Isolation of highly active photosystem II core complexes with a His-tagged Cyt b559 subunit from transplastomic tobacco plants.
Photosystem II (PSII) is a huge multi-protein-complex consisting, in higher plants and green algae, of the PS II core and the adjacent light harvesting proteins. In the study reported here, N-terminal His-tags were added to the plastome-encoded alpha-subunit of cytochrome b559, PsbE, in tobacco plants, thus facilitating rapid, mild purification of higher plant PSII. Biolistic chloroplast transformation was used to replace the wildtype psbE gene by His-tagged counterparts. Transgenic plants did not exhibit an obvious phenotype. However, the oxygen evolution capacity of thylakoids prepared from the mutants compared to the wildtype was reduced by 10-30% depending on the length of the His-tag, although Fv/Fm values differed only slightly. Homoplasmic F1 plants were used to isolate PSII cores complexes. The cores contained no detectable traces of LHC or PsaA/B polypeptides, but the main core subunits of PSII could be identified using immunodetection and mass spectroscopy. In addition, Psb27 and PsbS were detected. The presence of the former was presumably due to the preparation method, since PSII complexes located in the stroma are also isolated. In contrast to previous reports, PsbS was solely found as a monomer on SDS-PAGE in the PSII core complexes of tobacco.
Fey
Holger
H
Institute of Molecular Biosciences, University of Frankfurt, Siesmayerstr. 70, D60323 Frankfurt, Germany.
Piano
Dario
D
Horn
Ruth
R
Fischer
David
D
Schmidt
Matthias
M
Ruf
Stephanie
S
Schröder
Wolfgang P
WP
Bock
Ralph
R
Büchel
Claudia
C
D003573:Cytochrome b Group / Q000302:isolation & purification* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D054628:Genome, Chloroplast; D006639:Histidine / Q000378:metabolism*; D015151:Immunoblotting; D009154:Mutation / Q000235:genetics; D009842:Oligopeptides / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000302:isolation & purification* / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified; D018087:Plastids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D021122:Protein Subunits / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis; D020524:Thylakoids / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*
D003573:Cytochrome b Group; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006639:Histidine; C019313:cytochrome b559; D010100:Oxygen