Figure 3.

Effects of pretreatment with recombinant Pel1, PelA, and PG on mature cucumber hypocotyl walls' susceptibility to expansin. A, Nongrowing basal segments were frozen, thawed, abraded, pressed, and heated with water for 100 s in a microwave oven. These were then pretreated with 33 units mL⁻¹ (line 1, representative; line 4, mean) or 22 units mL⁻¹ (line 2, mean) of Pel1 in 50 mm sodium acetate buffer (buffer B, pH 5.0), or with cell extraction (line 3, mean) in buffer B (pH 5.0) from E. coli containing empty pET-28a (+) expression plasmid for 30 min at 30°C, washed with buffer B (pH 5.0) three times, and, finally, were stretched under tension in bathing buffer in an extensometer for 30 min, after which the bathing buffer was replaced with 0.1 mL of bathing buffer containing either 2 mg mL⁻¹ of crude expansin or bathing buffer alone (only line 4 for minus expansin control). B, Nongrowing basal segments were pretreated with 1.50 units mL⁻¹ (line 1, representative; line 4, mean) or 1.15 units mL⁻¹ (line 2, mean) of PelA, or cell extraction (line 3, mean) in buffer B (pH 5.5); others for extension assay as described in A. C, The nongrowing basal segments were pretreated with 0.08 units mL⁻¹ (line 1, representative; line 4, mean) or 0.06 units mL⁻¹ (line 2, mean) of PG, or cell extraction (line 3, mean) in buffer B (pH 5.0); others for extension assay as described in A. Curves are the mean (mean) or representative (representative, based on its breakage) of three independent experiments. Thick arrows indicate when the bathing buffer was replaced.