancenment strategies to improve the commercial feasibility of this approach. Attempts to enhance anthocyanin production by augnmenting anthocyanin biosynthesis alone, without considering the post-biosynthetic limitations (transport and storage) have been largely unsuccessful in the development of a commercial bioprocess.Theaimsofthisstudyweretocharacterisetheanthoncyanin transport pathway and storage sites in VitisviniferaL. suspension cells towards significantly improving anthocyanin production by rational enhancement strategies at the molecnular 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 susnpension cells, characterised by nano-LC MS/MS and Edman sequencing, with the coding sequences identified and cloned. Bombardment of anthocyanin transport-deficient maize kernnels with V.viniferaL. GST sequences indicated the potential involvement of two GSTs, GST1 and GST4, in anthocyanin transport.GeneexpressionanalysesbyQPCRindicatedastrong correlation of these two GSTs with anthocyanin accumulation. GST4 was enhanced 60-fold with vera aisoninShirazberryskins, while GST1 and to a lesser extent GST4, was induced in V.viniferaL. cv. Gamay Fra eaux suspension cells under elicitantion 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 susnpension 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 identinfied 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. AVIshavebeendocumentedin45ofthehighestanthocyaninnaccumulating 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 tanninsandotherunidentifiedorganiccompounds.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 anthoncyanin 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.viniferaL. 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.