Antisense suppression of 2-cysteine peroxiredoxin in Arabidopsis specifically enhances the activities and expression of enzymes associated with ascorbate metabolism but not glutathione metabolism

Abstract

The aim of this study was to characterize the effect of decreased 2-cysteine peroxiredoxin (2-CP) on the leaf anti-oxidative system in Arabidopsis. At three stages of leaf development, two lines of transgenic Arabidopsis mutants with decreased contents of chloroplast 2-CP were compared with wild type and a control line transformed with an empty vector. Glutathione contents and redox state were similar in all plants, and no changes in transcript levels for enzymes involved in glutathione metabolism were observed. Transcript levels for chloroplastic glutathione peroxidase were much lower than those for 2-CP, and both cytosolic and chloroplastic glutathione peroxidase were not increased in the mutants. In contrast, the foliar ascorbate pool was more oxidized in the mutants, although the difference decreased with plant age. The activities of thylakoid and stromal ascorbate peroxidase and particularly monodehydroascorbate reductase were increased as were transcripts for these enzymes. No change in dehydroascorbate reductase activity was observed, and effects on transcript abundance for glutathione reductase, catalase, and superoxide dismutase were slight or absent. The results demonstrate that 2-CP forms an integral part of the anti-oxidant network of chloroplasts and is functionally interconnected with other defense systems. Suppression of 2-CP leads to increased expression of other anti-oxidative genes possibly mediated by increased oxidation state of the leaf ascorbate pool.

Figure 1

Total contents of glutathione (A), ascorbate (B), and redox poise of ascorbate (C) in 2-CP antisense mutants, wild-type, and control plants of Arabidopsis 60 min after onset of illumination (mean ± sd; n = 7–8 [ascorbate]; n = 6–8 [glutathione]).

Figure 2

Transcript amounts of chloroplast and cytosolic enzymes involved in the anti-oxidant system of leaves in 2-, 4-, and 6-week-old 2-CP mutants, controls, and wild-type plants. Transcript level of cytosolic actin, chloroplast GPx1, cytosolic GPx2, thylakoid and stromal APx, MDHAR, chloroplast γ-glutamylcysteine synthetase, chloroplast GR, and cytosolic glutathione synthetase were detected by RT-PCR with gene-specific primers on agarose gels by ethidium bromide staining. The numbers above the bands show the induction level of thylakoid and stromal APx and MDHAR calculated from the differential integrated density of bands. Cycle numbers and amount of probe used for analysis are given in brackets. Chloroplast localization of the gene products is indicated by oval circles.

Figure 3

Total protein (A) and chlorophyll contents (B) and chlorophyll/protein ratio (C) of 2-, 4-, and 6-week-old 2-CP mutant, control, and wild-type plants (mean ± sd; n = 23).

Figure 4

Total SOD (A), soluble SOD activity (B), and percent fraction of soluble SOD (C) of 2-, 4-, and 6-week-old 2-CP mutant, control, and wild-type plants calculated from inhibition of formazan formation by the xanthine/xanthine oxidase system with 1 relative unit corresponding to 50% inhibition (mean ± sd; n = 6).

Figure 5

Total (A) and soluble APx activities (B) and percent fraction of soluble APx (C) of 2-, 4-, and 6-week-old 2-CP antisense mutant, control, and wild-type plants in aqueous extracts stabilized with 5 mm ascorbate (mean ± sd; n = 6).

Figure 6

Soluble NADPH-MDHAR (A) and NADH-MDHAR activities (B) of 2-, 4-, and 6-week-old 2-CP mutant, control, and wild-type plants in aqueous extracts (mean ± sd; n = 5–6).