RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass.

Sugarcane is a prime bioethanol feedstock. Currently, sugarcane ethanol is produced through fermentation of the sucrose, which can easily be extracted from stem internodes. Processes for production of biofuels from the abundant lignocellulosic sugarcane residues will boost the ethanol output from sugarcane per land area. However, unlocking the vast amount of chemical energy stored in plant cell walls remains expensive primarily because of the intrinsic recalcitrance of lignocellulosic biomass. We report here the successful reduction in lignification in sugarcane by RNA interference, despite the complex and highly polyploid genome of this interspecific hybrid. Down-regulation of the sugarcane caffeic acid O-methyltransferase (COMT) gene by 67% to 97% reduced the lignin content by 3.9% to 13.7%, respectively. The syringyl/guaiacyl ratio in the lignin was reduced from 1.47 in the wild type to values ranging between 1.27 and 0.79. The yields of directly fermentable glucose from lignocellulosic biomass increased up to 29% without pretreatment. After dilute acid pretreatment, the fermentable glucose yield increased up to 34%. These observations demonstrate that a moderate reduction in lignin (3.9% to 8.4%) can reduce the recalcitrance of sugarcane biomass without compromising plant performance under controlled environmental conditions.

[1]  M. Gallo-meagher,et al.  Effect of various growth regulators on shoot regeneration of sugarcane , 2001, In Vitro Cellular & Developmental Biology - Plant.

[2]  M. Bowman,et al.  Structure-Function Analyses of a Caffeic Acid O-Methyltransferase from Perennial Ryegrass Reveal the Molecular Basis for Substrate Preference[W][OA] , 2010, Plant Cell.

[3]  A. Campalans,et al.  Cellular and subcellular localization of the lignin biosynthetic enzymes caffeic acid-O-methyltransferase, cinnamyl alcohol dehydrogenase and cinnamoyl-coenzyme A reductase in two monocots, sugarcane and maize , 2003 .

[4]  C. Pikaard,et al.  Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations. , 2003, The Plant journal : for cell and molecular biology.

[5]  Peng Gao,et al.  Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom , 2009, BMC Bioinformatics.

[6]  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.

[7]  T. Klopfenstein,et al.  Comparative Effects of the Sorghum bmr -6 and bmr -12 Genes: I. Forage Sorghum Yield and Quality , 2005 .

[8]  T. Tew,et al.  Genetic Improvement of Sugarcane (Saccharum spp.) as an Energy Crop , 2008 .

[9]  Y. Barrière,et al.  Down-Regulation of Caffeic Acid O-Methyltransferase in Maize Revisited Using a Transgenic Approach1 , 2002, Plant Physiology.

[10]  R. Dixon,et al.  Stress-Induced Phenylpropanoid Metabolism. , 1995, The Plant cell.

[11]  Xu Li,et al.  Emerging strategies of lignin engineering and degradation for cellulosic biofuel production. , 2008, Current opinion in biotechnology.

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

[13]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[14]  J. Ralph,et al.  Syntheses of lignin-derived thioacidolysis monomers and their uses as quantitation standards. , 2012, Journal of agricultural and food chemistry.

[15]  R. Furbank,et al.  C4 plants as biofuel feedstocks: optimising biomass production and feedstock quality from a lignocellulosic perspective. , 2011, Journal of integrative plant biology.

[16]  J. Bouton,et al.  Improvement of Switchgrass as a Bioenergy Crop , 2008 .

[17]  G. C. Marten,et al.  Effect of the Brown Midrib-Allele on Maize Silage Quality and Yield 1 , 1983 .

[18]  N. Terashima,et al.  Formation and structure of lignin in monocotyledons. III. Heterogeneity of sugarcane (saccharum officinarum L.) Lignin with respect to the composition of structural units in different morphological regions , 1990 .

[19]  J. Saddler,et al.  Substrate and Enzyme Characteristics that Limit Cellulose Hydrolysis , 1999, Biotechnology progress.

[20]  N. Mosier,et al.  Current Technologies for Fuel Ethanol Production from Lignocellulosic Plant Biomass , 2008 .

[21]  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.

[22]  Prakash Lakshmanan,et al.  Sugarcane biotechnology: The challenges and opportunities , 2005, In Vitro Cellular & Developmental Biology - Plant.

[23]  Jian-Min Zhou,et al.  Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling and site-directed mutagenesis , 2010, BMC Plant Biology.

[24]  Chris Somerville,et al.  Feedstocks for Lignocellulosic Biofuels , 2010, Science.

[25]  R. Dixon,et al.  Silencing of 4-coumarate:coenzyme A ligase in switchgrass leads to reduced lignin content and improved fermentable sugar yields for biofuel production. , 2011, The New phytologist.

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

[27]  Jianping Xu,et al.  Generation of large numbers of independently transformed fertile perennial ryegrass (Lolium perenne L.) plants of forage- and turf-type cultivars , 2000, Molecular Breeding.

[28]  K. Shimamoto,et al.  RNA Silencing of Single and Multiple Members in a Gene Family of Rice1[w] , 2005, Plant Physiology.

[29]  R. Dixon,et al.  Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa: Impacts on Lignin Structure and Implications for the Biosynthesis of G and S Lignin , 2001, Plant Cell.

