Arabidopsis peptide methionine sulfoxide reductase2 prevents cellular oxidative damage in long nights

Abstract

Peptide methionine sulfoxide reductase (PMSR) is a ubiquitous enzyme that repairs oxidatively damaged proteins. In Arabidopsis (Arabidopsis thaliana), a null mutation in PMSR2 (pmsr2-1), encoding a cytosolic isoform of the enzyme, exhibited reduced growth in short-day conditions. In wild-type plants, a diurnally regulated peak of total PMSR activity occurred at the end of the 16-h dark period that was absent in pmsr2-1 plants. This PMSR activity peak in the wild-type plant coincided with increased oxidative stress late in the dark period in the mutant. In pmsr2-1, the inability to repair proteins resulted in higher levels of their turnover, which in turn placed an increased burden on cellular metabolism. This caused increased respiration rates, leading to the observed higher levels of oxidative stress. In wild-type plants, the repair of damaged proteins by PMSR2 at the end of the night in a short-day diurnal cycle alleviates this potential burden on metabolism. Although PMSR2 is not absolutely required for viability of plants, the observation of increased damage to proteins in these long nights suggests the timing of expression of PMSR2 is an important adaptation for conservation of their resources.

Figure 1.

The Localization and Characterization of the PMSR2 Knockout. (A) Genomic organization of the PMSR2 gene in Arabidopsis on chromosome 5 (BAC clone K11J9) and location of the dSpm insertion in the pmsr2-1 mutant. The black and white sections are exons and introns, respectively. (B) 3′ RACE analysis of the Arabidopsis PMSR2 gene in the wild type and the pmsr2-1 mutants. PCR products were analyzed by probing with a PMSR2 full-length cDNA. Primers for the ASCORBATE PEROXIDASE1 (APX1) gene were used as a control for cDNA synthesis. (C) Phenotype of the pmsr2-1 mutant compared with the wild-type and pmsr2-1/PMSR2 plants. Plants were grown under short-day conditions for 4 weeks.

Figure 2.

Total PMSR Activity Is Reduced in the pmsr2-1 Mutant. (A) Total PMSR activity during a 24-h period in the wild type (closed squares) and pmsr2-1 mutant (closed triangles). (B) PMSR activity during the second half of a 16-h dark period taken at the end of the 16-h dark period in the wild type (closed squares), pmsr2-1 mutant (closed triangles), and pmsr2-1/PMSR2 (open squares).

Figure 3.

The pmsr2-1 Mutant Shows Signs of Increased Oxidative Stress during the Dark. (A) Foliar H₂O₂ measured during a 24-h period in the wild type (closed squares), pmsr2-1/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles). (B) Lipid peroxidation during the dark-to-light transition in the wild type (closed squares), pmsr2/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles). (C) Redox state ([GSH]/ [GSH] + [GSSG]) of the glutathione pool during a 24-h period in the wild type (closed squares), pmsr2-1/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles).

Figure 4.

The pmsr2-1 Mutant Has Altered Sugar Metabolism. (A) Analysis of respiration (O₂ uptake) during a 24-h period in the wild type (closed squares), pmsr2-1/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles). (B) Foliar glucose levels during a 24-h period in the wild type (closed squares), pmsr2-1/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles). (C) Foliar starch levels during a 24-h period in the wild type (closed squares), pmsr2-1/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles).

Figure 5.

The pmsr2-1 Mutant Has a Reduced Energy Charge Due to Increased ADP Levels. (A) Energy charge ([ATP]/[ATP]+[ADP]+[AMP]) during a 24-h period in the wild type (closed squares), pmsr2-1/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles) (B) ADP plus AMP levels during a 24-h period in the wild type (closed squares), pmsr2-1/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles).

Figure 6.

Photosynthetic Capacity Is Altered in the pmsr2-1 Mutant. (A) Operating efficiency of PSII (Fq′/Fm′) during the dark-to-light transition in the wild type (closed squares) and pmsr2-1 mutant (closed triangles). (B) The effect of prolonged dark periods on photosynthesis was analyzed measuring maximum efficiency of PSII (Fv/Fm) during a 55-h dark period in the wild type (closed squares) and pmsr2-1 mutant (closed triangles). (C) The effect of prolonged dark periods on H₂O₂ levels during a 55-h dark period in the wild type (closed squares) and pmsr2-1 mutant (closed triangles).

Figure 7.

Evidence for Increased Protein Turnover in the pmsr2-1 Mutant. (A) Protein oxidation (carbamylation) after MG132 treatment during the dark in the wild type (closed squares) and pmsr2-1 mutant (closed triangles). (B) Incorporation of ³⁵S-Met into total protein during the dark in the wild type (closed squares), pmsr2-1/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles). (C) Protease activity measurements during a 24-h period in the wild type (closed squares), pmsr2-1/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles). (D) Elimination of ³⁵S-Met from total protein during the dark period in the wild type (closed squares), pmsr2-1/PMSR2 (open squares), and pmsr2-1 mutant (closed triangles). The arrow indicates the transfer of ³⁵S-labeled leaves into nonlabeled solution.