Fig. 2.

Extraction and characterization of FucAl-labeled cell wall components. (A) Fluorescently labeled FucAl is enriched in CDTA and urea extracts of cell walls. Seedlings were treated with 25 μM FucAl or 0.1% DMSO for 24 h and labeled with Alexa 594-azide. The labeled seedlings were homogenized and cellular components were sequentially extracted. Fluorescence at 585 nm (F₅₈₅) of 200-μL aliquots of each extract was measured, and the ratio of FucAl-treated to DMSO-treated F₅₈₅ was calculated. (B) Cell walls prepared from labeled FucAl-treated seedlings were digested with 2.5 U of PME/PG, 1,5-Ara, 1,4-Gal, 1,5-Ara/1,4-Gal, 1,3-Gal, and XEG. Fluorescence was measured as in A and standardized to no enzyme controls (Materials and Methods). Error bars represent SEM from three independent experiments. (C–H) Solubilized material from the indicated digests (red) or no enzyme control (blue) was fractionated on a Sephadex G-75 column, and F₅₈₅ of 1 mL fractions was measured. Chromatograms are representative of three repetitions of each experiment. Note different scales on y axes of graphs. v₀ = void volume, v_i = included volume.