Double knockout of OsWRKY36 and OsWRKY102 boosts lignification with altering culm morphology of rice

Breeding to enrich lignin, a major component of lignocelluloses, in plants contributes to enhanced applications of lignocellulosic biomass into solid biofuels and valuable aromatic chemicals. To collect information on enhancing lignin deposition in grass species, important lignocellulose feedstocks, we generated rice (Oryza sativa) transgenic lines deficient in OsWRKY36 and OsWRKY102, which encode putative transcriptional repressors for secondary cell wall formation. We used CRISPR/Cas9-mediated targeted mutagenesis and closely characterized their altered cell walls using chemical and nuclear magnetic resonance (NMR) methods. Both OsWRKY36 and OsWRKY102 mutations significantly increased lignin content by up to 28 % and 32 %, respectively. Additionally, OsWRKY36/OsWRKY102-double-mutant lines displayed lignin enrichment of cell walls (by up to 41 %) with substantially altered culm morphology over the single-mutant lines as well as the wild-type controls. Our chemical and NMR analyses showed that relative abundances of guaiacyl and p-coumarate units were slightly higher and lower, respectively, in the WRKY mutant lignins compared with those in the wild-type lignins. Our results provide evidence that both OsWRKY36 and OsWRKY102 are associated with repression of rice lignification.

Keywords: CRISPR/Cas9; Grass; Lignin; Lignocellulose; Oryza sativa; OsWRKY102; OsWRKY36.