Functional Complementation of Anthocyanin Sequestration in the Vacuole by Widely Divergent Glutathione S-Transferases

Abstract

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.

A Multidrug Resistance–Associated Protein Involved in Anthocyanin Transport in Zea mays

“…The simplest explanation is that these transport mechanisms are compound specific. In bronze2 and An9 mutants and in aZmMrp3 plants, anthocyanins are mislocalized (Marrs et al, 1995;Alfenito et al, 1998; Figure 5C), whereas in Arabidopsis tt12 mutants, the substrates in question are proanthocyanidins (Debeaujon et al, 2001). The fact that tt12 mutants do not affect anthocyanin pigmentation argues that the TT12 transporter is not involved in anthocyanin transport in Arabidopsis.…”

“…Indeed, maize, Arabidopsis, and petunia (Petunia hybrida) all require the activity of a specific GST for correct anthocyanin sequestration (Marrs et al, 1995;Alfenito et al, 1998;Kitamura et al, 2004). This strongly suggests a role for an MRP in this process because GST activity is intimately linked with transport by MRPs in all systems studied to date.…”

“…The presence of a MATE-type flavonoid transporter needs to be reconciled with the GST-mediated transport of anthocyanin in maize, petunia, and Arabidopsis. The finding that the tt19 gene in Arabidopsis encodes a GST that is required for anthocyanin pigmentation in the plant body (Kitamura et al, 2004) combined with evidence for strong upregulation of a close homolog of the anthocyanin-specific GST from petunia (Alfenito et al, 1998) in Ant1 overexpressing tomato plants (Matthews et al, 2003) argues for the presence of both MATE and MRP transport systems in tomato and Arabidopsis. The simplest explanation is that these transport mechanisms are compound specific.…”

“…Easily identified phenotypes and complete mutant viability have made anthocyanin production an ideal model for analysis of gene regulation, paramutation, and transposon biology. Recent work on the last genetically defined step in this pathway (Marrs et al, 1995;Alfenito et al, 1998) has also identified anthocyanin biosynthesis as an important model for studying the mechanisms for the detoxification and vacuolar sequestration of heterocyclic organic anions. In addition to anthocyanins, this class of compounds includes a vast array of endogenous and xenobiotic chemicals involved in pest and disease resistance, pigmentation, UV protection, intracellular and extracellular signaling, and weed control (Abell et al, 1993;Shirley, 1996;Hammerschmidt, 1999).…”

Genetic Control and Evolution of Anthocyanin Methylation

“…DIFa, DIFf, DIFg, and DIFi encode, respectively, anthocyanidin synthase, a cytochrome b5 required for hydroxylation by flavonoid 39,59 hydroxylase , a sugar-transferase encoded by RT (Kroon et al, 1994), and a glutathione-transferase encoded by AN9 (Alfenito et al, 1998). To identify additional enzymes involved in the decoration of anthocyanins, we analyzed the remaining DIF genes.…”