A Mutant Strain of Chlamydomonas reinhardtii Lacking the Chloroplast Photosystem II psbI Gene Grows Photoautotrophically

A sensitive and simple reverse phase HPLC purification scheme was developed for the rapid separation of the small protein subunits from photosystem II reaction center preparations. The precise molecular masses of the ␣-and ␤-subunits of cytochrome b 559 and the psbI gene product from pea plants, found to be 4394.6 ؎ 0.6, 9283.6 ؎ 0.7, and 4209.5 ؎ 0.5 Da, respectively, were then successfully determined for the first time by electrospray-and fast atom bombardment-mass spectrometry. Discrepancies between the molecular weights assigned and those calculated from the respective DNA sequences were observed for ␣-and ␤-subunits of cytochrome b 559 . Currently, the nucleotide sequence of the psbI gene product from pea plants is not available. Application of novel mapping and sequencing strategies has assured the elucidation of full primary structures of all of the purified subunits. The modifications identified here include the post-translational processing of the initiating methionine on both subunits of cytochrome b 559 , NH 2 -terminal acetylation and an mRNA editing site at residue 26 (Ser 3 Phe) on the ␤-subunit, and retention of the NH 2 -terminal formyl-Met on the psbI gene product. In addition, specific oxidation of a single amino acid residue was identified on the psbI gene product and the ␤-subunit purified from light-treated reaction center preparations. Overall, these studies provide the first detailed primary structural characterization of the small subunits of the reaction center complex and their associated light-induced modifications. Photosystem II (PSII)1 catalyzes the photoinduced splitting of water into molecular oxygen and reducing equivalents. This complex is embedded in the thylakoid membrane of plants, algae, and cyanobacteria and is composed of a large number of subunits (1, 2). At the heart of the complex is the reaction center where primary and secondary electron transfer processes occur. The cofactors that support these reactions are bound to the D1 and D2 proteins. These proteins are the products of the psbA and psbD genes, respectively, and are comparable with the L and M subunits of the reaction center of purple photosynthetic bacteria (3).In its isolated form, the reaction center of PSII contains, in addition to the D1 and D2 proteins, the ␣-and ␤-subunits of cytochrome b 559 , which are the products of the psbE and psbF genes, and the PSII-I protein, which is encoded by the psbI gene. In a recent paper (4), a sixth component has been claimed, which has an apparent molecular mass of about 6.5 kDa and is the product of the nuclear gene recently named psbW.Sites in the D2 and D1 proteins, named Q A and Q B respectively, bind plastoquinones that facilitate secondary electron flow. However, in the case of the isolated reaction center complex the plastoquinone binding affinity is significantly reduced, and no secondary electron flow occurs unless appropriate acceptors are added (5). In their absence, the photochemical reaction is restricted to the formation of the radical pair state P680 ϩ Phe...

Section: Discussionmentioning

confidence: 97%

Characterization of the Low Molecular Weight Photosystem II Reaction Center Subunits and Their Light-induced Modifications by Mass Spectrometry

Sharma

Panico

Barber

et al. 1997

“…Mutants deficient in cytochrome b 559 accumulate no detectable amount of PSII complex (30), whereas psbI-deficient mutants accumulate PSII complex only to 20 -30% of the wild-type level (31). Probably the absence of these polypeptides affects stability and/or synthesis of the PSII complex.…”

Section: Discussionmentioning

confidence: 99%

PsbT Polypeptide Is Required for Efficient Repair of Photodamaged Photosystem II Reaction Center

Ohnishi¹,

Takahashi²

2001

Self Cite

PsbT is a small chloroplast-encoded hydrophobic polypeptide associated with the photosystem II (PSII) core complex. A psbT-deficient mutant (⌬psbT) of the green alga Chlamydomonas reinhardtii grows photoautotrophically, whereas its growth is significantly impaired in strong light. To understand the photosensitivity of ⌬psbT, we have studied the effect of strong illumination on PSII activity and proteins. It is shown that the level of PSII activity and proteins is reduced in the ⌬psbT more significantly than in wild type under strong light. When recovery of the photodamaged PSII is inhibited by a chloroplast protein synthesis inhibitor, the light-induced inactivation and degradation of PSII occur similarly in wild-type and mutant cells. On the contrary, the recovery of PSII activity after partial photoinactivation is remarkably delayed in the ⌬psbT cells, suggesting that PsbT is required for efficient recovery of the photodamaged PSII complex. These results therefore present the first evidence for involvement of this small PSII polypeptide in the recovery process. Partial disintegration of the purified PSII core complex and localization of PSII proteins in the resulting PSII subcore complexes have revealed that PsbT is associated with D1/D2 heterodimer. A possible role of PsbT in the recovery process is discussed.

“…The Synechocystis ⌬psbI mutant was found to be slightly more sensitive to light than the corresponding WT (29). Deletion of psbI in C. reinhardtii caused a more severe effect compared with that in cyanobacteria (28). Although the C. reinhardtii ⌬psbI mutant grows photoautotrophically under low light, its growth rate was quite sensitive to high light.…”

mentioning

confidence: 95%

“…No knock-out strains for psbI are currently available from higher plants. Mutants lacking PsbI have been generated from Chlamydomonas reinhardtii (28) and Synechocystis (29). Inactivation of psbI in Synechocystis and Thermosynechococcus elongatus strain BP-1 caused some reduction of PSII activity, resulting in decreased oxygen evolution to 70 -80% of WT levels, but the mutants grew photoautotrophically.…”

mentioning

confidence: 99%

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

Schwenkert

Umate

Bosco

et al. 2006

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.