title: Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  These 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.
authors.affiliation: Biology Department, University of Massachusetts 221 Morrill Science Center III, Amherst, MA 01003, USA.
authors.forename: Gina M
authors.initials: GM
authors.lastname: Trabucco
authors.forename: Dominick A
authors.initials: DA
authors.lastname: Matos
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Lee
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Saathoff
authors.forename: Henry D
authors.initials: HD
authors.lastname: Priest
authors.forename: Todd C
authors.initials: TC
authors.lastname: Mockler
authors.forename: Gautam
authors.initials: G
authors.lastname: Sarath
authors.forename: Samuel P
authors.initials: SP
authors.lastname: Hazen
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D058431:Brachypodium / Q000201:enzymology* / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism; D015536:Down-Regulation; D000431:Ethanol / Q000378:metabolism; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D017343:Genes, Plant; D008031:Lignin / Q000096:biosynthesis; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018547:Plant Stems / Q000737:chemistry / Q000235:genetics; D030821:Plants, Genetically Modified / Q000201:enzymology; D016415:Sequence Alignment; D045868:Sorghum / Q000235:genetics; D019076:Transgenes; D003313:Zea mays / Q000235:genetics
chemical_list: D010940:Plant Proteins; D000431:Ethanol; D008031:Lignin; D000429:Alcohol Oxidoreductases; C018656:cinnamyl alcohol dehydrogenase; D008780:Methyltransferases; C019608:caffeate O-methyltransferase
references.citation: Plant Physiol. 2002 Dec;130(4):1675-85
references.pmid: 12481050
references.citation: Plant J. 2008 May;54(4):569-81
references.pmid: 18476864
references.citation: Phytochemistry. 2004 Feb;65(3):313-21
references.pmid: 14751302
references.citation: Plant Physiol. 2000 Aug;123(4):1363-74
references.pmid: 10938354
references.citation: Plant Cell Rep. 2008 Mar;27(3):471-8
references.pmid: 17999063
references.citation: Curr Biol. 2007 Feb 20;17(4):R115-9
references.pmid: 17307040
references.citation: Plant Biotechnol J. 2003 Nov;1(6):437-49
references.pmid: 17134402
references.citation: Transgenic Res. 2001 Oct;10(5):457-64
references.pmid: 11708655
references.citation: BMC Bioinformatics. 2008 Nov 01;9:465
references.pmid: 18976492
references.citation: Heredity (Edinb). 2002 Jun;88(6):450-7
references.pmid: 12180087
references.citation: Plant Mol Biol. 1999 Feb;39(3):437-47
references.pmid: 10092173
references.citation: J Vis Exp. 2010 Mar 11;(37):
references.pmid: 20224547
references.citation: Plant Physiol. 1994 Jan;104(1):75-84
references.pmid: 12232063
references.citation: Plant Physiol. 1996 Dec;112(4):1479-1490
references.pmid: 12226459
references.citation: Plant Cell Rep. 2012 Jan;31(1):91-101
references.pmid: 21912859
references.citation: PLoS One. 2008 Mar 19;3(3):e1829
references.pmid: 18350165
references.citation: Plant J. 1998 Jun;14(5):545-53
references.pmid: 9675900
references.citation: Phytochemistry. 2002 Oct;61(3):221-94
references.pmid: 12359514
references.citation: Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3803-8
references.pmid: 21321194
references.citation: Nature. 1974 Jul 19;250(463):194-9
references.pmid: 4368490
references.citation: Biotechnol Bioeng. 2006 Apr 20;93(6):1196-206
references.pmid: 16470880
references.citation: Nature. 2010 Feb 11;463(7282):763-8
references.pmid: 20148030
