Protective function of chloroplast 2-cysteine peroxiredoxin in photosynthesis. Evidence from transgenic Arabidopsis

Abstract

2-Cysteine peroxiredoxins (2-CPs) constitute a ubiquitous group of peroxidases that reduce cell-toxic alkyl hydroperoxides to their corresponding alcohols. Recently, we cloned 2-CP cDNAs from plants and characterized them as chloroplast proteins. To elucidate the physiological function of the 2-CP in plant metabolism, we generated antisense mutants in Arabidopsis. In the mutant lines a 2-CP deficiency developed during early leaf and plant development and eventually the protein accumulated to wild-type levels. In young mutants with reduced amounts of 2-CP, photosynthesis was impaired and the levels of D1 protein, the light-harvesting protein complex associated with photosystem II, chloroplast ATP synthase, and ribulose-1,5-bisphosphate carboxylase/oxygenase were decreased. Photoinhibition was particularly pronounced after the application of the protein synthesis inhibitor, lincomycin. We concluded that the photosynthetic machinery needs high levels of 2-CP during leaf development to protect it from oxidative damage and that the damage is reduced by the accumulation of 2-CP protein, by the de novo synthesis and replacement of damaged proteins, and by the induction of other antioxidant defenses in 2-CP mutants.

Figure 1

Detection of EcoRI fragments containing the barley 2-CP cDNA in genomic DNA extracted from mutants bas-23 and bas-24 and control (702-3) plants. Ten micrograms of genomic DNA was digested with EcoRI and separated on a 0.9% (w/v) agarose gel. The nucleic acids were blotted on a nylon membrane and hybridized with the 273-bp barley 2-CP cDNA fragment under high-stringency conditions.

Figure 2

2-CP expression in transgenic and control plants. A, Determination of 2-CP and psbA mRNA levels on northern blots with nucleic acids (20 μg per lane) from rosettes of 6-week-old mutants and control (702-3) plants grown on soil. The 2-CP mRNA was detected with radiolabeled 2-CP cDNA. Duplicate filters were hybridized with pea psbA. B, Detection of 2-CP protein in western blots. Protein extracts of 6-week-old mutants, wild-type (wt), and control (702-3) plants corresponding to 20 mg fresh weight were separated by PAGE and blotted on nitrocellulose membranes. The 2-CP was detected using an antibody against the mature form of barley 2-CP. Depending on the redox state, the protein was detected as monomer or dimer, respectively.

Figure 3

Western-blot analysis of 2-CP in 14-week-old plants (late rosette stage). 2-CP protein was detected in leaves of different ages (A) using an antibody against heterologously expressed 2-CP (B). Quantification of the protein amounts (C) was performed by evaluating band density.

Figure 4

Quantum yield of PSII electron transport of the mutant bas-23 (○) and control 702-3 (•). Chlorophyll a fluorescence was measured in 4-, 9-, 14-, and 42-d-old, dark-adapted plants during a 30-min illumination period (white bars) followed by a subsequent 30-min dark period (black bars).

Figure 5

Quantum yield of PSII electron transport of the mutant bas-23 (○) and control line 702-3 (•) after feeding lincomycin for 4 and 8 h. Chlorophyll a fluorescence was measured in dark-adapted 8-d-old plants grown on soil. A 30-min illumination period (white bars) was followed by a 30-min dark period (black bars).

Figure 6

Western-blot analysis of D1, Rubisco, LHCII, CF1, and SUE protein amounts in extracts of 6-week-old mutants and control plants grown on soil. Protein extracts corresponding to 20 mg (fresh weight) were separated by PAGE, blotted on nitrocellulose membranes, and analyzed with the respective antibody.

Figure 7

Total peroxidase activity in 2- and 6-week-old Arabidopsis mutant seedlings grown on soil. fw, Fresh weight.