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Pigment composition and functional state of the thylakoid membranes during preparation of samples for pigment-protein complexes separation by nondenaturing gel electrophoresis

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Photosynthetica

Abstract

The present study was conducted to examine changes in photosynthetic pigment composition and functional state of the thylakoid membranes during the individual steps of preparation of samples that are intended for a separation of pigmentprotein complexes by nondenaturing polyacrylamide gel electrophoresis. The thylakoid membranes were isolated from barley leaves (Hordeum vulgare L.) grown under low irradiance (50 μmol m−2 s−1). Functional state of the thylakoid membrane preparations was evaluated by determination of the maximal photochemical efficiency of photosystem (PS) II (FV/FM) and by analysis of excitation and emission spectra of chlorophyll a (Chl a) fluorescence at 77 K. All measurements were done at three phases of preparation of the samples: (1) in the suspensions of osmotically-shocked broken chloroplasts, (2) thylakoid membranes in extraction buffer containing Tris, glycine, and glycerol and (3) thylakoid membranes solubilized with a detergent decyl-β-D-maltosid. FV/FM was reduced from 0.815 in the first step to 0.723 in the second step and to values close to zero in solubilized membranes. Pigment composition was not pronouncedly changed during preparation of the thylakoid membrane samples. Isolation of thylakoid membranes affected the efficiency of excitation energy transfer within PSII complexes only slightly. Emission and excitation fluorescence spectra of the solubilized membranes resemble spectra of trimers of PSII light-harvesting complexes (LHCII). Despite a disrupted excitation energy transfer from LHCII to PSII antenna core in solubilized membranes, energy transfer from Chl b and carotenoids to emission forms of Chl a within LHCII trimers remained effective.

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Abbreviations

Cars:

carotenoids

Chl:

chlorophyll

Dm:

decyl-β-D-maltosid

FV/FM :

maximal photochemical efficiency of photosystem II

LHCII:

light-harvesting complexes of photosystem II

PPCs:

pigment-protein complexes

PSI and PSII:

photosystem I and II

RES1 and RES2:

resuspension medium 1 and 2

VAZ:

the pool of xanthophyll cycle pigments (violaxanthin + antheraxanthin + zeaxanthin)

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Acknowledgements

We thank B. Piskořová for technical assistance. This work was supported by the Grant Agency of Czech Republic (522/07/P246), Ministry of Education, Youth and Sports of the Czech Republic (MSM 6198959215) and by the Palacky University (student project PrF_2010_050).

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Authors and Affiliations

  1. Laboratory of Biophysics, Faculty of Science, Palacký University, tř. Svobody 26, CZ-771 46, Olomouc, Czech Republic

    V. Karlický

  2. Department of Physics, Faculty of Science, Ostrava University, 30. dubna 22, CZ-701 03, Ostrava 1, Czech Republic

    V. Karlický, J. Podolinská, L. Nadkanská, M. Štroch, M. Čajánek & V. Špunda

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  1. V. Karlický
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  2. J. Podolinská
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  3. L. Nadkanská
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  4. M. Štroch
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  5. M. Čajánek
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  6. V. Špunda
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Correspondence to M. Štroch.

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Karlický, V., Podolinská, J., Nadkanská, L. et al. Pigment composition and functional state of the thylakoid membranes during preparation of samples for pigment-protein complexes separation by nondenaturing gel electrophoresis. Photosynthetica 48, 475–480 (2010). https://doi.org/10.1007/s11099-010-0063-y

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