C-terminal processing of reaction center protein D1 is essential for the function and assembly of photosystem II in Arabidopsis

Abstract

Photosystem II (PSII) reaction center protein D1 is synthesized as a precursor (pD1) with a short C-terminal extension. The pD1 is processed to mature D1 by carboxyl-terminal peptidase A to remove the C-terminal extension and form active protein. Here we report functional characterization of the Arabidopsis gene encoding D1 C-terminal processing enzyme (AtCtpA) in the chloroplast thylakoid lumen. Recombinant AtCtpA converted pD1 to mature D1 and a mutant lacking AtCtpA retained all D1 in precursor form, confirming that AtCtpA is solely responsible for processing. As with cyanobacterial ctpa, a knockout Arabidopsis atctpa mutant was lethal under normal growth conditions but was viable with sucrose under low-light conditions. Viable plants, however, showed deficiencies in PSII and thylakoid stacking. Surprisingly, unlike its cyanobacterial counterpart, the Arabidopsis mutant retained both monomer and dimer forms of the PSII complexes that, although nonfunctional, contained both the core and extrinsic subunits. This mutant was also essentially devoid of PSII supercomplexes, providing an unexpected link between D1 maturation and supercomplex assembly. A knock-down mutant expressing about 2% wild-type level of AtCtpA showed normal growth under low light but was stunted and accumulated pD1 under high light, indicative of delayed C-terminal processing. Although demonstrating the functional significance of C-terminal D1 processing in PSII biogenesis, our study reveals an unsuspected link between D1 maturation and PSII supercomplex assembly in land plants, opening an avenue for exploring the mechanism for the association of light-harvesting complexes with the PSII core complexes.

Keywords: photoinhibition; photosynthesis.

Fig. 1.

AtCtpA (At4g17740) localization. (A) AtCtpA is present in chloroplasts. Protein samples (corresponding to 4 µg chlorophyll or fractions of this amount—1/2, 1/4, 1/10, or 1/20) from total leaf extract or chloroplasts were separated by 12% SDS/PAGE and probed with antibodies against AtCtpA, Rubisco (a marker for chloroplast proteins), or ISE1 (a marker for mitochondria proteins). (B) AtCtpA is located in the thylakoid lumen. L, thylakoid lumen fraction; M, thylakoid membrane fraction; S, stromal fraction. Immunoblot analysis was conducted using antibodies against ClpC (a stromal protein), D2, and PC. (C) AtCtpA is not associated with high molecular PSII complexes. BN-PAGE and 2D SDS/PAGE immunoblot with an antibody against AtCtpA. Cytf, cytochrome f; B6, cytochrome b6. Auto-peroxidase activity of heme group in Cytf and Cytb6 produced signals in this assay.

Fig. 2.

Characterization of atctpa mutants. (A) Location of T-DNA insertion sites in the AtCtpA gene of atctpa-1 and atctpa-2 mutants. Solid bars indicate exons that are separated by introns (black lines). The 5′- and 3′-UTRs are indicated by white boxes. The horizontal arrows denote primers used in B. (B) RT-PCR analysis of AtCtpA transcripts in the atctpa mutants. (a) RT-PCR products using forward and reverse primers (P1F and P1R) indicated in A. (b) RT-PCR analysis performed with primers P2F and P2R shown in A. (C) Immunoblot analysis of AtCtpA protein in WT and atctpa mutants. Protein samples equivalent to 1 µg chlorophyll as 1/1 or fractions of 1/2, 1/4, 1/8, or a 5× multiple of this amount were analyzed from WT or mutants. (A–C) atctpa-1 grown under 15 µE in 1/2 MS medium containing 2% sucrose for 5 wk; atctpa-2 and WT grown under 350 µE in soil for 4 wk. (D) Phenotypes of 5-wk-old WT atctpa-1 and 4-wk-old WT atctpa-2 plants. atctpa-1 and its WT control were grown in 1/2 MS medium containing 2% sucrose under 15 or 80 µE light conditions; atctpa-2 and its WT counterpart were grown in soil under 100- or 350-µE light intensity. (E) Electron micrographs of chloroplasts were taken as described elsewhere (34) from 5-wk-old WT and atctpa-1 grown in 1/2 MS medium containing 2% sucrose under 15 µE.

