Inactivation of LACCASE8 and LACCASE5 genes in Brachypodium distachyon leads to severe decrease in lignin content and high increase in saccharification yield without impacting plant integrity

Abstract

Background Dedicated lignocellulosic feedstock from grass crops for biofuel production is extensively increasing. However, the access to fermentable cell wall sugars by carbohydrate degrading enzymes is impeded by lignins. These complex polymers are made from reactive oxidized monolignols in the cell wall. Little is known about the laccase-mediated oxidation of monolignols in grasses, and inactivation of the monolignol polymerization mechanism might be a strategy to increase the yield of fermentable sugars. Results LACCASE5 and LACCASE8 are inactivated in a Brachypodium double mutant. Relative to the wild type, the lignin content of extract-free mature culms is decreased by 20–30% and the saccharification yield is increased by 140%. Release of ferulic acid by mild alkaline hydrolysis is also 2.5-fold higher. Interfascicular fibers are mainly affected while integrity of vascular bundles is not impaired. Interestingly, there is no drastic impact of the double mutation on plant growth. Conclusion This work shows that two Brachypodium laccases with clearly identified orthologs in crops are involved in lignification of this model plant. Lignification in interfascicular fibers and metaxylem cells is partly uncoupled in Brachypodium . Orthologs of these laccases are promising targets for improving grass feedstock for cellulosic biofuel production. Electronic supplementary material The online version of this article (10.1186/s13068-019-1525-5) contains supplementary material, which is available to authorized users.

Substrate Specificity of LACCASE8 Facilitates Polymerization of Caffeyl Alcohol for C-Lignin Biosynthesis in the Seed Coat of Cleome hassleriana

“…Laccases are a large group of multicopper oxidases that are widely distributed in bacterial, fungi, animals, and plants. Recent advances in high-throughput sequencing technology and molecular biology have allowed several laccases that participate in lignin polymerization to be characterized in multiple plant species (Cheng et al, 2019;He et al, 2019;Le Bris et al, 2019;Wang et al, 2019;Simões et al, 2020).…”

“…Like peroxidases, the essential functions of laccases in lignification have been revealed by loss of function approaches, using Arabidopsis, poplar (Populus trichocarpa) and Brachypodium distachyon plants (Berthet et al, 2011;Wang et al, 2015b; Le Bris et al, 2019). The laccase triple mutant (lac4 lac11 lac17) of Arabidopsis showed severe growth defects and lack of lignin in vascular tissues and fibers, but its Casparian strip structure was not affected, suggesting that laccases are essential for lignin polymerization and have non-redundant roles with peroxidases in lignification in vascular tissues (Zhao et al, 2013).…”

LACCASE14 is required for the deposition of guaiacyl lignin and affects cell wall digestibility in poplar

“…S1). Multiple sequence alignment revealed that PtoLAC14 shared high identity with characterized laccase proteins, including AtLAC4 and AtLAC17 from Arabidopsis [24], PtLAC2 and PtLAC3 from poplar [25,26], BdLAC5 from Brachypodium distachyon [32,33], and SofLAC from sugarcane [34] (Additional file 1: Fig. S1, Table S2).…”

Grass secondary cell walls, Brachypodium distachyon as a model for discovery

“…Once oxidised, the monolignols radically polymerise into the branched lignin polymer with multiple bond types resulting from the various positions of the oxygen radical on the monolignol subunit. There are 17 laccases in A. thaliana and 29 in B. distachyon (Berthet et al, 2011;Le Bris et al, 2019). Brachypodium distachyon LACCASE 5 and 8 were identified as orthologues of AtLAC17 and were shown to be responsible for lignification in interfascicular fibres (Wang et al, 2015;Le Bris et al, 2019).…”

“…There are 17 laccases in A. thaliana and 29 in B. distachyon (Berthet et al, 2011;Le Bris et al, 2019). Brachypodium distachyon LACCASE 5 and 8 were identified as orthologues of AtLAC17 and were shown to be responsible for lignification in interfascicular fibres (Wang et al, 2015;Le Bris et al, 2019). A laccase gene from sugarcane (SofLAC) also genetically complemented an A. thaliana lac17 mutant (Cesarino et al, 2013).…”