BioEnergy Research

, Volume 5, Issue 4, pp 855–865

Identification and Characterization of Four Missense Mutations in Brown midrib 12 (Bmr12), the Caffeic O-Methyltranferase (COMT) of Sorghum

  • Scott E. Sattler
  • Nathan A. Palmer
  • Ana Saballos
  • Ann M. Greene
  • Zhanguo Xin
  • Gautam Sarath
  • Wilfred Vermerris
  • Jeffrey F. Pedersen
Article

Abstract

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.

Keywords

Brown midrib bmr12 Caffeic O-methyltransferase (COMT) Lignin Sorghum 

Supplementary material

12155_2012_9197_Fig6_ESM.jpg (25 kb)
Supplementary Fig. 1

Expression of recombinant Bmr12 proteins in E. coli. The recombinant proteins were expressed E. coli cells and purified using an N-terminal 6 His tag. See “Materials and Methods” section for further details. a SDS-PAGE analysis of the proteins prior to purification from the induced E. coli cells Lane 1 molecular weight marker, lane 2 WT Bmr12, lane 3 Ala225Asp. Arrows denote the Bmr12 protein and a truncated form occurring in the recombinant protein Ala225Asp, which corresponds to bmr12-30. b SDS-PAGE analysis of the purified proteins from the induced E. coli cells. Lane 1 molecular weight marker, lane 2 WT Bmr12, lane 3 Gly325Ser, lane 4 Pro150Leu, lane 5 Ala71Val, lane 6 Ala71Val Pro150Leu. The recombinant protein containing Gly325Ser corresponds to bmr12-35. The recombinant protein containing both Ala71Val and Pro150Leu amino acid changes corresponds to bmr12-34/820. The molecular weights of the markers are as follows: 200, 116.25, 97.4, 66.2 45.0, 31.0, and 21.5 kDa (JPEG 24 kb)

12155_2012_9197_MOESM1_ESM.eps (1.8 mb)
High resolution image file (EPS 1,872 kb)
12155_2012_9197_MOESM2_ESM.doc (36 kb)
Supplementary Table 1(DOC 36 kb)

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Copyright information

© Springer Science+Business Media, LLC (outside the USA) 2012

Authors and Affiliations

  • Scott E. Sattler
    • 1
    • 2
  • Nathan A. Palmer
    • 1
    • 2
  • Ana Saballos
    • 3
  • Ann M. Greene
    • 3
  • Zhanguo Xin
    • 4
  • Gautam Sarath
    • 1
    • 2
  • Wilfred Vermerris
    • 3
  • Jeffrey F. Pedersen
    • 1
    • 2
  1. 1.Grain Forage and Bioenergy Research UnitUSDA-ARSLincolnUSA
  2. 2.Department of Agronomy and HorticultureUniversity of Nebraska–LincolnLincolnUSA
  3. 3.Agronomy Department and Genetics InstituteUniversity of FloridaGainesvilleUSA
  4. 4.Plant Stress and Germplasm Development Unit, USDA-ARSLubbockUSA

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