Impact of PsbTc on Forward and Back Electron Flow, Assembly, and Phosphorylation Patterns of Photosystem II in Tobacco  

Abstract

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn 4 Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in DpsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electronaccepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in DpsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in DpsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.PSII, the oxygen-evolving pigment-protein complex of the thylakoid membrane system, is present in photosynthetic organisms from cyanobacteria to vascular plants. The proteins of the photochemically active reaction center (RC), the heterodimeric polypeptides PsbA (D1) and PsbD (D2) and the lowmolecular-weight (LMW) subunits PsbE and PsbF, and the a-and b-chains of cytochrome b 559 coordinate the redox cofactors necessary for primary PSII photochemistry. Electron flow following charge separation occurs via several electron carriers of the RC, including pheophytin a, as well as the primary and secondary quinones, Q A and Q B . The striking similarities between cyanobacterial and land plant PSII core components, including the intrinsic light-harvesting antennae, the chlorophyll-binding proteins CP43 and CP47, and their interactions during photosynthetic charge separation, suggest a high degree of structural and functional conservation. Thus, much of the information obtained from crystallization of a cyanobacterial PSII can be applied to that of higher plants (Zouni et al., 2001;Kamiya and Shen, 2003;Ferreira et al., 2004;Loll et al., 2005;Nield and Barber, 2006).Besides the RC, the PSII core contains a remarkably high number (16) of intrinsic or peripheral LMW peptides. Five of them are encoded in eukaryotes by nuclear genes (psbR, psbTn, psbW, ps...

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

“…psbB and psbTc are part of a prominent 2.0-kb dicistronic transcript that is not processed further to monocistronic transcripts in tobacco (Umate et al, 2008;Krech et al, 2013). Expression of this mRNA remained unchanged in ΔpsbN-F and ΔpsbN-R ( Figure 3C).…”

“…Although no visible side effects on the phenotype were apparent when the aadA cassette was introduced into the psbB gene cluster to knock out psbTc (Umate et al, 2008), we inserted the cassette in both directions in order to judge the possible effect of its orientation on expression of other genes present in the psbB gene cluster ( Figure 3A). The homoplastomic state and the orientation of the insertion of the aadA cassette in the transplastomic ΔpsbN-F and ΔpsbN-R lines were confirmed by PCR analysis using the three primers psbTc-for, psbH-rev, and aadA-for (Figures 3A and 3B; for sequences of oligonucleotides used in this work, see Supplemental Table 1).…”

“…The resulting fragment was cloned into a pDrive cloning vector (Qiagen) to produce plasmid pDN ( Figure 3A). A similar strategy has been used to generate knockouts of psbTc, also present in the psbB gene cluster (Umate et al, 2008). A SmaI-HindIII fragment of 916 bp containing the chimeric terminator-less aadA cassette was excised, blunted, and ligated to the HpaI site of pDN.…”

“…CP43, PsbH, and PsaA antisera were obtained from Agrisera. Other antibodies used in this study were described elsewhere (Umate et al, 2008;Stoppel et al, 2011). Loading was estimated by staining with Coomassie Brilliant Blue prior to immunohybridization.…”

The Low Molecular Weight Protein PsaI Stabilizes the Light-Harvesting Complex II Docking Site of Photosystem I

“…Wild-type tobacco plants appeared pale when germinated on selection medium. Tobacco lines carrying the aadA selection cassette in a neutral insertion site of the plastome were used in this work as wild-type control plants (Umate et al, 2008). These wild-type control plants and the two independent DpsaI mutants showed uniform resistance to spectinomycin and streptomycin (Supplemental Fig.…”