[30]  Wei E Huang,et al.  When single cell technology meets omics, the new toolbox of analytical biotechnology is emerging. , 2012, Current opinion in biotechnology.

[31]  B. Dien,et al.  Downregulation of Cinnamyl-Alcohol Dehydrogenase in Switchgrass by RNA Silencing Results in Enhanced Glucose Release after Cellulase Treatment , 2011, PloS one.

[32]  B. Meyers,et al.  Construction of small RNA cDNA libraries for deep sequencing. , 2007, Methods.

[33]  J. Seemann,et al.  An efficient method for isolation of RNA from tissue cultured plant cells. , 1991, Nucleic acids research.

[34]  C. Wyman,et al.  Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose , 2004, Biotechnology and bioengineering.

[35]  M. Davey,et al.  Genetic Transformation – Biolistics , 2010 .

[36]  Mukesh Jain,et al.  Agronomic Evaluation of Sugarcane Lines Transformed for Resistance to Sugarcane mosaic virus Strain E , 2005 .

[37]  J. Rigau,et al.  Molecular cloning of cDNAs coding for three sugarcane enzymes involved in lignification , 1999 .

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

[39]  W. F. Thompson,et al.  Rapid isolation of high molecular weight plant DNA. , 1980, Nucleic acids research.

[40]  C. Chapple,et al.  The genetics of lignin biosynthesis: connecting genotype to phenotype. , 2010, Annual review of genetics.

[41]  T. Umezawa,et al.  Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[44]  R. Dixon,et al.  Chapter two Structural, functional, and evolutionary basis for methylation of plant small molecules , 2003 .

[45]  C. Felby,et al.  Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities , 2007 .

[46]  R. Birch,et al.  RNAi Mediated Down-Regulation of PDS Gene Expression in Sugarcane (Saccharum), a Highly Polyploid Crop , 2009, Tropical Plant Biology.

[47]  F. Altpeter,et al.  Comparison of direct and indirect embryogenesis protocols, biolistic gene transfer and selection parameters for efficient genetic transformation of sugarcane , 2012, Plant Cell, Tissue and Organ Culture (PCTOC).

[48]  Gaëtan Droc,et al.  High homologous gene conservation despite extreme autopolyploid redundancy in sugarcane. , 2011, The New phytologist.

[49]  Andrew R. Robinson,et al.  Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling. , 2009, The Plant journal : for cell and molecular biology.

[50]  R. Wing,et al.  Diploid/Polyploid Syntenic Shuttle Mapping and Haplotype-Specific Chromosome Walking Toward a Rust Resistance Gene (Bru1) in Highly Polyploid Sugarcane (2n ∼ 12x ∼ 115) , 2008, Genetics.

[51]  J. Irvine,et al.  Herbicide Resistant Transgenic Sugarcane Plants Containing the bar Gene , 1996 .

[52]  W. Vermerris,et al.  A candidate-gene approach to clone the sorghum Brown midrib gene encoding caffeic acid O-methyltransferase , 2003, Molecular Genetics and Genomics.

[53]  M. R. Hemm,et al.  New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[54]  Michael R. Ladisch,et al.  Molecular breeding to enhance ethanol production from corn and sorghum stover. , 2007 .

[55]  R. Dixon,et al.  Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass , 2011, Proceedings of the National Academy of Sciences.

[56]  W. Vermerris,et al.  Allelic Association, Chemical Characterization and Saccharification Properties of brown midrib Mutants of Sorghum (Sorghum bicolor (L.) Moench) , 2008, BioEnergy Research.

[57]  R. Birch,et al.  Transgenic sugarcane plants via microprojectile bombardment , 1992 .

[58]  C. Wyman,et al.  Features of promising technologies for pretreatment of lignocellulosic biomass. , 2005, Bioresource technology.

[59]  F. Altpeter,et al.  Rapid production of transgenic sugarcane with the introduction of simple loci following biolistic transfer of a minimal expression cassette and direct embryogenesis , 2011, In Vitro Cellular & Developmental Biology - Plant.

[60]  S. Neate,et al.  A single backcross effectively eliminates agronomic and quality alterations caused by somaclonal variation in transgenic barley , 2008 .

[61]  V. Srivastava,et al.  Accelerated production of transgenic wheat (Triticum aestivum L.) plants , 1996, Plant Cell Reports.

[62]  A. Klyosov,et al.  Adsorption of high-purity endo-1,4-β-glucanases from Trichoderma reesei on components of lignocellulosic materials: Cellulose, lignin, and xylan , 1988 .

[63]  M. Cotta,et al.  Improved Sugar Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents , 2009, BioEnergy Research.

[64]  W. Vermerris,et al.  Isolation and Identification of Phenolic Compounds , 2008 .

[65]  M. Chan,et al.  An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens , 1998, Transgenic Research.

[66]  B. Keller,et al.  RNA Interference-Based Gene Silencing as an Efficient Tool for Functional Genomics in Hexaploid Bread Wheat1[W][OA] , 2006, Plant Physiology.

[67]  Stephen P. Long,et al.  Meeting US biofuel goals with less land: the potential of Miscanthus , 2008 .

[68]  P. Moore,et al.  Sugarcane for bioenergy production: an assessment of yield and regulation of sucrose content. , 2010, Plant biotechnology journal.

[69]  R. S. Simpson,et al.  Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane , 2007, Plant Molecular Biology Reporter.