Downregulation of Cinnamyl Alcohol Dehydrogenase (CAD) Leads to Improved Saccharification Efficiency in Switchgrass

The bioconversion of carbohydrates in the herbaceous bioenergy crop, switchgrass (Panicum virgatum L.), is limited by the associated lignins in the biomass. The cinnamyl alcohol dehydrogenase (CAD) gene encodes a key enzyme which catalyzes the last step of lignin monomer biosynthesis. Transgenic switchgrass plants were produced with a CAD RNAi gene construct under the control of the maize ubiquitin promoter. The transgenic lines showed reduced CAD expression levels, reduced enzyme activities, reduced lignin content, and altered lignin composition. The modification of lignin biosynthesis resulted in improved sugar release and forage digestibility. Significant increases of saccharification efficiency were obtained in most of the transgenic lines with or without acid pretreatment. A negative correlation between lignin content and sugar release was found among these transgenic switchgrass lines. The transgenic materials have the potential to allow for improved efficiency of cellulosic ethanol production.

[1]  Michael Ladisch,et al.  Loosening lignin's grip on biofuel production , 2007, Nature Biotechnology.

[2]  Robert B. Mitchell,et al.  Chemical composition and response to dilute-acid pretreatment and enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass , 2006 .

[3]  M. Campbell,et al.  The genetic control of lignin deposition during plant growth and development. , 2004, The New phytologist.

[4]  Scott E. Sattler,et al.  A Nonsense Mutation in a Cinnamyl Alcohol Dehydrogenase Gene Is Responsible for the Sorghum brown midrib6 Phenotype1[W][OA] , 2009, Plant Physiology.

[5]  Q. Qian,et al.  GOLD HULL AND INTERNODE2 Encodes a Primarily Multifunctional Cinnamyl-Alcohol Dehydrogenase in Rice1 , 2006, Plant Physiology.

[6]  K. Moore,et al.  Describing and Quantifying Growth Stages of Perennial Forage Grasses , 1991 .

[7]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[8]  W. Boerjan,et al.  Field and pulping performances of transgenic trees with altered lignification , 2002, Nature Biotechnology.

[9]  Mariam B. Sticklen,et al.  Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol , 2010, Nature Reviews Genetics.

[10]  Cathie Martin,et al.  Engineering plants with increased levels of the antioxidant chlorogenic acid , 2004, Nature Biotechnology.

[11]  Y. Ge,et al.  Recent advances in genetic transformation of forage and turf grasses , 2006, In Vitro Cellular & Developmental Biology - Plant.

[12]  Richard A Dixon,et al.  Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa (Medicago sativa L.). , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[13]  T. K. Ghose Measurement of cellulase activities , 1987 .

[14]  Richard A Dixon,et al.  Lignin modification improves fermentable sugar yields for biofuel production , 2007, Nature Biotechnology.

[15]  S. Sattler,et al.  Switchgrass Contains Two Cinnamyl Alcohol Dehydrogenases Involved in Lignin Formation , 2011, BioEnergy Research.

[16]  J. Ralph,et al.  Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: some cautionary notes. , 1999, Journal of agricultural and food chemistry.

[17]  A. Boudet,et al.  Impact of different levels of cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenic tobacco plants , 1997, Planta.

[18]  M. Ferrarese,et al.  High performance liquid chromatography method for the determination of cinnamyl alcohol dehydrogenase activity in soybean roots. , 2006, Plant physiology and biochemistry : PPB.

[19]  A. Cornu,et al.  Cell Wall Degradability of Transgenic Tobacco Stems in Relation to Their Chemical Extraction and Lignin Quality , 1996 .

[20]  Deborah Goffner,et al.  Lignins and lignocellulosics: a better control of synthesis for new and improved uses. , 2003, Trends in plant science.

[21]  Xirong Xiao,et al.  Developmental Control of Lignification in Stems of Lowland Switchgrass Variety Alamo and the Effects on Saccharification Efficiency , 2009, BioEnergy Research.

[22]  Lloyd W Sumner,et al.  Quantification of saponins in aerial and subterranean tissues of Medicago truncatula. , 2005, Journal of agricultural and food chemistry.

[23]  Sudhir Kumar,et al.  MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment , 2004, Briefings Bioinform..

