FUNCTIONAL COMPLEMENTATION OF ANTHOCYANIN SEQUESTRATION IN THE VACUOLE BY WIDELY DIVERGENT GLUTATHIONE S-TRANSFERASES

Glutathione S-transferases (GSTs) traditionally have been studied in p lants and other organisms for their ability to detoxify chemically div erse herbicides and other toxic organic compounds. Anthocyanins are am ong the few endogenous substrates of plant GSTs that have been identif ied. The Bronze2 (Bz2) gene encodes a type III GST and performs the la st genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C 3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the compar able step in the petunia anthocyanin pathway is controlled by the Anth ocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST, Bz2 and An9 have evolved independently fro m distinct types of GSTs, but each is regulated by the conserved trans criptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. A mong several other GSTs tested, only soybean GmGST26A (previously call ed GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been ass ociated with the anthocyanin pathway. Requirements for An9 and Bz2 gen e function were investigated by sequencing functional and nonfunctiona l germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1.