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. 2022 Mar 9;8(10):eabm8145. doi: 10.1126/sciadv.abm8145

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Fig. 8. — (A) Activity of recombinant Cleome LAC4, LAC5, LAC8, and LAC15 against caffeyl alcohol. (B) Effects of water- and ethyl acetate–soluble extracts from poplar fibers on the oxidation of coniferyl alcohol by ChLAC4. Data are the areas of the coniferyl alcohol peak determined by HPLC. mAU, milli–absorbance units. (C) Activity of recombinant Cleome LAC4, LAC5, LAC8, and LAC15 against coniferyl alcohol in the presence of water-soluble extract (WS) from poplar fibers. (D) Activity of recombinant Cleome LAC4, LAC5, LAC8, and LAC15 against caffeyl alcohol in the presence of WS. (E) Effects of caffeyl alcohol on the oxidation of coniferyl alcohol by recombinant Cleome LAC4, LAC5, LAC8, and LAC15 in the presence of WS. Data are the areas of the coniferyl alcohol peak determined by HPLC. (F) Oxidation of caffeyl alcohol by ChLAC4, ChLAC5, ChLAC8, and ChLAC15 in the presence of coniferyl alcohol and WS. C, caffeyl alcohol; G, coniferyl alcohol; ES, ethyl acetate–soluble fraction from poplar fibers; N/A, no activity. Reactions (100 μl) contained 50 μM of each monolignol substrate and 300 ng of purified recombinant protein. Activity was calculated by substrate disappearance as monitored by HPLC. Water- and ethyl acetate–soluble fractions from poplar fibers were prepared as described in Materials and Methods and added at a level of 1 μl per 100 μl of reaction. Recombinant LAC protein purified from infiltrated tobacco (N. benthamiana) leaves harvested from three plants was counted as one biological replicate. Data are means ± SE derived from three biological replicates. The different letters above the bars represent statistically significant differences determined by ANOVA (Duncan, P ≤ 0.05) with SPSS Statistics (version 27; IBM). nkat, nano katals.