Abstract
The chloroplasts contain an extensive system of internal membranes or thylakoids in which all the light-harvesting and energy-transducing processes of the photosynthesis are located. Thylakoids are differentiated into stacked membrane regions (or grana thylakoids) and nonstacked membranes (or stroma thylakoids), each with a specialized structure and function. Both kinds of thylakoids can be separated by detergent-based methods or mechanical fragmentation such as sonication. We describe the fractionation of thylakoid membranes into grana and stroma thylakoids by treatment with the detergent digitonin and successive ultracentrifugation of the resultant vesicles. After their separation, the thylakoid fractions retain electron transport and enzymatic activities and are characterized using various parameters. The stroma thylakoids have higher chlorophyll a/chlorophyll b and protein/total chlorophyll ratios, and greater photosystem I and NADH dehydrogenase activities than the grana thylakoids. In the conditions used and on a protein basis of total thylakoids, the yield of stroma thylakoids is 5%, which is considerable taking into account that the stroma thylakoids are a minor component of total thylakoids.
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References
Staehelin, L. A. and van der Staay, G. W. M. (1996) Structure, composition, functional organization and dynamic properties of thylakoid membranes, in Oxygenic Photosynthesis: The Light Reactions (Ort, D. R. and Yocum, C. F., eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 11–30.
Albertsson, P. A. (1995) The structure and function of the chloroplast photosynthetic membrane—a model for the domain organization. Photosynth. Res. 46, 141–149.
Albertsson, P. A. (2001) A quantitative model of the domain structure of the photosynthetic membrane. Trends Plant Sci. 6, 349–354.
Andersson, B. and Anderson, J. M. (1985) The chloroplast thylakoid membrane—isolation, subfractionation and purification of its supramolecular complexes, in Cell Components (Linskens, H. F. and Jackson, J. F., eds.), Springer-Verlag, Berlin, pp. 231–258.
Albertsson, P. A., Andreasson, E., Stefánsson, H., and Wollenberger, L. (1994) Fractionation of Thylakoid Membrane. Methods Enzymol. 228, 469–482.
Gadjieva, R., Mamedov, F., and Albertsson, P. A. (1999) Fractionation of the thylakoid membranes from tobacco. A tentative isolation of ‘end membrane’ and purified’ stroma lamellae’ membranes. Biochim. Biophys. Acta 1411, 92–100.
Leto, K. J., Bell, E., and McIntosh, L. (1985) Nuclear mutation leads to an accelerated turnover of chloroplast-encoded 48 kd and 34.5 kd polypeptides in thylakoids lacking photosystem II. EMBO J. 4, 1645–1653.
Lichtenthaler, H. K. and Wellburn, A. R. (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 11, 591–592.
Ford, R. C., Chapman, D. J., Barber, J., Pedersen, J. Z., and Cox, R. P. (1982) Fluorescence polarization and spin-label studies of the fluidity of stromal and granal chloroplast membranes. Biochim. Biophys. Acta 681, 145–151.
Wollenberger, L., Weibull, C., and Albertsson, P. A. (1995) Further characterization of the chloroplast grana margins: the non-detergent preparation of granal Photosystem I cannot reduce ferredoxin in the absence of NADP+ reduction. Biochim. Biophys. Acta 1230, 10–22.
Wilhelmová, N. and KutÍk, J. (1995) Influence of exogenously applied 6-benzylaminopurine on the structure of chloroplasts and arrangement of their membranes. Photosynthetica 31, 559–570.
Quiles, M. J., GarcÍa, A., and Cuello, J. (1999) Localization of the chloroplast NAD(P)H dehydrogenase complex in stroma thylakoids from barley. Plant Sci. 146, 17–25.
Quiles, M. J., Molina, N. C., and Cuello, J. (2002) Isolation of an NADH dehydrogenase complex not associated to ferredoxin-NADP oxidoreductase from oat stroma thylakoids. J. Plant Physiol. 159, 457–464.
Sazanov, L. A., Burrows, P. A., and Nixon, P. J. (1998) The plastid ndh genes code for an NADH-specific dehydrogenase: isolation of a complex I analogue from pea thylakoid membranes. Proc. Natl. Acad. Sci. USA 95, 1319–1324.
Ellis, R. J. and Hartley, M. R. (1982) Preparation of higher plant chloroplasts active in protein and RNA synthesis, in Methods in Chloroplast Molecular Biology (Edelman, M., Hallick, R. B., and Chua, R.-H., eds.), Elsevier Biomedical Press, Amsterdam, pp. 169–188.
Shikanai, T. and Endo, T. (2000) Physiological function of a respiratory complex, NAD(P)H dehydrogenase in chloroplasts: dissection by chloroplast reverse genetics. Plant Biotechnol. 17, 79–86.
Cuello, J. and Quiles, M. J. (2001) The NAD(P)H dehydrogenase complex of higher plant chloroplasts, in Recent Research Developments in Plant Physiology 2 (Pandalai, S. G., ed.), Research Signpost, Trivandrum, India, pp. 139–156.
Teicher, H. B. and Scheller, H. V. (1998) The NAD(P)H dehydrogenase in barley thylakoids is photoactivatable and uses NADPH as well as NADH. Plant Physiol. 117, 525–532.
Cuello, J. and Quiles, M. J. (2000) Effects of photoperiod and plant developmental stage on NADH dehydrogenase and photosystem activities of isolated chloroplasts. Biol. Plant. 43, 393–398.
Cuello, J., Quiles, M. J., Albacete, M. E., and Sabater, B. (1995) Properties of a large complex with NADH dehydrogenase activity from barley thylakoids. Plant Cell Physiol. 36, 265–271.
Arnon, D. I. (1949) Copper enzymes in isolated chloroplasts: polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15.
Cuello, J. (1997) Differential effects of linolenic acid and methyl jasmonate on the degradation of chlorophylls and carotenoids of senescing barley leaves. Acta Bot. Neerl. 46, 303–314.
Quiles, M. J., GarcÍa, A., and Cuello J. (2000) Separation by blue-native PAGE and identification of the whole NAD(P)H dehydrogenase complex from barley stroma thylakoids. Plant Physiol. Biochem. 38, 225–232.
Ellis, R. J. (1977) Protein synthesis by isolated chloroplasts. Biochim. Biophys. Acta 463, 185–215.
Quiles, M. J. and Cuello, J. (1998) Association of ferredoxin-NADP oxidoreductase with the chloroplastic pyridine nucleotide dehydrogenase complex in barley leaves. Plant Physiol. 117, 235–244.
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Cuello, J., Quiles, M.J. (2004). Fractionation of Thylakoid Membranes Into Grana and Stroma Thylakoids. In: Carpentier, R. (eds) Photosynthesis Research Protocols. Methods In Molecular Biology™, vol 274. Humana Press. https://doi.org/10.1385/1-59259-799-8:001
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DOI: https://doi.org/10.1385/1-59259-799-8:001
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