In vivo grapevine anthocyanin transport involves vesicle‐mediated trafficking and the contribution of anthoMATE transporters and GST

Abstract

SUMMARYIn cells, anthocyanin pigments are synthesized at the cytoplasmic surface of the endoplasmic reticulum, and are then transported and finally accumulated inside the vacuole. In Vitis vinifera (grapevine), two kinds of molecular actors are putatively associated with the vacuolar sequestration of anthocyanins: a glutathione-Stransferase (GST) and two MATE-type transporters, named anthoMATEs. However, the sequence of events by which anthocyanins are imported into the vacuole remains unclear. We used MYBA1 transformed hairy roots as a grapevine model tissue producing anthocyanins, and took advantage of the unique autofluorescence of anthocyanins to study their cellular trafficking. In these tissues, anthocyanins were not only visible in the largest vacuoles, but were also present at higher concentrations in several vesicles of different sizes. In the cell, small vesicles actively moved alongside the tonoplast, suggesting a vesicular trafficking to the vacuole. Subcellular localization assays revealed that anthoMATE transporters were closely related with these small vesicles, whereas GST was localized in the cytoplasm around the nucleus, suggesting an association with the endoplasmic reticulum. Furthermore, cells in hairy roots expressing anthoMATE antisense did not display small vesicles filled with anthocyanins, whereas in hairy roots expressing GST antisense, anthocyanins were accumulated in vesicles but not in the vacuole. This suggests that in grapevine, anthoMATE transporters and GST are involved in different anthocyanin transport mechanisms.

The multidrug and toxic compound extrusion (MATE) family in plants

“…Suppression of GST reduced anthocyanin accumulation in vacuoles, while having no effect on the colors of small vesicles. In contrast, reduction of VvAMs decreased the number of small vesicles, but did not affect vacuolar anthocyanin, suggesting that MATE transporters and GST function independently in regulating anthocyanin accumulation in grapevines (Gomez et al 2011).…”

“…In contrast, these MATE transporters showed no transport activity for non-acylated anthocyanins, such as Cy3G ). Subcellular localization analysis using MYBA1 transformed hairy roots revealed that both MATE transporters are localized to small vesicles (Cutanda-Perez et al 2009;Gomez et al 2011). Analysis of these hairy roots showed that anthocyanins accumulated not only in vacuoles, but in small vesicles that actively moved around the tonoplast, suggesting that VvAM1 and VvAM3 are involved in the vesiclemediated transport of anthocyanins into vacuoles ( Figure 4).…”

ABCC1, an ATP Binding Cassette Protein from Grape Berry, Transports Anthocyanidin 3-O-Glucosides

“…Alternatively, it was suggested that GSTs might act as escort proteins/ligandins of anthocyanins that deliver these compounds to the transporter (Mueller et al, 2000). Using a grapevine HR system that produces anthocyanins but is silenced for a GST known to be required for efficient anthocyanin accumulation, Gomez et al (2011) observed that vesicles containing anthocyanins were still visible, but anthocyanin accumulation in the large vacuole was impaired. This may be interpreted in two ways: Either vacuolar anthocyanin transport depends on a GST, or GST absence forces the accumulation of anthocyanins in vesicles due to an impaired fusion of anthocyanin-containing vesicles with the large, central vacuole.…”

“…Scientific evidence supports both models. The vesicle-mediated transport system is mostly based on microscopy observations (Irani and Grotewold, 2005;Zhang et al, 2006;Gomez et al, 2011). Two distinct vesicular structures were described as having a putative role in anthocyanin transport.…”

Vacuolar Sorting Mechanisms are Differently Influenced by Detoxification Processes

“…This pathway needs to be supported by transporters on the tonoplast and by an adequate vacuolar compartmentalization; in fact it shares many features and problematics with the pathway for vacuolar secondary metabolites accumulation. As for secondary metabolites, such as anthocyanins [1,2], specific compartmentalization adaptations could be observed and the sequence of traffic events remains unclear [3]. Here we investigated the alteration induced by GST overexpression and/or glyphosate treatment on the vacuolar sorting pathway using two known vacuolar markers: AleuGFP (Sar1 dependent sorting) and GFPChi (Sar1 independent sorting) [4].…”