[24]  H. Hisano,et al.  Genetic modification of lignin biosynthesis for improved biofuel production , 2009, In Vitro Cellular & Developmental Biology - Plant.

[25]  F. Smith,et al.  COLORIMETRIC METHOD FOR DETER-MINATION OF SUGAR AND RELATED SUBSTANCE , 1956 .

[26]  J. Oszmiański,et al.  Lignin deficiency in transgenic flax resulted in plants with improved mechanical properties. , 2007, Journal of biotechnology.

[27]  R. Dixon,et al.  Improving Saccharification Efficiency of Alfalfa Stems Through Modification of the Terminal Stages of Monolignol Biosynthesis , 2008, BioEnergy Research.

[28]  S. Sattler,et al.  Genetic background impacts soluble and cell wall-bound aromatics in brown midrib mutants of sorghum , 2008, Planta.

[29]  C. Halpin,et al.  Effect of down-regulation of cinnamyl alcohol dehydrogenase on cell wall composition and on degradability of tobacco stems , 1998 .

[30]  K. Shimamoto,et al.  Simple RNAi vectors for stable and transient suppression of gene function in rice. , 2004, Plant & cell physiology.

[31]  Marc Van Montagu,et al.  Biosynthesis and genetic engineering of lignin , 1998 .

[32]  Pollet,et al.  Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O-methyltransferase activity have an opposite impact on the efficiency of industrial kraft pulping , 1999, Plant physiology.

[33]  J. Bouton Molecular breeding of switchgrass for use as a biofuel crop. , 2007, Current opinion in genetics & development.

[34]  Deepak R. Keshwani,et al.  Switchgrass for bioethanol and other value-added applications: a review. , 2009, Bioresource technology.

[35]  L. A. Kszos,et al.  Development of switchgrass (Panicum virgatum) as a bioenergy feedstock in the United States. , 2005 .

[36]  R. Perrin,et al.  Net energy of cellulosic ethanol from switchgrass , 2008, Proceedings of the National Academy of Sciences.

[37]  Zengyu Wang,et al.  Lignin deposition and associated changes in anatomy, enzyme activity, gene expression, and ruminal degradability in stems of tall fescue at different developmental stages. , 2002, Journal of agricultural and food chemistry.

[38]  M. Rogers,et al.  A real-time PCR assay to estimate Leishmania chagasi load in its natural sand fly vector Lutzomyia longipalpis , 2008, Transactions of the Royal Society of Tropical Medicine and Hygiene.

[39]  K. Edwards,et al.  Brown-midrib maize (bm1)--a mutation affecting the cinnamyl alcohol dehydrogenase gene. , 1998, The Plant journal : for cell and molecular biology.

[40]  Yaxin Ge,et al.  Agrobacterium-Mediated Transformation of Switchgrass and Inheritance of the Transgenes , 2009, BioEnergy Research.

[41]  D. Inzé,et al.  Red Xylem and Higher Lignin Extractability by Down-Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar , 1996, Plant physiology.

[42]  W. Vermerris,et al.  A Genomewide Analysis of the Cinnamyl Alcohol Dehydrogenase Family in Sorghum [Sorghum bicolor (L.) Moench] Identifies SbCAD2 as the Brown midrib6 Gene , 2009, Genetics.

[43]  J. Gominho,et al.  Down regulation of Cinnamyl Alcohol Dehydrogenase, a lignification enzyme, in Eucalyptus camaldulensis , 2003, Molecular Breeding.

[44]  Richard A Dixon,et al.  Improved forage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase. , 2003, Plant biotechnology journal.

[45]  A. Moorman,et al.  Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data , 2003, Neuroscience Letters.

[46]  C. Lapierre,et al.  New insights into the molecular architecture of hardwood lignins by chemical degradative methods , 1995 .

[47]  Brigitte Chabbert,et al.  Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility , 1999, Plant Molecular Biology.

[48]  D. Bedgar,et al.  Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[49]  John Ralph,et al.  Effects of Coumarate 3-Hydroxylase Down-regulation on Lignin Structure* , 2006, Journal of Biological Chemistry.

[50]  Gautam Sarath,et al.  Internode structure and cell wall composition in maturing tillers of switchgrass (Panicum virgatum. L). , 2007, Bioresource technology.