A gamma-glutamyl transpeptidase-independent pathway of glutathione catabolism to glutamate via 5-oxoproline in Arabidopsis

Abstract

The degradation pathway of glutathione (GSH) in plants is not well understood. In mammals, GSH is predominantly metabolized through the gamma-glutamyl cycle, where GSH is degraded by the sequential reaction of gamma-glutamyl transpeptidase (GGT), gamma-glutamyl cyclotransferase, and 5-oxoprolinase to yield glutamate (Glu) and dipeptides that are subject to peptidase action. In this study, we examined if GSH is degraded through the same pathway in Arabidopsis (Arabidopsis thaliana) as occurs in mammals. In Arabidopsis, the oxoprolinase knockout mutants (oxp1-1 and oxp1-2) accumulate more 5-oxoproline (5OP) and less Glu than wild-type plants, suggesting substantial metabolite flux though 5OP and that 5OP is a major contributor to Glu steady-state levels. In the ggt1-1/ggt4-1/oxp1-1 triple mutant with no GGT activity in any organs except young siliques, the 5OP concentration in leaves was not different from that in oxp1-1, suggesting that GGTs are not major contributors to 5OP production in Arabidopsis. 5OP formation strongly tracked the level of GSH in Arabidopsis plants, suggesting that GSH is the precursor of 5OP in a GGT-independent reaction. Kinetics analysis suggests that gamma-glutamyl cyclotransferase is the major source of GSH degradation and 5OP formation in Arabidopsis. This discovery led us to propose a new pathway for GSH turnover in plants where GSH is converted to 5OP and then to Glu by the combined action of gamma-glutamyl cyclotransferase and 5-oxoprolinase in the cytoplasm.

Figure 1.

Models for GSH degradation in animals and plants. The solid lines are experimentally confirmed pathways, and the dashed lines are proposed pathways. A, The γ-glutamyl cycle as proposed for animals. GSH is degraded in the extracellular space by GGT, and the constituent amino acids are transported back to the cytoplasm to resynthesize GSH. The GSH hydrolase activity of GGT predominates over the transpeptidase reaction. B, GSH metabolism proposed in Arabidopsis. GSH degradation occurs in the cytoplasm by GGC, with only minor GGT involvement in total GSH turnover. Roles of the GGTs are limited to the extracellular space, such as the degradation of oxidized GSH (GSSG) by GGT1 in the apoplast and the degradation of GSH conjugates by GGT4 in the vacuole.

Figure 2.

Organ-specific expression pattern for OXP1. The accumulation of OXP1 mRNAs in Columbia wild-type plants is shown. Rosette leaves from soil-grown 3-, 4-, 5-, and 7-week-old plants, cauline leaves, stems, and flowers from soil-grown 7-week-old plants, and siliques (2–4 d after flowering [DAF]) and roots of liquid-cultured 12-d-old plants were harvested and total RNA was extracted. RNA was reverse transcribed and subjected to real-time PCR analysis to monitor the amplification of OXP1 cDNAs. The accumulation of OXP1 mRNAs relative to that of ACTIN was determined. Values are normalized with the means of rosette leaves from 3-week-old plants. Means and sd values of three independent biological replications are shown.

Figure 3.

The oxp1-1 and oxp1-2 knockout mutants. A, Structure of the OXP1 gene in Arabidopsis. The white boxes represent untranslated regions, the black boxes represent coding regions, and the black lines represent introns. The T-DNA insertions in oxp1-1 and oxp1-2 are shown. B, OXP1 transcript accumulation. RNA was isolated from wild-type (WT) and knockout mutant plants and subjected RT-PCR.

Figure 4.

5OP and Glu concentrations in wild-type (WT) and oxp1 plants. A, 5OP in Columbia (Col) wild-type and oxp1-1 plants. B, 5OP in Wassilewskija (Ws) wild-type and oxp1-2 plants. C, Glu in Col wild-type and oxp1-1 plants. D, Glu in Ws wild-type and oxp1-2 plants. Roots of liquid-cultured 12-d-old plants, rosette leaves from soil-grown 20-, 30-, and 50-d-old plants, and cauline leaves, stems, and flowers from soil-grown 50-d-old plants and siliques (2–4 d after flowering [DAF]) were harvested from Col wild-type and oxp1-1 plants and assayed for 5OP and Glu. Rosette leaves from soil-grown 20-d-old plants and flowers from soil-grown 45-d-old plants were harvested from Ws wild-type and oxp1-2 plants and assayed for 5OP and Glu. The means ± sd of three (5OP) or four (Glu) biological replicates are shown. Asterisks indicate significant differences between Col wild-type and oxp1-1 plants or between Ws wild-type and oxp1-2 plants. (P < 0.05, one-tailed Student's t test). N.D., Not detected.

Figure 5.

GGC enzyme activity. GGC activities in 10-d-old liquid-cultured ggt1-1/ggt4-1 double knockout plants were determined with GSH (black circles) or γ-EC (white circles) as a substrate. The means ± sd of three experiments are shown.