A chloroplast cyclophilin functions in the assembly and maintenance of photosystem II in Arabidopsis thaliana

Abstract

Photosynthetic light reactions rely on the proper function of large protein complexes (including photosystems I and II) that reside in the thylakoid membrane. Although their composition, structure, and function are known, the repertoire of assembly and maintenance factors is still being determined. Here we show that an immunophilin of the cyclophilin type, CYP38, plays a critical role in the assembly and maintenance of photosystem II (PSII) supercomplexes (SCs) in Arabidopsis. Mutant plants with the CYP38 gene interrupted by T-DNA insertion showed stunted growth and were hypersensitive to high light. Leaf chlorophyll fluorescence analysis and thylakoid membrane composition indicated that cyp38 mutant plants had defects in PSII SCs. Sucrose supplementation enabled the rescue of the mutant phenotype under low-light conditions, but failed to mitigate hypersensitivity to high-light stress. Protein radiolabeling assays showed that, although individual thylakoid proteins were synthesized equally in mutant and wild type, the assembly of the PSII SC was impaired in the mutant. In addition, the D1 and D2 components of the mutant PSII had a short half-life under high-light stress. The results provide evidence that CYP38 is necessary for the assembly and stabilization of PSII.

Fig. 1.

Genetic characterization and phenotype of cyp38 mutant plants. (A) Localization of T-DNA insertion sites in CYP38 genomic DNA of three cyp38 alleles: cyp38-1, cyp38-2, and cyp38-3. The CYP38 genomic DNA includes seven exons (black boxes) and six introns (bold lines between black boxes). ATG, initiation codon; TAA, stop codon. (B) CYP38 mRNA levels in wild type (WT), cyp38-1, cyp38-2, and cyp38-3 as shown by RT-PCR. (C) Phenotype of wild type, cyp38-1, cyp38-2, cyp38-3, and cyp38-2 transformed with CYP38 genomic DNA (cyp38-2C) grown for 7 weeks under low light (25–35 μmol·m⁻²·s⁻¹) with a long-day cycle (16–8 h). (D) The plants shown in C were exposed to illumination (300 μmol·m⁻²·s⁻¹) for 2 days after 7 weeks of growth in low light (25–35 μmol·m⁻²·s⁻¹).

Fig. 2.

Chlorophyll fluorescence analyses of wild-type (WT) and cyp38-2 plants. Plants were grown under 25–35 μmol·m⁻²·s⁻¹ for 7 weeks. Data represent means ± SE (n = 6). ΦPSII, efficiency of PSII photochemistry; 1-qL, parameter estimating the fraction of PSII in close states or excitation pressure of PSII based on a lake mode (21).

Fig. 3.

BN/PAGE and subsequent analysis of thylakoid membrane complexes in 2D SDS/PAGE. (A) Thylakoid membranes (5 μg of chlorophyll) were solubilized with 1% dodecyl-β-maltoside and separated by 5–13.5%. Native gel slices were horizontally laid on the top of 2D SDS/PAGE. PSI-M, PSI monomer; PSII-D, PSII dimer; LHCII, PSII light-harvesting complex; LHCII-T, PSII light-harvesting complex trimer; LHCII monomer, PSII light-harvesting complex monomer. (B) Thylakoid membrane complexes separated by BN/PAGE in A were further subjected to 12.5% SDS/PAGE and stained with silver. Protein identification was based on ref. .

Fig. 4.

Immunodetection of thylakoid membrane proteins. Protein samples (1 μg of chlorophyll) were resolved by 12.5% SDS/PAGE, transferred to a nitrocellulose membrane, and probed with antibodies against the indicated thylakoid membrane proteins. WTT, total protein sample from wild-type plants; 38-2T, total protein sample from cyp38-2; WTM, thylakoid membrane sample from wild-type plants; 38-2M, thylakoid membrane sample from cyp38-2.

Fig. 5.

Effect of sucrose treatment on wild-type and cyp38-2 plants. (A) Wild-type and cyp38-2 plants were grown vertically on half-strength MS medium without sucrose for 4 weeks under low-light conditions (25–35 μmol·m⁻²·s⁻¹). (B) Wild-type and cyp38-2 plants were grown vertically on half-strength MS medium supplemented with 2% sucrose under low-light conditions (25–35 μmol·m⁻²·s⁻¹) for 4 weeks. (C) Plants grown as described in B were exposed to high light (300 μmol·m⁻²·s⁻¹) for 5 days. (D) BN/PAGE of thylakoid membrane complexes from wild-type (WT) and cyp38-2 plants after 4 weeks of growth on 2% sucrose half-width MS medium. PSI-M, PSI monomer; PSII-D, PSII dimer. (E) Immunoanalysis of thylakoid proteins (1 μg of chlorophyll) from wild-type total protein (WTT), cyp38-2 total protein (38-2T), wild-type membrane protein (WTM), and cyp38-2 membrane protein (38-2M). Plants were grown on 2% sucrose medium for 4 weeks.

Fig. 6.

In vivo synthesis of plastid-encoded membrane proteins. (A) Primary leaves from wild-type (WT) and cyp38-2 (38-2) sucrose-grown plants were fed [³⁵S]Met in the presence of cycloheximide. After a 20-min incubation, thylakoid membrane proteins (5 μg of chlorophyll) were isolated and separated by 12.5% SDS/PAGE. Proteins were then transferred to a nitrocellulose membrane and visualized autoradiographically. (B) After 20 min of labeling leaves with [³⁵S]Met (as in A), thylakoid membrane complexes (5 μg of chlorophyll) were separated by BN/PAGE. (Left) BN/PAGE pattern. (Right) Autoradiography image of the BN/PAGE. PSI-M, PSI monomer; PSII-D, PSII dimer; RC47, PSII reaction center complex without CP43 subunit (41); RC, PSII reaction center complex without CP47 and CP43 subunits (41).

Fig. 7.

Half-life of thylakoid membrane proteins in wild-type and cyp38 plants. (A) Arabidopsis leaves from sucrose-grown wild-type (WT) and cyp38-2 plants were incubated with lincomycin and cycloheximide for 30 min and illuminated under 25–35 μmol·m⁻²·s⁻¹ for 0, 2, and 4 h. Thylakoid membrane proteins (1 μg of chlorophyll) were resolved by SDS/PAGE, and the gel was immunoblotted to assess protein half-life. (B) Conditions as in A, except that leaves were illuminated under 300 μmol·m⁻²·s⁻¹ for 0 and 2 h.