Fig. 3.

AtCtpA is responsible for D1 C-terminal processing. (A) Western blot analysis of D1 protein in WT, atctpa-1, and atctpa-2 mutant plants (Upper). Photographs of WT, atctpa-1, and atctpa-2 plants (Lower). Thylakoid protein samples equivalent to 2 µg of chlorophyll were separated by 10% SDS/PAGE containing 8M urea, blotted and probed with D1 antibody. (B) D1 C-terminal processing activity assay in vitro. WT thylakoid sample was used as a control for mD1 in the left lane. Processing of pD1 into D1 in the presence of 5 µg of recombinant GST-AtCtpA is shown at different times. GST-FKBP13 (FK506-binding protein 13) protein served as a negative control. (C) Western blot analysis of other PSII subunits in WT, atctpa-1, and atctpa-2 plants. Immunoblot analysis was performed as in A with antibodies against the indicated thylakoid membrane proteins. Plant growth conditions: when WT was compared with atctpa-2, both plants were grown in the soil under 350 µE for 4 wk. When WT was compared with atctpa-1, plants were grown in 1/2 MS medium with 2% sucrose under 15 µE for 5 wk.

Fig. 4.

PSII activity in WT and mutant plants. (A) Chlorophyll fluorescence parameters of 5-wk-old WT and atctpa-1 plants grown in 1/2 MS plates with 2% sucrose (15 µE). (B) Chlorophyll fluorescence parameters of 4-wk-old WT and atctpa-2 plants grown in soil (350 µE). Data are presented as means ± SD (n = 4). PFD, photon flux density. WT and mutant data are significantly different (P < 0.05). (C) Oxygen evolution rate (OER) of WT, atctpa-1, and atctpa-2 plants. atctpa-1 was grown under 15 µE in 1/2 MS medium containing 2% sucrose for 5 wk; atctpa-2 and WT were grown under 350 µE in soil for 4 wk. Data shown as means ± SD (n = 4). WT and mutant data are significantly different (P < 0.05).

Fig. 5.

Protein complexes in the thylakoid membranes of WT and mutant plants. (A) BN gel analysis of thylakoid membrane protein complexes (7.5 µg chlorophyll) of WT and atctpa-1 grown under 15 µE in 1/2 MS medium with 2% sucrose for 5 wk and WT, atctpa-2 plants grown under 350 µE in soil for 4 wk. Protein complexes were identified as described previously (31). (B) Thylakoid proteins of WT and atctpa-1 separated by BN gel in A were further subjected to SDS/PAGE and immunoblotted with antibodies as indicated. (C) Thylakoid proteins of WT and atctpa-2 separated by BN gel in A were further subjected to SDS/PAGE and immunoblotted with antibodies as indicated. Plant growth conditions were the same as described in Fig. 3. LHCII-M, PSII LHC monomers; LHCII-T: PSII LHC trimers; NDH, NADPH dehydrogenase complexes; PSI-M, PSI monomers; PSI SC, PSI supercomplexes; PSII-M, PSII monomers.

Fig. 6.

PSII activity and D1 processing during photoinhibition and repair. (A) PSII activity (F_V/F_M) was recorded using detached leaves from 4-wk-old WT and atctpa-2 plants grown under 350 µE and monitored during exposure to high light (1,800 µE) for 4 h in the presence or absence of 1.5 mM lincomycin. Data for WT and atctpa-2 are not significantly different (P > 0.1) in the presence of lincomycin but are significantly different (P < 0.05) without lincomycin. (B) Photoinhibition recovery of WT and atctpa-2. Plants with 50% photoinhibition recovered under low light (15 µE) for up to 20 h. Data represent means ± SD (n = 3). WT and atctpa-2 data are not significantly different (P > 0.1). (C) D1 status during photoinhibition treatment. Samples were taken from plants identical to those used in Fig. 6A. Total proteins (equivalent to 2 µg chlorophyll) were subjected to immunoblotting with antibodies against D1 and D2, respectively. (D) D1 status during photoinhibition recovery. Samples were taken from plants as used in Fig. 6B and subjected to the same assay as that in C.