Figure 6. Test of CUS1's CCT activity in situ and ex situ.

(a) Comparison CCT activity (in-situ formation of ester-bonded [³H]HHA; extract f of Figure 2) in epidermis of wild-type tomato (cv. M82) and the isogenic cus1 mutant (diminished in cutin synthase) at pH 5.5. Native or heat-denatured, blot-dried epidermis (50 mg) was incubated with 0.16 or 0.92 kBq [³H]HHA in 300 µl buffer at 20°C for 24 h. Bars indicate range (n = 2) of two independent experiments. (b) Testing whether N. benthamiana-produced CUS1 catalyses [³H]HHA incorporation into the total methanol-insoluble fraction (residue B) of heat-denatured pea epicotyl epidermis (10 mg) ex situ at pH 5.5. Epidermis samples were incubated with 1.03, 1.16 or 1.38 kBq [³H]HHA with or without the cobalt-purified His₆-tagged CUS1 (1 µg) in 300 µl buffer at 20°C for 1 day. Bars indicate standard errors (n = 3; results from three independent experiments).