Abstract
BackgroundLignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production.ResultsWe developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants.ConclusionThese results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency.
Highlights
Lignin is a significant barrier in the conversion of plant biomass to bioethanol
Characterization and phylogenetic analysis of BdCAD and BdCOMT gene families Given their importance to monolignol synthesis in other plants, Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) genes with a potential role in secondary cell wall lignification were identified and perturbed in order to evaluate their role in B. distachyon (Figure 1)
Seven putative BdCADs were identified by BLAST search of the B. distachyon genome with the Oryza sativa CAD protein family [55]
Summary
Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. We identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production. The efficiency of existing approaches to generating biofuels can be improved through research into plant feedstock attributes, namely biomass yield and recalcitrance to conversion [4]. The composition and interaction among these three constituents largely dictate the amenability of a plant feedstock for conversion to simple sugars and to biofuels. There has been strong interest in the engineering of bioenergy crops, namely grasses, that are more amenable to feedstock conversion, notably by the manipulation of lignin biosynthesis, in order to improve efficiency of the pretreatment process and obtain maximum fuel yield [8]
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