Effects of feruloyl-CoA 6′-hydroxylase 1 overexpression on lignin and cell wall characteristics in transgenic hybrid aspen

In plant cell walls, lignin, cellulose, and the hemicelluloses form intricate three-dimensional structures. Owing to its complexity, lignin often acts as a bottleneck for the efficient utilization of polysaccharide components as biochemicals and functional materials. A promising approach to mitigate and/or overcome lignin recalcitrance is the qualitative and quantitative modification of lignin by genetic engineering. Feruloyl-CoA 6'-hydroxylase (F6'H1) is a 2-oxoglutarate-dependent dioxygenase that catalyzes the conversion of feruloyl-CoA, one of the intermediates of the lignin biosynthetic pathway, into 6'-hydroxyferuloyl-CoA, the precursor of scopoletin (7-hydroxy-6-methoxycoumarin). In a previous study with <i>Arabidopsis thaliana</i>, we demonstrated that overexpression of <i>F6'H1</i> under a xylem-preferential promoter led to scopoletin incorporation into the cell wall. This altered the chemical structure of lignin without affecting lignin content or saccharification efficiency. In the present study, the same <i>F6'H1</i> construct was introduced into hybrid aspen (<i>Populus tremula × tremuloides</i> T89), a model woody plant, and its effects on plant morphology, lignin chemical structure, global gene expression, and phenolic metabolism were examined. The transgenic plants successfully overproduced scopoletin while exhibiting severe growth retardation, a phenotype not previously observed in <i>Arabidopsis</i>. Scopoletin accumulation was most pronounced in the secondary walls of tracheary elements and the compound middle lamella, with low levels in the fiber cell walls. Overexpression of <i>F6'H1</i> also affected the metabolism of aromatics, including lignin precursors. Heteronuclear single-quantum coherence (HSQC) NMR spectroscopy revealed that scopoletin in cell walls was bound to lignin, leading to a reduction in lignin content and changes in its monomeric composition and molar mass distribution. Furthermore, the enzymatic saccharification efficiency of the transgenic cell walls was more than three times higher than that of the wild-type plants, even without pretreatment. Although addressing growth inhibition remains a priority, incorporating scopoletin into lignin demonstrates significant potential for improving woody biomass utilization.