Compensatory expression and substrate inducibility of gamma-glutamyl transferase GGT2 isoform in Arabidopsis thaliana

Abstract

γ-Glutamyl transferases (GGT; EC 2.3.2.2) are glutathione-degrading enzymes that are represented in Arabidopsis thaliana by a small gene family of four members. Two isoforms, GGT1 and GGT2, are apoplastic, sharing broad similarities in their amino acid sequences, but they are differently expressed in the tissues: GGT1 is expressed in roots, leaves, and siliques, while GGT2 was thought to be expressed only in siliques. It is demonstrated here that GGT2 is also expressed in wild-type roots, albeit in very small amounts. GGT2 expression is enhanced in ggt1 knockout mutants, suggesting a compensatory effect to restore GGT activity in the root apoplast. Supplementation with 100 μM glutathione (GSH) resulted in the up-regulation of GGT2 gene expression in wild-type and ggt1 knockout roots, and of GGT1 gene expression in wild-type roots. Glutathione recovery was hampered by the GGT inhibitor serine/borate, suggesting a major role for apoplastic GGTs in this process. These findings can explain the ability of ggt1 knockout mutants to retrieve exogenously added glutathione from the growth medium.

Fig. 1.

Arabidopsis thaliana wild-type (left) and ggt1 knockout mutant plants (right) grown in hydroponics.

Fig. 2.

GGT gene expression patterns in different tissues. Relative abundance of each GGT transcript in leaf (L), root (R), and silique (S). The number of PCR cycles is indicated on the right. All reactions were performed at 30 cycles, but the At4g39650 PCR was extended to 40 cycles to highlight the presence of its transcripts in root tissues. The same quantity of mRNA from different tissues was used to obtain cDNA, and the products were amplified with each GGT gene-specific primer pair. β-Actin was used as the internal standard. GGT1, At4g39640; GGT2, At4g39650; GGT3, At1g69820; GGT4, At4g29210; β-ACT, β-actin.

Fig. 3.

Enzyme histochemical detection of GGT activity in different tissues of A. thaliana. Tissue sections were incubated in a solution containing γ-glutamyl-p-nitroanilide and stained with Fast Garnet GBC. Reddish to purplish-brown staining indicates deposition of diazonium salts at the site of GGT activity: (A) root tip, longitudinal section; (B) vascular bundle, leaf cross-section; (C) silique, longitudinal section; (D) silique, seed magnified; (E) leaf, cross-section; (F) leaf cross-section in 10 mM serine/borate to inhibit GGT activity. Scale bars: A–D=100 μm; E, F=200 μm.

Fig. 4.

Enzyme histochemical detection of GGT activity in wild-type and ggt1 knockout roots and leaves. (A) Root apex transversal section, wild type; (B) wounded leaf, wild type; (C) root apex transversal section, ggt1; (D) wounded leaf, ggt1. Inset pictures in B and D are enlargements of a wounded area. Scale bars=100 μm.

Fig. 5.

Timing of glutathione depletion from the incubation medium. Wild-type, with or without 10 mM serine/borate inhibitor, and ggt1 roots were incubated in 100 μM glutathione; the solution was sampled at different times and used for thiol quantification by HPLC analysis. (A) Glutathione content; (B) cysteinyl-glycine content; (C) cysteine content in the external solution. Data are expressed as nmol ml⁻¹ solution. Filled squares, wild-type roots; filled triangles, ggt1 roots; open squares, wild-type roots with 10 mM serine/borate inhibitor (n=10).

Fig. 6.

In vivo measurement of GGT activity in wild-type and ggt1 knockout A. thaliana roots. GGT activity is measured in vivo as the release of p-nitroaniline by Arabidopsis roots placed in a spectrophotometric cuvette containing 4.6 mM γ-glutamyl-p-nitroanilide; the solution was cycled through home-made piping from the cuvette to the spectrophotometer cell using a peristaltic pump (see Materials and methods for details). Plants incubated with 100 μM glutathione were harvested after 3 h and 6 h of incubation; single plant roots were transferred to a 5 ml tube containing 4.4 ml of bioassay solution. The change in absorbance at 407 nm induced by releasing p-nitroaniline into the assay medium was recorded for 10 min. Inset graph: spectrophotometric recording of the assay solution in contact with wild-type or ggt1 roots. Values are means ±SE of at least five replicates.

Fig. 7.

Gene expression analysis by semi-quantitative PCR of the four ggt genes in Arabidopsis thaliana wild-type and ggt1 knockout mutants, after 100 μM glutathione supplementation. Roots and leaves were harvested before treatment and then 1, 3, and 6 h after adding 100 μM glutathione to the growth medium. Gene expression analysis of the four ggt genes: PCR amplification products and relative expression levels in roots (A, C) and leaves (B, D). GGT1, At4g39640; GGT2, At4g39650; GGT3, At1g69820, GGT4, At4g29210; β-ACT, At3g12110. Data are presented as means ±SD from at least five independent measurements; error bars represent the SD. Different letters indicate significant differences between the wild type and ggt1 knockout lines (P <0.05); significance was evaluated with the Tukey test. See Materials and methods for further details.