2-cysteine peroxiredoxins and thylakoid ascorbate peroxidase create a water-water cycle that is essential to protect the photosynthetic apparatus under high light stress conditions

Abstract

Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H2O2)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H2O2, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H2O2- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX.

Figure 1.

HL sensitivity and photosynthetic impairment of 2cpa 2cpb and 2cpa 2cpb tapx mutant plants. Wild-type and mutant plants were grown under ML (160 µmol m⁻² s⁻¹; 8 h d⁻¹) and treated with HL (900 µmol m⁻² s⁻¹; 8 h d⁻¹). A, 2cpa 2cpb and 2cpa 2cpb tapx plants displayed a smaller habitus with pale green leaves compared with wild-type and tapx plants. Leaves of wild-type and tapx plants were tolerant to HL, whereas 2cpa 2cpb and 2cpa 2cpb tapx plants became partially photobleached after 1 d (2cpa 2cpb tapx) or 2 d (2cpa 2cpb) of HL treatment. B, Optimal quantum yield (F_v/F_m) of wild-type (black bars), tapx (dark-gray bars), 2cpa 2cpb (white bars), and 2cpa 2cpb tapx (light-gray bars) plants grown under ML or HL conditions. Shown are means ± sd (n = 6). C, Carbon dioxide fixation rate in wild-type (black bars) and 2cpa 2cpb mutant (white bars) plants grown under ML or HL conditions. The rate of carbon dioxide uptake was determined by gas exchange measurements using an infrared gas analyzer. Shown are means ± sd (n = 3). Significant differences between mean values are indicated by asterisks using Student’s t test. WT, Wild type; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

Light-induced superoxide anion radical accumulation in wild-type and mutant plants. O₂·⁻ accumulation in dark-adapted leaves of wild-type (black bars), tapx (dark-gray bars), 2cpa 2cpb (white bars), and 2cpa 2cpb tapx (light-gray bars) mutant plants was visualized by NBT staining in the dark or after 1 h of ML (160 µmol m⁻² s⁻¹) or HL (900 µmol m⁻² s⁻¹) treatment. A, Representative images of stained leaves after chlorophyll extraction. B, Relative NBT staining (integration of blue color density per leaf area) was determined using the ImageJ image processing software. Shown are means ± se (n = 10). Significant differences between mean values are indicated by asterisks using Student’s t test. D, Dark; WT, wild type; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

Sensitivity of wild-type (WT) and mutant plants to HL stress. A, Autoluminescence of wild-type, tapx, 2cpa 2cpb, and 2cpa 2cpb tapx mutant plants after 8 h of ML (160 µmol m⁻² s⁻¹) or 1 or 2 d of HL (900 µmol m⁻² s⁻¹; 8 h d⁻¹) treatment. Images were taken with a CCD camera after 40 min of dark adaptation. Representative pictures from nine measurements were converted to false-color images indicating the relative autoluminescence intensity. B, Effect of light treatment on protein carbonylation in wild-type (1), tapx (2), 2cpa 2cpb (3), and 2cpa 2cpb tapx (4) plants. Proteins (15 µg of total protein) were derivatized with 2,4-dinitrophenylhydrazine and subjected to SDS-PAGE. Immunoblotting was performed using an antidinitrophenylhydrazone antibody. Representative blots from three experiments are shown.

Figure 4.

Lipid peroxide reduction by 2-Cys PRX. A, The initial rate of peroxide reduction by 2CPA was determined in vitro using 100 µm DTT as reductant and the following substrates: H₂O₂ (25 µm), t-BOOH (25 µm), 13-hydroperoxy octadecatrienoic acid (HOO-18:3; 25 µm), and peroxidized MGDGs (HOO-MGDG; 2.5 µm). Purified recombinant His-tagged 2CPA (5 µm protein) was used in the assays. B, Wild-type (black bars) and 2cpa 2cpb (white bars) mutant plant leaves were exposed to ML (160 µmol m⁻² s⁻¹; 8 h d⁻¹) or HL (900 µmol m⁻² s⁻¹; 8 h d⁻¹). The ratios of MGDG peroxides (MGDG-OOH) and hydroxides (MGDG-OH) relative to their nonoxidized MGDG precursors were determined for the indicated MGDG species. C, The ratio of MGDG peroxides relative to total oxidized MGDG is shown for the indicated MGDG species. All values shown are means ± sd (n = 3).

Figure 5.

Light-induced H₂O₂ levels in different genotypes. At the end of the night, leaves of wild-type (WT), tapx, 2cpa 2cpb, and 2cpa 2cpb tapx plants were kept in the dark (black bars) for 3 h or treated with ML (gray bars; 160 µmol m⁻² s⁻¹) or HL (white bars; 900 µmol m⁻² s⁻¹) for the times indicated. Whole-leaf H₂O₂ levels were determined using the homovanillic acid fluorescence assay. All values shown are means ± sd (n = 10). Significant differences between mean values are indicated by asterisks using Student’s t test. FW, Fresh weight; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

HL-induced expression of redox-regulated genes in wild-type and mutant plants. Expression of HSFA2, HSP101, OXI1, ZAT12, At1g49150, and BAP1 in wild-type (black bars), tapx (dark-gray bars), 2cpa 2cpb (white bars), and 2cpa 2cpb tapx (light-gray bars) plants after ML (160 µmol m⁻² s⁻¹) or HL (900 µmol m⁻² s⁻¹) treatment for the times indicated. Expression was normalized to the actin gene ACTIN2/ACTIN8. All values shown are means ± sd (n = 3). Significant differences between mean values of wild-type and mutant plants are indicated by asterisks using Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7.

Light-induced anthocyanin content and anthocyanin-related gene expression in wild-type and 2cpa 2cpb plants. A, Anthocyanin accumulation in wild-type (black bars) and 2cpa 2cpb mutant (white bars) plants after the dark period (16 h), ML (8 h d⁻¹; 160 µmol m⁻² s⁻¹), or HL (8 h d⁻¹; 900 µmol m⁻² s⁻¹). B, Quantitative reverse transcription PCR analysis of regulatory and structural genes contributing to anthocyanin biosynthesis in wild-type (black bars) and 2cpa 2cpb mutant (white bars) plants after darkness, ML, and HL treatment. Expression of the PAP1, PAP2, CHS, and FSH genes was normalized to the ACTIN2/ACTIN8 genes. Expression in wild-type leaves after dark was arbitrarily set to one, and all other expression values were expressed relative to it. All values shown are means ± sd (n = 3). Significant differences between mean values are indicated by asterisks using Student’s t test. D, Dark; FW, fresh weight; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 8.

Model of the photoprotective function of 2-Cys PRX and tAPX. The WWC and other alternative electron flows (AEFs) are proposed to prevent overreduction of the linear electron transport chain (LET) and protect the photosystems from oxidative damage. Under HL conditions, 2CPs are essential for maintaining a WWC and more important than plastid APX involved in the classical WWC for protection of the photosystems. 2-Cys PRX and tAPX synergistically inhibit activation of redox-responsive genes and prevent overaccumulation of H₂O₂ as well as photooxidative damage under HL conditions. FNR, Fd NADP oxidoreductase; SOD, superoxide dismutase.