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One-step isolation and biochemical characterization of a highly active plant PSII monomeric core

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Abstract

We describe a one-step detergent solubilization protocol for isolating a highly active form of Photosystem II (PSII) from Pisum sativum L. Detailed characterization of the preparation showed that the complex was a monomer having no light harvesting proteins attached. This core reaction centre complex had, however, a range of low molecular mass intrinsic proteins as well as the chlorophyll binding proteins CP43 and CP47 and the reaction centre proteins D1 and D2. Of particular note was the presence of a stoichiometric level of PsbW, a low molecular weight protein not present in PSII of cyanobacteria. Despite the high oxygen evolution rate, the core complex did not retain the PsbQ extrinsic protein although there was close to a full complement of PsbO and PsbR and partial level of PsbP. However, reconstitution of PsbP and PsbPQ was possible. The presence of PsbP in absence of LHCII and other chlorophyll a/b binding proteins confirms that LHCII proteins are not a strict requirement for the assembly of this extrinsic polypeptide to the PSII core in contrast with the conclusion of Caffarri et al. (2009).

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Abbreviations

BN-PAGE:

Blue native polyacrylamide gel electrophoresis

Chl:

Chlorophyll

DCBQ:

2,6-Dichlorobenzoquinone

DCMU:

3-(3,4-Dichloropheny1)-l,l-dimethylurea

EM:

Electron microscopy

ESI:

Electrospray ionization

K3Fe(CN)6 :

Potassium ferricyanide

LC:

Liquid chromatography

LHC:

Light harvesting complex

MALDI-TOF:

Matrix-assisted laser desorption/ionization-time of flight

MS:

Mass spectrometry

OEC:

Oxygen evolving complex

PEG6000:

Polyethylene glycol Mr 6000

PSII:

Photosystem II

SDS-PAGE:

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

α-DM:

alpha-Dodecylmaltoside

β-DM:

beta-Dodecylmaltoside

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Acknowledgments

The authors kindly thank Prof. Roberto Barbato (University of Piemonte Orientale, Italy) for supplying antibodies against CP47, CP43, D2, D1 and LHCII polypeptides as well as for very useful discussions about the manuscript. We are very grateful to Prof. Miwa Sugiura (Ehime University, Japan) for MALDI‐TOF mass analysis and Dr. Tim Grant (Imperial College, London, UK) for EM analysis. This work was supported by Regione Piemonte (PROESA project, DGR. 36‐8559 7/04/2008) and by European Commission (SOLHYDROMICS project (227192), FP‐7‐Energy‐2008‐FET).

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Authors and Affiliations

  1. Department of Materials Science and Chemical Engineering – BioSolar Lab, Politecnico di Torino, Viale T. Michel 5, 15121, Alessandria, Italy

    Cristina Pagliano, Fabiana Chimirri, Guido Saracco & James Barber

  2. Department of Environmental and Life Sciences-Proteomic Facility, University of Piemonte Orientale, Viale T. Michel 11, 15121, Alessandria, Italy

    Francesco Marsano

  3. Wolfsons Laboratories, Imperial College London, London, SW7 2AZ, United Kingdom

    James Barber

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  1. Cristina Pagliano
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  2. Fabiana Chimirri
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  3. Guido Saracco
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  4. Francesco Marsano
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  5. James Barber
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Correspondence to Cristina Pagliano.

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Pagliano, C., Chimirri, F., Saracco, G. et al. One-step isolation and biochemical characterization of a highly active plant PSII monomeric core. Photosynth Res 108, 33–46 (2011). https://doi.org/10.1007/s11120-011-9650-4

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