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The defense-related plant metabolites known as glucosinolates play important roles in agriculture, ecology, and human health. Despite an advanced biochemical understanding of the glucosinolate pathway, the source of the reduced sulfur atom in the core glucosinolate structure remains unknown. Recent evidence has pointed toward GSH, which would require further involvement of a GSH conjugate processing enzyme. In this article, we show that an Arabidopsis thaliana mutant impaired in the production of the γ-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. We also show that the double mutant is impaired in the production of camalexin and accumulates high amounts of the camalexin intermediate GS-IAN upon induction. In addition, we demonstrate that the cellular and subcellular localization of GGP1 and GGP3 matches that of known glucosinolate and camalexin enzymes. Finally, we show that the purified recombinant GGPs can metabolize at least nine of the 10 glucosinolate-related GSH conjugates as well as GS-IAN. Our results demonstrate that GSH is the sulfur donor in the biosynthesis of glucosinolates and establish an in vivo function for the only known cytosolic plant γ-glutamyl peptidases, namely, the processing of GSH conjugates in the glucosinolate and camalexin pathways.
INTRODUCTION
Among the most well-studied defense-related compounds in plants is the group of metabolites known as glucosinolates. Glucosinolates are sulfur-containing secondary metabolites characteristic of the order Brassicales, which includes the agriculturally important oilseed rape (Brassica napus), the cruciferous vegetables, and the model plant Arabidopsis thaliana (Fahey et al., 2001). Together with the enzyme myrosinase, glucosinolates constitute the so-called mustard oil bomb, which is a binary defense system against generalist insects (Hopkins et al., 2009) and has also been implicated in defense against nonadapted pathogens (Bednarek et al., 2009; Clay et al., 2009). Apart from their ecological and agricultural importance, glucosinolates have been proposed to have cancer-preventive properties (Higdon et al., 2007; Hayes et al., 2008), which has sparked great interest in their metabolic engineering and heterologous production (Gasper et al., 2005; Geu-Flores et al., 2009). After a decade of glucosinolate research in the Arabidopsis postgenomic era, the biosynthetic pathway is well understood and most biosynthetic genes are known (Sønderby et al., 2010). A notable exception is the step involving the incorporation of reduced sulfur, where...