Demurtas et al. developed a high-throughput approach to identifying vacuolar transporters and used it to explore transport of crocins in saffron. Plant Cell https://doi.org/10.1105/tpc.19.00193
By Olivia C. Demurtas and Giovanni Giuliano
Background: The saffron spice is made of dried stigmas from the plant Crocus sativus. The red color of the spice is due to crocins, glycosylated metabolites that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. Crocins are polar compounds and are synthesized in the cytosol; therefore, a transporter protein is needed to make them cross the vacuolar membrane (the tonoplast).
Question: We searched the saffron stigma for transcripts encoding ABCC and MATE transporters, which are known to mediate vacuolar transport. Three of the candidate transcripts were strongly co-expressed with crocins and/or with the first dedicated enzyme catalyzing crocin biosynthesis. Since crocins are a complex mixture of compounds, instead to purifying them and testing their transport one by one, we set up an assay (named “transportomics”). This assay essentially consists of expressing the candidate transporters in yeast, isolating vacuoles from the yeast cells, incubating them with a stigma metabolite extract, and testing which of the metabolites in the extract get transported into the vacuoles.
Findings: We characterized 2 ABCC and 3 MATE transporters. The 2 ABCC transporters were able to transport crocins in yeast vacuoles, while the MATE transporters transported flavonoids. We were also able to show that, in Nicotiana benthamiana leaf cells engineered to produce crocins, one of the ABBC transporters was able to boost crocin accumulation.
Next steps: The approach described here has a general application for the discovery of vacuolar transporters mediating high-level accumulation of specialized metabolites in plant tissues, such as those mediating the accumulation of steviosides in Stevia leaves.
Olivia Costantina Demurtas, Rita de Brito Francisco, Gianfranco Diretto, Paola Ferrante, Sarah Frusciante, Marco Pietrella, Giuseppe Aprea, Lorenzo Borghi, Mistianne Feeney, Lorenzo Frigerio, Adriana Coricello, Giosuè Costa, Stefano Alcaro, Enrico Martinoia, Giovanni Giuliano. (2019). ABCC Transporters Mediate the Vacuolar Accumulation of Crocins in Saffron Stigmas. Plant Cell. https://doi.org/10.1105/tpc.19.00193.
Keywords: Crocus sativus, stigma, ATP Binding Cassette (ABC) transporters, Multidrug And Toxic Compound Extrusion (MATE) transporters, crocins, in vitro transport assay, microsomes, confocal microscopy.