| Abstract |
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.
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