Fig. 5
From: 4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase
Biochemical assays to study the fate of caffeate in Brachypodium and Arabidopsis. a Specific activities of individual caffeate 3-O-methyltransferase (COMT) and 4-hydroxycinnamate:CoA ligase (4CL) reactions in crude stem protein extracts from 1-month-old Brachypodium and Arabidopsis plants, performed at 10 and 50 μM caffeate concentrations (left panel), and double reactions performed by co-incubating caffeate with both cofactors required to perform the CoA activation (CoA+ATP) and 3-methoxylation (S-adenosyl methionine, SAM) reactions (right panel). The final common product of both parallel activities is feruloyl CoA. b Scheme of the studied reactions including cofactors and showing the proposed most favored pathways in the model monocot Brachypodium (red arrows) and the model dicot Arabidopsis (black arrows). The bar plot displays the activity ratios of competing enzymatic activities for both species and substrate (caffeate) concentrations calculated from the data shown in panel a. c Labeling patterns of lignin monomers in isotopic feeding experiments (m/z, mass-to-charge ratio). d Percentage of 13C-labeled ferulate incorporated into different monolignols (H-, G-, and S-units) and total lignin (T) in roots of Brachypodium and Arabidopsis seedlings. C3H 4-coumarate 3-hydroxylase, CSE caffeoyl shikimate esterase, CCoAOMT caffeoyl CoA 3-O-methyltransferase. Error bars indicate mean ± SD, two-sided unpaired t-test. n = 3
