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The PSBP2 protein of Chlamydomonas reinhardtii is required for singlet oxygen-dependent signaling

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Abstract

In the green alga Chlamydomonas reinhardtii, the cytosolic GLUTATHIONE PEROXIDASE 5 gene (GPX5) is known to be transcriptionally up-regulated in response to singlet oxygen (1O2). As demonstrated by previous studies, fusion of the promoter region of GPX5 to the ARYLSULFATASE 2 gene (ARS2) creates an effective reporter system that can be used to monitor 1O2-driven GPX5 expression. This system was also used in this study to generate a stably transformed C. reinhardtii strain which expresses ARS2 in a 1O2-dependent manner, resulting in the synthesis of a functional protein with detectable activity. Using the strain of C. reinhardtii harboring a 1O2-sensitive reporter construct, a secondary mutagenic screen was performed. This allowed identification of mutant cell lines that were unable to up-regulate expression of the GPX5–ARS2 fusion in response to 1O2. In one of these lines, the mutation was subsequently localized to the first exon of the PSBP-like gene (PSBP2). The PSBP2 gene is part of a small protein family in C. reinhardtii, also present in all angiosperms studied thus far. While each member of the PSBP protein family contains a similar domain to the PSBP1 protein, which is a member of the oxygen evolving complex of photosystem II (PSII), the PSBP2 protein does not appear to be involved in PSII function, but may function as a sensor and/or signal mediating molecule of the 1O2 generated in the chloroplast.

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Abbreviations

1 − q L :

Excitation pressure

1O2 :

Singlet oxygen

ARS2 :

ARYLSULPHATASE 2

DCMU:

3-(3,4-dichlorophenyl)-1,1-dimethylurea

F v/F m :

Maximal quantum efficiency of PSII

GL:

Growth light

GPX5 :

GLUTATHIONE PEROXIDASE 5

H2O2 :

Hydrogen peroxide

HL:

High light

ML:

Moderate light

NR:

Neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride)

NPQ:

Non-photochemical quenching

PPD:

PSBP domain

PPL:

PSBP-like

PSI:

Photosystem I

PSII:

Photosystem II

RACE:

Rapid amplification of cDNA ends

RB:

Rose Bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein)

RESDA:

Restriction-enzyme site-directed amplification

ROS:

Reactive oxygen species

sqRT:

Semi-quantitative reverse transcriptase

TAP:

Tris–acetate–phosphate

t-BOOH:

tert-Butyl hydroperoxide

TP:

Tris–phosphate

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Acknowledgments

The authors would like to acknowledge the Chlamydomonas Center for supplying algal strains and plasmids. They would also like to thank Dr. Wolfgang Mages (Universität Regensburg) for the gift of the pHYG3 plasmid, Dr. Saul Purton (University College London) for the gift of the pSP124s plasmid, and Dr. Jean-David Rochaix (University of Geneva) for the wild-type 6C+. The work in KEW’s laboratory was supported by Discovery, and Research Tools and Instruments Grants from the Natural Science and Engineering Research Council (NSERC) of Canada, a New Opportunities Grant from the Canadian Foundation for Innovation and by institutional funding from the University of Saskatchewan. GRG is also supported by an NSERC Discovery grant. PB was supported, in part, by the Department of Biology, University of Saskatchewan.

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  1. Pawel Brzezowski

    Present address: Institute of Biology/Plant Physiology, Humboldt University Berlin, Philippstraße 13, Building 12, 10115, Berlin, Germany

Authors and Affiliations

  1. Department of Biology, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada

    Pawel Brzezowski & Kenneth E. Wilson

  2. Department of Plant Sciences, University of Saskatchewan, Saskatoon, SK, S7N 5A8, Canada

    Gordon R. Gray

  3. Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada

    Gordon R. Gray

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  1. Pawel Brzezowski
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  2. Kenneth E. Wilson
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  3. Gordon R. Gray
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Correspondence to Gordon R. Gray.

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Brzezowski, P., Wilson, K.E. & Gray, G.R. The PSBP2 protein of Chlamydomonas reinhardtii is required for singlet oxygen-dependent signaling. Planta 236, 1289–1303 (2012). https://doi.org/10.1007/s00425-012-1683-1

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