Exploring the function of laccases from Miscanthus sinensis in lignin biosynthesis

Abstract

Miscanthus, a fast-growing C4-grass with high water use efficiency and low fertilizer demand is an attractive source of lignocellulosic biomass for bioeconomy. While the lignin component causes cell wall recalcitrance towards saccharification, it also offers a novel source for aromatic platform chemicals, thus performing as friend or foe depending on biomass use. Consequently, changing amount and/or quality of Miscanthus lignin are important breeding goals. Transcriptional regulation of monolignol biosynthesis genes in Miscanthus has been previously studied. However, monolignol polymerization is as of yet poorly understood. Laccases are phenol-oxidizing enzymes which can convert monolignols into free radicals, leading to the formation of lignin polymers. In plants, a number of laccases have been reported to be involved in lignin polymerization in secondary cell walls and middle lamellas, not only affecting lignin content, but also influencing the composition of lignin monomers. Here, we have explored the link between the lignification process and different laccase isoforms from Miscanthus. A comprehensive analysis has been performed including expression profiling, characterization of recombinant MsLAC1 protein, subcellular localization, activation of the MsLAC1-5 promoters by lignification-promoting R2R3 MYB transcription factors, effective complementation of an Arabidopsis laccase double mutant (lac4-2 lac17), and impact of ectopic expression. MsLAC1, 2 and 5 are co-expressed with the regulators of secondary cell wall biosynthesis in Miscanthus. Those laccases localize to cell walls and are regulated by the Miscanthus lignin-related transcription factors. We also established that recombinant MsLAC1 is able to oxidize monolignol in vitro, confirming the catalytic ability of laccases for lignin polymerization. The semi-dwarf phenotype and collapsed xylem phenotypes of the Arabidopsis lac4-2 lac17 knockout mutant were also complemented by MsLAC1 and MsLAC2. Furthermore, transgenic Arabidopsis plants over-expressing MsLAC1 showed higher G-lignin content, while expressing MsLAC5 in lac4-2 lac17 double mutant significantly increased the H-lignin content in stem. The results provide compelling evidence for the involvement of Miscanthus laccases in monolignol polymerization. Miscanthus laccases are regulated by secondary cell wall MYBs and MsLAC1 is probably involved in lignification of xylem fibers. The role of Miscanthus laccases in orchestrating the lignification process is discussed. Its ability to modify lignin content and composition identifies the Miscanthus laccase genes as promising breeding targets in Miscanthus for biofuel and biomaterial applications.