Fig. 4

ADE/POD activity and protein levels during leaf maturation of Excoecaria and Osmanthus. A-B Anthocyanin degradation enzyme (ADE) activities. ADE activities of the three leaf developmental stages as indicated in Fig. 1A and B were determined with the anthocyanin substrates partially purified from Excoecaria and Osmanthus leaves respectively, at pH 4.0, with or without H₂O₂. C Activities of PODs. POD (peroxidase) activities were measured with guaiacol and H₂O₂ as substrates at pH 7.0. D-E In gel ADE activity assays. Ten micrograms of total proteins were separated by semi-native PAGE. The ADE activities were visualized by using the anthocyanin substrates as indicated in (A-B). Lane M indicated the protein markers, while Lane 1–3 represented the leaf samples as in (A-B). F In-gel POD activity assays. POD activity was visualized by immersing the gel in the substrate as described in (C). Lanes are as described in (D-E). G-H In-gel activity assay of the purified POD. POD was extracted and purified from Osmanthus leaves. The POD activity and its activity on anthocyanin degradation (ADE/POD) was visualized by using guaiacol (G) and partially purified Osmanthus anthocyanins (H) respectively as substrates in the presence of 2 mM H₂O₂. Lane M is as described in (D-E). I POD mediated degradation of the major anthocyanins. The major anthocyanins of Excoecaria and Osmanthus, as indicated in Fig. 3(A-C) and Fig. 2F, were purified respectively as described in Fig. 2A and B and added as substrates to the ADE/POD reactions as in (C), with denatured enzyme as control. The remained anthocyanin contents were determined after the reaction for 20 min. J Immuno-detection of PODs. POD protein abundance was detected in both soluble and insoluble fraction proteins from the leaf samples as in (A). The red frame indicates the size of the POD band as about 110 kDa. The details of the values presented in (A-C, I) are as described in Fig. 1