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Differential Autoradiography and Spin-Column Purification of Sugars on Activated Charcoal

Dep. of Soil and Crop Science, Texas A&M Univ., College Station, TX 77843-2474

^* Corresponding author (tarpley{at}csrumsu.ars.ag.gov).

Methods utilizing activaled charcoal for separating radiolabeled sugars from each other and from water are described. The first method alleviales a practical limitation for use of tritiated sugars as a tracer in planta. Tritium exchange with waler can now be readily quanlified because lhe method allows a more rapid separalion of sugars and water than has been previously described. A spin column conlaining activaled charcoal and powdered cellulose relained (>99%) sucrose during elution (>99%) of ³H₂O in waler rinses. The retained sucrose could then be eluled (95%) from the column by rinsing with aqueous methanol. The second melhod partially resolves anolher limitation in the use of radiolabeled sugars as tracers in planta. We can now ascertain the spatial distribution of various radiolabeled sugars within particular organs. Charcoal filler paper was used to adsorb sucrose and hexose sugars for development of differential autoradiographs of sugars blotted from stem cross-sections of sorghum (Sorghum bicolor [L.] Moench). The hexose sugars were selectively removed during rinsing of the charcoal paper in water. Autoradiography was performed before and after rinsing. The autoradiographic image obtained before rinsing reflected the presence on the blot of both radiolabeled sucrose and hexose sugars. In comparison, the image obtained after rinsing no longer reflected the presence of radiolabeled hexose sugars. These methods help refine studies of carbon dynamics in plants by enhancing our ability to detect in planta movement of sugars through particular metabolic pathways and to detect lhe spatial distribution within organs of particular radiolabeled sugars.

Received for publication August 12, 1996.