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Isolation of chestnut chloroplasts: membrane potentials of chestnut and spinach thylakoids

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Photosynthetica

Abstract

Typical chestnut thylakoid extracts isolated by mechanical disruption of leaf tissues had an equivalent of 0.28 kg m−3 chlorophyll (Chl) which is six times less than in thylakoids obtained from spinach, although Chl content in leaves was only half as small. According to optical microscopy, the vesicles showed a good integrity, exhibiting at 21 °C a high capacity of photon-induced potential membrane generation, which was demonstrated by the almost full 9-amino-6-chloro-2-methoxyacridine fluorescence quenching in a hyper-saline medium containing 150 mM KCl and having osmotic potential of −1.5 MPa. The half-time of the thylakoid potential generation was 11.7 s with the time of dissipation around 8.9 s. In such conditions, spinach thylakoids showed an increased swelling and also differences in the half-time generation which was almost four times faster than was observed in chestnut. However, when spinach thylakoids were incubated in a typical hypo-saline medium without KCl with osmotic potential −0.8 MPa, no additional swelling was observed. Consequently the half-time of potential dissipation was 35 s. Studies with nigericin suggested a chestnut thylakoid ΔpH significantly smaller than that observed in spinach, which was confirmed by the measurements of the ATP driven pumping activity.

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Abbreviations

ACMA:

9-amino-6-chloro-2-methoxyacridine

BHT:

butylated hydroxytoluene

BSA:

bovine serum albumin

EDTA:

disodium ethylenediamine tetraacetic acid

F:

ACMA fluorescence quenching under dark conditions

MV:

methyl viologen

PS:

photosystem

Q:

light-induced ACMA fluorescence quenching

R:

ACMA fluorescence recuperation

t0.5l:

half-time fluorescence quenching

t0.5d:

half-time fluorescence recuperation

Tricine:

N-tris (hydroxymethyl) methylglycine

Δp:

electric membrane potential

ΔpH:

electrochemical potential difference of protons

Δψ:

transmembrane potential

Ψπ :

osmotic potential

Ψw :

water potential

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Authors and Affiliations

  1. Centre for Technological Studies on Environment and Life, University of Tras-os-Montes and Alto Douro, 5001-911, Vila Real, Portugal

    J. Gomes-Laranjo & P. Salgado

  2. Department of Structural Biology, BioCentrum Amsterdam, Faculty of Earth and Life Sciences, Free University of Amsterdam, De Boelelaan 1087, 1081 HV, Amsterdam, The Netherlands

    H. W. Wong Fong Sang & R. Kraayenhof

Authors
  1. J. Gomes-Laranjo
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  2. P. Salgado
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  3. H. W. Wong Fong Sang
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  4. R. Kraayenhof
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  5. J. Torres-Pereira
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Correspondence to J. Gomes-Laranjo.

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Gomes-Laranjo, J., Salgado, P., Wong Fong Sang, H.W. et al. Isolation of chestnut chloroplasts: membrane potentials of chestnut and spinach thylakoids. Photosynthetica 43, 237–246 (2005). https://doi.org/10.1007/s11099-005-0039-5

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  • DOI: https://doi.org/10.1007/s11099-005-0039-5

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