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Extra info for Annual European Symposium on Photomorphogenesis. Photochemistry and Photobiology
Preliminary experiments demonstrated (a) that the light doses used were saturating for phytochrome photoconversions and (b) that the centrifugation procedure adopted pelleted the same amount of phytochrome as the 20,000 x g for 30 min commonly used in the literature. Phytochrome measurements. Phytochrome was assayed as previously described with a modified (Marme, 1969) Ratiospect using C a C 0 as a scattering agent (Butler and 3 Norris, 1960). "Percent pelletable phytochrome" represents the amount in the 50,000 x g pellet as a percentage of the combined total of supernatant and pellet.
Tissue irradiated and incubated at 0°C was held bathed in extraction medium in tubes on ice during both operations. Tissue irradiated and incubated at 25°C was held in tubes in a water bath at that temperature and rapidly cooled at the times indicated by addition of ice-cold extraction medium and transfer of the tubes to ice. Pre-chilled tissue (2 g) was homogenized for 5 s in 6 mi of ice-cold extraction medium using an Ultra Turrax blender at 6700 rpm and the homogenate filtered through nylon cloth.
Shoot tissue was irradiated at 25°C as in+ addition of ice-cold extraction dicated and chilled 2 by medium (minus M g ) either immediately (where the terminal irradiation was far red) or after 3 min dark incubation (where the terminal irradiation was red). After a further 3 min on ice the tissue was homogenized for 5 s. 2+ given immediThe 5 s in vitro red irradiation indicated was ately following the homogenization. M g was added to the crude homogenates at the times indicated to a final concentration of 10 mM.