Range of cell-wall alterations enhance saccharification in Brachypodium distachyon mutants

Significance Bioethanol produced from waste biomass from crops has the potential to provide a sustainable alternative to petroleum-based transportation fuel that does not compete with human food supply. The main obstacle to this approach is the resistance of this biomass to digestion. Thus, expensive energetic pretreatment and high enzyme inputs are needed to increase digestion. In this study, we screened a population of randomly mutated plants for digestibility with the aim of identifying novel factors that impact on this trait. We found a number of mutants with high digestibility and no impairments in growth or fitness. These mutants show a range of alterations in cell-wall composition, and we have mapped and characterized the mutant with the highest increase in digestibility. Lignocellulosic plant biomass is an attractive feedstock for the production of sustainable biofuels, but the commercialization of such products is hampered by the high costs of processing this material into fermentable sugars (saccharification). One approach to lowering these costs is to produce crops with cell walls that are more susceptible to hydrolysis to reduce preprocessing and enzyme inputs. To deepen our understanding of the molecular genetic basis of lignocellulose recalcitrance, we have screened a mutagenized population of the model grass Brachypodium distachyon for improved saccharification with an industrial polysaccharide-degrading enzyme mixture. From an initial screen of 2,400 M ₂ plants, we selected 12 lines that showed heritable improvements in saccharification, mostly with no significant reduction in plant size or stem strength. Characterization of these putative mutants revealed a variety of alterations in cell-wall components. We have mapped the underlying genetic lesions responsible for increased saccharification using a deep sequencing approach, and here we report the mapping of one of the causal mutations to a narrow region in chromosome 2. The most likely candidate gene in this region encodes a GT61 glycosyltransferase, which has been implicated in arabinoxylan substitution. Our work shows that forward genetic screening provides a powerful route to identify factors that impact on lignocellulose digestibility, with implications for improving feedstock for cellulosic biofuel production.