references.citation: Nat Biotechnol. 2008 Feb;26(2):169-72
references.pmid: 18259168
references.citation: Plant Cell. 2001 Jan;13(1):73-88
references.pmid: 11158530
references.citation: Plant Biotechnol J. 2013 Aug;11(6):709-16
references.pmid: 23551338
references.citation: Plant Cell. 1995 Jul;7(7):1001-1013
references.pmid: 12242395
references.citation: Plant Physiol. 1999 Jan;119(1):153-64
references.pmid: 9880356
references.citation: Mol Plant. 2012 Jul;5(4):817-30
references.pmid: 22147756
references.citation: Genetics. 2009 Feb;181(2):783-95
references.pmid: 19087955
references.citation: Plant Physiol. 1998 Jul;117(3):761-70
references.pmid: 9662519
references.citation: J Exp Bot. 2012 Oct;63(17):6223-36
references.pmid: 23028019
references.citation: Funct Plant Biol. 2004 May;31(3):235-245
references.pmid: 32688895
references.citation: Mol Biol Evol. 2011 Oct;28(10):2731-9
references.pmid: 21546353
references.citation: Nat Biotechnol. 2007 Jul;25(7):759-61
references.pmid: 17572667
references.citation: BMC Evol Biol. 2002;2:2
references.pmid: 11835688
references.citation: Plant Cell. 2005 Jul;17(7):2059-76
references.pmid: 15937231
references.citation: Mol Genet Genomics. 2003 May;269(2):205-14
references.pmid: 12756532
references.citation: PLoS One. 2011 Jan 27;6(1):e16416
references.pmid: 21298014
references.citation: Annu Rev Plant Biol. 2009;60:165-82
references.pmid: 19014348
references.citation: Plant Physiol. 2009 Jun;150(2):584-95
references.pmid: 19363091
references.citation: Planta. 2005 Mar;220(5):678-88
references.pmid: 15452707
references.citation: Plant Cell. 1995 Apr;7(4):407-16
references.pmid: 7773015
references.citation: Annu Rev Genet. 2010;44:337-63
references.pmid: 20809799
references.citation: Plant Mol Biol. 1991 Dec;17(6):1203-15
references.pmid: 1932694
references.citation: Planta. 1997 Mar;201(3):311-8
references.pmid: 19343409
references.citation: Curr Opin Plant Biol. 2002 Jun;5(3):224-9
references.pmid: 11960740
references.citation: Curr Opin Plant Biol. 2008 Jun;11(3):278-85
references.pmid: 18434238
references.citation: Methods Mol Biol. 2012;918:341-9
references.pmid: 22893298
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: Plant Physiol. 2011 Sep;157(1):3-13
references.pmid: 21771916
references.citation: J Exp Bot. 2009;60(9):2763-71
references.pmid: 19451187
references.citation: Transgenic Res. 2002 Oct;11(5):495-503
references.pmid: 12437080
references.citation: C R Biol. 2004 Sep-Oct;327(9-10):847-60
references.pmid: 15587076
references.citation: Plant Physiol. 2006 Mar;140(3):972-83
references.pmid: 16443696
references.citation: Plant Cell. 2010 Oct;22(10):3357-73
references.pmid: 20952635
references.citation: Curr Opin Plant Biol. 2008 Jun;11(3):301-7
references.pmid: 18434239
references.citation: Int J Plant Genomics. 2013;2013:423189
references.pmid: 23431288
references.citation: Nature. 2008 May 15;453(7193):379-82
references.pmid: 18480821
references.citation: Plant J. 2013 Feb;73(3):496-508
references.pmid: 23078216
references.citation: Biotechnol Biofuels. 2012 Feb 08;5:5
references.pmid: 22316115
references.citation: Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
references.pmid: 7984417
references.citation: Front Plant Sci. 2012 Apr 23;3:74
references.pmid: 22639662
references.citation: Plant Mol Biol. 1998 Jul;37(4):663-74
references.pmid: 9687070
references.citation: Heredity (Edinb). 1999 Aug;83 ( Pt 2):171-8
references.pmid: 10469205
references.citation: Org Biomol Chem. 2006 May 7;4(9):1687-97
references.pmid: 16633561