Compensatory Guaiacyl Lignin Biosynthesis at the Expense of Syringyl Lignin in 4CL1-Knockout Poplar

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4 - coumarate : CoA ligases ( 4CLs ) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar ( Populus tremula × alba ) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5 - hydroxylases ( F5Hs ). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1 Upregulation of caffeoyl - CoA O - methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1 -knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.