Flowering plants synthesize diverse metabolites, many of which are stored in the vacuoles. Many such secondary metabolites are toxic or highly reactive and must be sequestered from the cell to prevent damage; interestingly, many of these stored compounds are useful drugs, because of their specific interactions with enzymes and other cellular constituents. To understand how plants manage toxic metabolites the mechanism of vacuolar sequestration for anthocyanin pigments and the precursor flavonoid compounds will be analyzed using a combination of genetics and biochemistry. Anthocyanins are ideal model compounds because so much is already known about their biochemistry and the genetic control of their synthesis. These pigments confer the red/blue/purple colors in many flowers; the pigments are members of a large chemical class, the flavonoids, widely distributed in green plants. Flavonoids are natural sunscreens against UV damage, attractants for pollinators, signals in plant-microbe interaction, and molecules required for successful pollen-stigma interaction. Flavonoids are also an important component of a healthy human diet. Despite their good features, flavonoids cause cellular damage by oxidizing proteins and intercalating into DNA increasing mutation frequency; such damage is prevented if the flavonoids are sequestered efficiently into the vacuolar compartment. Presently, it is known that the BZ2 protein of maize and the AN9 protein of Petunia are required at an early step in the transfer of anthocyanin from the cytoplasm to the vacuole; mutant plants lacking these proteins accumulate anthocyanin in the cytoplasm. In this project the role of MRP29 in moving anthocyanin across the tonoplast membrane will be tested. The current model is that BZ2 is a carrier protein for anthocyanin; it delivers this cargo to the MRP29 transporter, which in turn transfers the cargo into the vacuole. MRP29, an ATP Cassette Binding Protein transporter, is regulated by the same transcription factors that are required to activate the biosynthetic genes of the anthocyanin pathway. Research will focus on establishing whether MRP29 is localized to the tonoplast membrane of the vacuole, on the impact of mutation at MRP29 on anthocyanin sequestration, on the interaction of MRP29 with BZ2 and related proteins in vivo and in vitro, and on the requirements for anthocyanin sequestration in an in vitro assay that will be established during the research project. Mutant yeast cells, lacking all ABC transporters, will be transformed to express the maize MRP29 protein; using vacuoles prepared from such yeast strains, tonoplasts containing just one plant ABC transporter can be purified. This material will be used to test the model at a more refined biochemical level.
