AN9 is a glutathione S-transferase from petunia (Petunia hybrida) required for efficient anthocyanin export from the site of synthesis in the cytoplasm into permanent storage in the vacuole. For many xenobiotics it is well established that a covalent glutathione (GSH) tag mediates recognition of molecules destined for vacuolar sequestration by a tonoplast-localized ATP-binding cassette pump. Here we inquired whether AN9 catalyzes the formation of GSH conjugates with flavonoid substrates. Using high-performance liquid chromatography analysis of reaction mixtures containing enzyme, GSH, and flavonoids, including anthocyanins, we could detect neither conjugates nor a decrease in the free thiol concentration. These results suggest that no conjugate is formed in vitro. However, AN9 was shown to bind flavonoids using three assays: inhibition of the glutathione S-transferase activity of AN9 toward the common substrate 1-chloro 2,4-dinitrobenzene, equilibrium dialysis, and tryptophan quenching. We conclude that AN9 is a flavonoid-binding protein, and propose that in vivo it serves as a cytoplasmic flavonoid carrier protein.

Anthocyanin biosynthesis is one of the most thoroughly studied enzymatic pathways in biology, but little is known about the molecular mechanisms of its final stage: the transport of the anthocyanin pigment into the vacuole. We have identified a multidrug resistance-associated protein (MRP), ZmMrp3, that is required for this transport process in maize (Zea mays). ZmMrp3 expression is controlled by the regulators of anthocyanin biosynthesis and mirrors the expression of other anthocyanin structural genes. Localization of ZmMRP3 in vivo shows its presence in the tonoplast, the site at which anthocyanin transport occurs. Mutants generated using antisense constructs have a distinct pigmentation phenotype in the adult plant that results from a mislocalization of the pigment as well as significant reduction in anthocyanin content, with no alteration in the anthocyanin species produced. Surprisingly, mutant plants did not show a phenotype in the aleurone. This appears to reflect the presence of a second, highly homologous gene, ZmMrp4, that is also coregulated with the anthocyanin pathway but is expressed exclusively in aleurone tissue. This description of a plant MRP with a role in the transport of a known endogenous substrate provides a new model system for examining the biological and biochemical mechanisms involved in the MRP-mediated transport of plant secondary metabolites.

Glutathione S -transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 ( Bz2 ) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 ( An9 ) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional 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. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2 / 4 ) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529 , bz2::Ds , and bz2::Mu1. INTRODUCTIONGlutathione S -transferases (GSTs; EC 2.5.1.18) conjugate the glutathione tripeptide ( ␥ -Glu-Cys-Gly; GSH) to a broad variety of substrates. They are an ancient and ubiquitous gene family encoding ‫ف‬ 25-to 29-kD proteins that form both homodimers and heterodimers in vivo. GSTs are as plentiful as they are diverse. As a group, maize GSTs are among the most abundant nonphotosynthetic enzymes in plant cells, making up as much as 1% of the soluble leaf protein (SariGorla et al., 1993). At least 38 plant GSTs have been identified (Marrs, 1996), and a search of the Arabidopsis expressed sequence tag (EST) database identifies Ͼ 200 distinct ESTs with substantial homology to GSTs. It is striking that the GST family is so highly divergent; only a motif of ‫ف‬ 15 amino acids is moderately conserved in sequence and position among all plant GSTs (Marrs, 1996).Mammalian GST genes are divided into five groups ( ␣ , , , , and ) based on sequence similarities, immunological cross-reactivity, and substrate specificity. All identified plant GSTs fall into the most ancient class, that of theta. Droog et al. (1995) recently proposed three subdivisions of plant GSTs-type I, type II, and type III-based on a combination of sequence conservation, immunological cross-reactivity, and intron/exon structure of the gene. Type I GSTs have two introns, and many are induced, both transcriptionally and translationally, by environmental perturbations, ...

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Expression of a bacterial transporter protein in Toxoplasma gondii results in parasite susceptibility to Formidomycin, a drug targeting isoprenoid precursor synthesis.