LPA19, a Psb27 homolog in Arabidopsis thaliana, facilitates D1 protein precursor processing during PSII biogenesis

Abstract

The biogenesis and assembly of photosystem II (PSII) are mainly regulated by the nuclear-encoded factors. To further identify the novel components involved in PSII biogenesis, we isolated and characterized a high chlorophyll fluorescence low psii accumulation19 (lpa19) mutant, which is defective in PSII biogenesis. LPA19 encodes a Psb27 homolog (At1g05385). Interestingly, another Psb27 homolog (At1g03600) in Arabidopsis was revealed to be required for the efficient repair of photodamaged PSII. These results suggest that the Psb27 homologs play distinct functions in PSII biogenesis and repair in Arabidopsis. Chloroplast protein labeling assays showed that the C-terminal processing of D1 in the lpa19 mutant was impaired. Protein overlay assays provided evidence that LPA19 interacts with D1, and coimmunoprecipitation analysis demonstrated that LPA19 interacts with mature D1 (mD1) and precursor D1 (pD1). Moreover, LPA19 protein was shown to specifically interact with the soluble C terminus present in the precursor and mature D1 through yeast two-hybrid analyses. Thus, these studies suggest that LPA19 is involved in facilitating the D1 precursor protein processing in Arabidopsis.

FIGURE 1.

Phenotypes and chlorophyll fluorescence analysis. A, 5-week-old plants grown in the growth chamber under medium light (ML) conditions of 120 μmol m⁻² s⁻¹. B, 2-week-old plants grown in a growth chamber under a photon flux density of 120 μmol m⁻² s⁻¹ and then transferred to low light (LL) conditions of 10 μmol m⁻² s⁻¹ for another 3 weeks. C, 2-week-old plants grown in a growth chamber under a photon flux density of 120 μmol m⁻² s⁻¹ and then transferred to a greenhouse with high light (HL) conditions of maximum photon flux density at noon of about 1,000 μmol m⁻² s⁻¹. D, chlorophyll fluorescence induction. Fm, maximum fluorescence yield when all PSII centers are closed, Fo, minimum fluorescence yield when all PSII centers are open. The ratios of variable to maximum fluorescence were calculated by Fv/Fm = (Fm-Fo)/Fm. SL, saturating light.

FIGURE 2.

Identification of the lpa19 mutation. A, schematic diagram of the LPA19 gene. Exons (black bars) and introns (black lines) are indicated. The positions of the mutation corresponding to lpa19-1 and lpa19-2 are shown. ATG indicates the start codon, and TGA indicates the stop codon. LB, left border; RB, right border. The diagram is not drawn to scale. The numbers indicate the positions of the nucleic acid relative to 5′-untranslated region. B, RT-PCR analysis of lpa19. The analysis was performed with specific primers for At1g05385 and actin. C, immunoblot analysis of LPA19. The thylakoids (2 μg of chlorophyll) and total leaf proteins (20 μg of total proteins), isolated from wild-type, lpa19-1, lpa19-2, and complemented plants, were separated by SDS-urea-PAGE and immunodetected with antibodies raised against LPA19.

FIGURE 3.

Immunolocalization analysis. Intact (A) and sonicated (B) thylakoid membranes from mature wild-type leaves were incubated with trypsin for 0, 5, and 10 min at 25 °C. C, sonicated thylakoid membranes were incubated with 1 m NaCl, 1 m CaCl₂, and 6 m urea for 30 min at 4 °C. After these treatments, the membrane fractions were separated by SDS-PAGE and immunodetected with anti-LPA19, anti-PsbO, and anti-CP47. Sonicated membrane preparations that had not been subjected to any of these treatments (CK) were used as controls. The experiments were repeated three times independently and similar results were obtained. Results from a representative experiment are shown.

FIGURE 4.

Immunodetection of chloroplast proteins. Total proteins (20 μg) from 5-week-old wild-type and lpa19-1 leaves were separated by SDS-urea-PAGE, and the blots were probed with specific anti-D1, anti-D2, anti-CP43, anti-CP47, anti-PsaA/B, anti-CF1β, anti-LHCII, and anti-Cyt f antibodies.

FIGURE 5.

In vivo chloroplast protein labeling assays. After a 20-min pulse in wild-type and mutant young seedlings in the presence of cycloheximide, the samples of lpa19-1 (A) and wild-type (B) were chased in 10 mm cold methionine. After labeling, the thylakoid membranes were isolated, and the proteins were separated by SDS-urea-PAGE and visualized by autoradiography.

FIGURE 6.

Protein overlay assay of LPA19 interactions with PSII proteins. Wild-type thylakoid membrane proteins (10 μg of chlorophyll) solubilized in 1% DM were separated by BN/SDS-PAGE two-dimensional electrophoresis (A) and then were transferred to nitrocellulose membranes. The positions of protein complexes representing PSII supercomplexes (band I), monomeric PSI and dimeric PSII (band II), monomeric PSII (band III), CP43 minus PSII (band IV), trimeric LHCII/PSII reaction center (band V), and monomeric LHCII (band VI) were assigned as previously described (19). B, nitrocellulose membrane was incubated with recombinant His-tagged LPA19 protein that subsequently reacted with His antibodies. C, nitrocellulose membrane was incubated with D2 and D1 antibodies, as indicated to the left. D, superposition of B and C. The signal of B was shown by thick line loops and denoted by arrowhead; the signal of C, which was immunodetection with anti-D2 and anti-D1 antibodies was shown by gray black line loops and labeled by D1 and D2. E, membrane was incubated with His antibody.

FIGURE 7.

Yeast two-hybrid interactions between LPA19 and different D1 peptides. Growth of cells cotransformed with prey vector AD-X (where X represents D1 soluble precursor C terminus, mature C terminus, D1 AB loop, CD loop, and all four constructs fused with the activation domain of Gal4) and bait vector LPA19-BD (mature full-length LPA19 fused with the binding domain of Gal4) could grow on SD-His-Leu-Trp-Ade/X-α-Gal/AbA plates. Cotransformed yeast cells were grown to the logarithmic phase, and 5-ml portions of 1:10 series dilutions were spotted on SD-His-Leu-Trp-Ade/X-α-Gal/AbA plates and incubated at 30 °C for 3 days. Yeast cells cotransformed with pGADT7-T (which encodes the Gal4 AD fused with SV40 large T-antigen) and pGBKT7–53 (which encodes the Gal4 DNA-BD fused with murine p53) were used as positive controls; those constructs cotransformed with pGADT7-T and pGBKT7-Lam (which encodes the Gal4 BD fused with lamin) were used as negative controls.

FIGURE 8.

Coimmunoprecipitation assays of LPA19 interacts with pD1 and mD1. After pulse labeling for 10 min, the thylakoid membranes were isolated and solubilized with DM. The solubilized membranes were incubated with preimmune serum and anti-LPA19 for 1 h. The immunoprecipitates were separated by SDS-urea-PAGE and visualized by autoradiography. Radiolabeled pD1 and mD1 are indicated.