PsbI Affects the Stability, Function, and Phosphorylation Patterns of Photosystem II Assemblies in Tobacco

Abstract

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q A binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redoxregulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in ⌬psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.

PsbN Is Required for Assembly of the Photosystem II Reaction Center in Nicotiana tabacum

“…The accumulation of LHCA2/LHCA3 subcomplexes not associated with PSI in ΔpsbN mutants most likely reflects a secondary effect as revealed by comparison with ΔpsbI, which is primarily affected in PSII dimer stability, but shows a much milder reduction in PSII accumulation and activity compared with ΔpsbN mutants (Schwenkert et al, 2006) (Supplemental Figure 3).…”

“…Thus, Blue-Native (BN) PAGE analysis was performed to investigate the effect of the psbN mutations on the assembly of thylakoid membrane complexes (Schwenkert et al, 2006). In the first dimension, PSII supercomplexes were clearly detectable in the wild type but not (or only in minute amounts) in ΔpsbN-F and ΔpsbN-R, indicating that assembly of higher order PSII complexes is severely abolished in psbN mutants ( Figure 6A).…”

“…To provide evidence that this is caused by a secondary effect, we analyzed a previously described PSII mutant in tobacco, which lacks the PSII low molecular weight subunit PsbI. The ΔpsbI line is much less affected in PSII function than ΔpsbN mutants since they exhibit an Fv/Fm ratio of ;0.74 and their PSII proteins are reduced by only 50% (Schwenkert et al, 2006). BN-PAGE analysis of ΔpsbI plants showed a similar accumulation of LHCa2 and LHCa3 in smaller subcomplexes, affirming that this effect is independent of the absence of PsbN and appears when PSII activity is diminished in tobacco (Supplemental Figure 3).…”

“…Growth under preferential state I condition was achieved by covering the light source (HE 21W/60; Osram) with a dark-red filter (027 Medium Red; Lee Filters). Before transferring the plants into glass jars, seeds were surface sterilized, incubated at 4°C in the dark for 2 d, and grown for 8 d at 25°C under 10 µmol photons m 22 s 21 continuous light in a Petri dish with Murashige and Skoog medium (Schwenkert et al, 2006).…”

“…Thylakoid membrane proteins were isolated, solubilized, and subjected to SDS-PAGE (15% acrylamide) as described (Schwenkert et al, 2006). PsbN antisera were generated against the synthetic peptide QPSQ-QLRDPFEEHGD of the hydrophilic C terminus (Agrisera).…”