Transcriptional regulation of monolignol O-methylation in Cleome hassleriana

Catechyl (C) lignin, a linear homopolymer restricted to seed coats of a limited number of plant species, has favorable properties for chemical and biomaterial manufacture. In the seed coat of Cleome hassleriana, lignin composition switches from guaiacyl (G-) lignin to C-lignin during development. This involves loss of expression of caffeoyl-CoA 3-O-methyltransferase (ChCCoAOMT) and caffeic acid 3-O-methyltransferase (ChCOMT), and upregulation of a cinnamyl alcohol dehydrogenase (ChCAD5) and laccase (ChLAC8) with substrate preference for the caffeyl moiety. How the loss of monolignol O-methylation is regulated is not understood. We reconstructed coexpression networks of the above enzymes with transcription factor (TF) candidates based on RNA sequencing data. Using a dual-luciferase reporter assay, we identified ChMYB20 as an activator of the ChCCoAOMT and ChCOMT promoters, and ChMYB6-like as a repressor. ChMYB5 and ChMYB42 induced ChCAD5, ChLAC8, and ChMYB6-like, and ChNAC92-like induced ChMYB5 and ChMYB42. The roles of the TFs in the network were further confirmed by expressing them in Cleome hairy roots. ChMYB5 and ChMYB42 suppressed monolignol O-methyltransferase genes, activated downstream C-lignin-associated genes, and also activated each other, suggesting they are players in the switch from G- to C-lignin biosynthesis via blockage of monolignol O-methylation. Models for the gene regulatory networks are presented.