Lay summary
Flavonoids represent a structurally multifaceted, prominent subgroup of secondary metabolites that play many roles in important processes including stress protection, defence, pollination and seed dispersal. Recent evidence suggests prime cellular functions in plants including the regulation of auxin transport.In order to achieve many of these functions, flavonoids accumulate at high concentrations. Yet, they are toxic to the cytosol primarily due to the denaturing properties of phenolic substances. Consequently, flavonoids accumulate either in the vacuole or undergo secretion into the extracellular space. In spite of their overall relevance and detailed knowledge of the biosynthetic pathway, our knowledge of transport mechanisms involved in the accumulation of flavonoids is limited. Most importantly, Arabidopsis, which accumulates flavonols, anthocyanins and proanthocyanidins (PAs) in different tissues and organs, has not been used extensively as a model to investigate flavonoid transport.In barley, glucosylated flavonoids are deposited in vacuoles via a flavonoid/H+-antiporter. Analysis of the Arabidopsis transparent testa12 (tt12) which fails to accumulate PAs in the seed coat endothelium revealed the first gene involved in PA precursor transport. TT12 is a membrane protein belonging to the multidrug and toxic efflux (MATE) transporter family, which are either Na+- or H+-dependent antiporters in prokaryotes. These results suggest that MATE proteins are involved in flavonoid transport. Our expression, localization and transport experiments demonstrate that TT12 acts as a vacuolar flavonoid/H+-antiporter which is expressed exclusively in the seed coat cells actively producing PAs. Thus, TT12 expression corresponds to the defined expression pat-tern of the BANYULS (BAN) promoter. In vitro, heterologously expressed TT12 catalyzes transport of glu-cosylated anthocyanins. Due to the ease of phenotypic analysis, tt12 is an elegant model to investigate different features of MATE transporters in higher plants. Consequently, we propose to use TT12 for an in-depth analysis of this poorly defined transporter family.Two strategies are presented that aim at the identification of (A) functionally important amino acid residues in TT12 and its transmembrane topology and (B) novel flavonoid transporters within the MATE family. In (A), twelve TT12 mutants in conserved amino acids, which were generated by site-directed mutagenesis, will be investigated for their capacity to (i) revert the tt12 PA-related seed phenotype, (ii) transport flavonoids after heterologous expression in yeast and (iii) properly reach the tonoplast as visual-ized as translational fusions with YFP. A novel experimental strategy is proposed to use bimolecular fluo-rescence complementation of split YFP fragments positioned in different structural parts of the TT12 transporter to analyse the topology of MATE proteins.In (B), two complementary screening strategies are proposed to identify unknown vacuolar transporters.