Glutathione-Indole-3-Acetonitrile Is Required for Camalexin Biosynthesis in Arabidopsis thaliana    

Abstract

Camalexin, a major phytoalexin in Arabidopsis thaliana, consists of an indole ring and a thiazole ring. The indole ring is produced from Trp, which is converted to indole-3-acetonitrile (IAN) by CYP79B2/CYP79B3 and CYP71A13. Conversion of Cys(IAN) to dihydrocamalexic acid and subsequently to camalexin is catalyzed by CYP71B15. Recent studies proposed that Cys derivative, not Cys itself, is the precursor of the thiazole ring that conjugates with IAN. The nature of the Cys derivative and how it conjugates to IAN and subsequently forms Cys(IAN) remain obscure. We found that protein accumulation of multiple glutathione S-transferases (GSTs), elevation of GST activity, and consumption of glutathione (GSH) coincided with camalexin production. GSTF6 overexpression increased and GSTF6-knockout reduced camalexin production. Arabidopsis GSTF6 expressed in yeast cells catalyzed GSH(IAN) formation. GSH(IAN), (IAN)CysGly, and gGluCys(IAN) were determined to be intermediates within the camalexin biosynthetic pathway. Inhibitor treatments and mutant analyses revealed the involvement of g-glutamyl transpeptidases (GGTs) and phytochelatin synthase (PCS) in the catabolism of GSH(IAN). The expression of GSTF6, GGT1, GGT2, and PCS1 was coordinately upregulated during camalexin biosynthesis. These results suggest that GSH is the Cys derivative used during camalexin biosynthesis, that the conjugation of GSH with IAN is catalyzed by GSTF6, and that GGTs and PCS are involved in camalexin biosynthesis.

DEXH Box RNA Helicase–Mediated Mitochondrial Reactive Oxygen Species Production in Arabidopsis Mediates Crosstalk between Abscisic Acid and Auxin Signaling

“…The GSH content was measured using a Glutathione Colorimetric Detection Kit (BioVision), with the following minor modifications (Su et al, 2011). The root tips were ground to powder using liquid nitrogen, ;50 mg of each sample was then dissolved in 400 mL GSH buffer, and 100 mL 5% sulfosalicylic acid was added and mixed gently.…”

Assigning Gene Function in Biosynthetic Pathways: Camalexin and Beyond

“…However, two recent studies published in this journal arrived at different conclusions regarding the conversion of the glutathione conjugate (g-Glu-Cys[IAN]-Gly) to the Cys conjugate (Cys [IAN]), particularly regarding the family of enzymes cleaving off the g-Glu residue. Su et al (2011) reported that known members of the g-glutamyl transpeptidase (GGT) family conducted this reaction, whereas we (Geu-Flores et al, 2011) found that members of the newly found g-glutamyl peptidase (GGP) family performed the same reaction. This has created some confusion in the literature, with several subsequent depictions of the pathway containing both GGTs and GGPs (Ahuja et al, 2012;Saga et al, 2012), although some have made a clear distinction (Bednarek, 2012).…”

“…Subsequently, CYP71A13 converts the aldoxime to IAN (Nafisi et al, 2007), which, after an unknown activation step, is conjugated to the tripeptide glutathione (g-Glu-Cys-Gly) by glutathione Stransferases, including GSTF6 (Bö ttcher et al, 2009;Su et al, 2011). The glutathione conjugate g-Glu-Cys(IAN)-Gly is then hydrolyzed by two consecutive peptidase reactions (discussed in detail below) to Cys(IAN), which is finally converted to camalexin by a two-step reaction catalyzed by CYP71B15 (PAD3) (Zhou et al, 1999;Schuhegger et al, 2006;Bö ttcher et al, 2009) (Figure 1).…”

“…In search for missing camalexin biosynthetic genes, Su et al (2011) performed proteomics on Arabidopsis seedlings with camalexin production induced via a dexamethasone-stimulated mitogen-activated protein kinase 9 (MAPK9) signaling cascade. Protein profiling by two-dimensional gel electrophoresis identified 25 proteins significantly upregulated in the seedling producing camalexin, including GSTF6 and GGP1 (annotated as defense-related protein, At4g30530) (Su et al, 2011).…”

“…The pathway leading to this model phytoalexin has been almost fully elucidated, and recent focus has been on the biosynthetic origin of the thiazole ring. Several publications have shown that glutathione, and not Cys, is the direct source of the heterocycle and have demonstrated that both glutathione and Cys conjugates of indole-3-acetonitrile (IAN) are intermediates in the pathway (Bö ttcher et al, 2009;Geu-Flores et al, 2011;Su et al, 2011). However, two recent studies published in this journal arrived at different conclusions regarding the conversion of the glutathione conjugate (g-Glu-Cys[IAN]-Gly) to the Cys conjugate (Cys [IAN]), particularly regarding the family of enzymes cleaving off the g-Glu residue.…”