Impact of lignin composition on biomass recalcitrance of engineered Arabidopsis

Lignin plays important biological functions and contributes to the biomass recalcitrance of plant cell wall. Genetic modification of lignin synthesis pathways has become one of the primary targets of cell wall engineering in recent years. In this study, we used a combination of approaches to characterize the structural and compositional features of wild-type Arabidopsis and the mutants with distinct lignin monomer compositions: fah1-2 (G-lignin dominant), pC4H-F5H (S-lignin dominant), AtCOMT1 (G/G’-lignin dominant) and a newly developed ref4/rfr1/ref8 (H-lignin dominant).  ELISA based glycome profiling was conducted to study the impact of lignin modification on lignin-carbohydrate complex characteristics by employing a toolkit of 155 monoclonal antibodies.  Furthermore, we sought to understand how does lignin modification affect the biomass recalcitrance, substrate reactivity and cellulose accessibility and their correlations with saccharification efficiency under a mild [C₂mim][OAc] ionic liquid (IL) pretreatment. Size exclusion chromatography, pyro-GC/MS, ³¹P, 2D HSQC NMR techniques revealed distinct chromatographic and spectroscopic patterns, reflecting the variations of lignin monomer composition in raw biomass and the different mode of lignin dissolution and depolymerization during IL pretreatment.  Results also show that the cleavages of β-O-4, 5–5’, and β-5 linkages in H- or S- lignin dominant mutant were greater than those of G-lignin dominant mutants. Furthermore, density functional theory (DFT) based calculations indicate higher chemical reactivity of the linkages between H- and S-lignin monomers, a possible cause of the reduced recalcitrance of H- or S- lignin dominant mutants.