Release of a “light” thylakoid membrane fragment with high F730/ F685 fluorescence emission ratio (77 K) by digitonin disruption from “low-salt”-destacked or phosphorylated thylakoids of pea

Abstract

Digitonin disruption of chloroplasts, phosphorylated in the light, results in the release of a light thylakoid fragment, sedimenting above 50 000× g and coprecipitating with the stroma lamellar subchloroplast fraction isolated by differential centrifugation; this fragment can be isolated as a zone, resolved between the grana and stroma lamellar zones, on sucrose density gradients upon ultracentrifugation; the extent of release depends on the light intensity during phosphorylation. The fragment is also released by digitonin action from isolated grana after phosphorylation, or from chloroplasts suspended in “low-salt” Tricine (0.5 mM MgCl 2). It is characterized by a high F 730/ F 685 fluorescence emission ratio at 77 K, and is enriched in the supramolecular complex of photosystem I (CPIa) and also contains light-harvesting complex II (LHC-II). The results suggest that the fragment originates in destacked, peripheral, grana thylakoids, and may contaminate the light stroma lamellar subchloroplast fraction isolated by differential centrifugation from digitonin-disrupted, phosphorylated or “low-salt” chloroplasts. Therefore, the enrichment in LHC-II components of the stroma lamellar fraction isolated from digitonin-disrupted, phosphorylated or “low-salt” chloroplasts cannot be considered as evidence for the lateral movement hypothesis. The enhancement of the F 730/ F 685 fluorescence emission ratio at 77 K, observed in chloroplasts on phosphorylation, may reflect the association of granal LHC-II with granal photosystem I (PSI).