Identification and Characterization of Four Missense Mutations in Brown midrib 12 (Bmr12), the Caffeic O-Methyltranferase (COMT) of Sorghum
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Modifying lignin content and composition are targets to improve bioenergy crops for cellulosic conversion to biofuels. In sorghum and other C4 grasses, the brown midrib mutants have been shown to reduce lignin content and alter its composition. Bmr12 encodes the sorghum caffeic O-methyltransferase, which catalyzes the penultimate step in monolignol biosynthesis. From an EMS-mutagenized TILLING population, four bmr12 mutants were isolated. DNA sequencing identified the four missense mutations in the Bmr12 coding region, which changed evolutionarily conserved amino acids Ala71Val, Pro150Leu, Gly225Asp, and Gly325Ser. The previously characterized bmr12 mutants all contain premature stop codons. These newly identified mutants, along with the previously characterized bmr12-ref, represent the first allelic series of bmr12 mutants available in the same genetic background. The impacts of these newly identified mutations on protein accumulation, enzyme activity, Klason lignin content, lignin subunit composition, and saccharification yield were determined. Gly225Asp mutant greatly reduced protein accumulation, and Pro150Leu and Gly325Ser greatly impaired enzyme activity compared to wild type (WT). All four mutants significantly reduced Klason lignin content and altered lignin composition resulting in a significantly reduced S/G ratio relative to WT, but the overall impact of these mutations was less severe than bmr12-ref. Except for Gly325Ser, which is a hypomorphic mutant, all mutants increased the saccharification yield relative to WT. These mutants represent new tools to decrease lignin content and S/G ratio, possibly leading toward the ability to tailor lignin content and composition in the bioenergy grass sorghum.
KeywordsBrown midrib bmr12 Caffeic O-methyltransferase (COMT) Lignin Sorghum
We thank Tammy Gries, Nathan Peroutka-Bigus and John Toy for their technical assistance with experimental data presented in this manuscript, Dr. Lisa Durso for the use of her real-time PCR instrument, and Dr. Heather Van Buskirk for critically reviewing the manuscript. This research was supported by National Institute of Food & Agriculture, Grant 2011-67009-30026 (S.E.S, J.F.P), the Office of Science (BER), U.S. Department of Energy, grant DE-FG02-07ER64458 (W.V., S.E.S, J.F.P), and additional funding from USDA-ARS, CRIS project 5440-21220-024-00D (S.E.S, J.F.P).
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