ABCC transporters mediate the vacuolar accumulation of crocins in saffron stigmas

Compartmentation is a key strategy enacted by higher plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast, and in vitro "transportomic" assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and used it to identify saffron transporters mediating vacuolar crocin accumulation. Three transporters, belonging to both the Multidrug And Toxic Compound Extrusion (MATE) and ATP Binding Cassette C (ABCC) families, were co-expressed with crocins and/or with the first dedicated gene in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in Crocus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in N. benthamiana leaves.