FAEB activity assay, PCR validation of faeB and immunocytochemical localization of FAEB. (A) FAEB activity as indicated by the change in absorbance over 60 min as a result of the hydrolysis of ethyl ferulate. Data represents three absorbance readings from the same extract from a single plant. Error bars indicate relative standard error. Apoplast (A), transgenic line 3A; control, wild type plant; endoplasmic reticulum (ER), transgenic line 28ER and vacuole (V), transgenic line 2 V. (B) PCR validation of faeB gene in transgenic lines. Apoplast-expressing sample: lane 1, nptII (apoplast); lane 2, faeB-apoplast and lane 3, wild type negative control for faeB-apoplast. ER-expressing sample: lane 4, nptII (ER); lane 5, faeB-ER and lane 6, wild type negative control for faeB-ER. Vacuole-expressing sample: lane 7, nptII (vacuole); lane 8, faeB-vacuole and lane 9, wild type negative control for faeB-vacuole. (C) Immunocytochemical localization to confirm recombinant protein expression in: (a) apoplast; (b) endoplasmic reticulum; (c) vacuole and (d) non-transgenic control in alfalfa leaves. A, apoplast; ER, endoplasmic reticulum; FAEB, type B ferulic acid esterase; V, vacuole.