Figure 4. Chromatographic analysis of enzymic products from pea epicotyl in-situ assays with [³H]HHA.

Blot-dried pea epicotyl epidermis (200 mg, cutin donor source; native or heat-denatured) was incubated with 12.1 kBq [³H]HHA in 1.2 ml buffer (pH 5.5) for 24 h. The plots show TLCs of reaction products solubilised from the epidermis by, sequentially extractants a, acidified methanol; b, toluene; c, chloroform/methanol (CM, 2 : 1); d, chloroform/methanol/water (CMW, 10 : 10 : 3); f, CMNaOH (CMW containing 0.52 M sodium hydroxide). Between d and f, the insoluble material was digested with proteinase, which, however, released negligible ³H. Replicate TLCs were performed for (f) and the two profiles are superimposed. Each ‘marker’ profile (∼1 kBq [³H]HHA) refers to the sample(s) shown below it. TLC was in toluene/acetic acid, 9 : 1, with three ascents. Figures given above peaks in black are R_F values. Figures given in red report the ³H in the HHA peak (red line) as a percentage of total ³H in the profile (corrected for the background, which is shown as a dashed blue line; 13 counts/60 min/channel in Figure 4a).