Functional characterization of the switchgrass (Panicum virgatum) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks.

• The major obstacle for bioenergy production from switchgrass biomass is the low saccharification efficiency caused by cell wall recalcitrance. Saccharification efficiency is negatively correlated with both lignin content and cell wall ester-linked p-coumarate: ferulate (p-CA : FA) ratio. In this study, we cloned and functionally characterized an R2R3-MYB transcription factor from switchgrass and evaluated its potential for developing lignocellulosic feedstocks. • The switchgrass PvMYB4 cDNAs were cloned and expressed in Escherichia coli, yeast, tobacco and switchgrass for functional characterization. Analyses included determination of phylogenetic relations, in situ hybridization, electrophoretic mobility shift assays to determine binding sites in target promoters, and protoplast transactivation assays to demonstrate domains active on target promoters. • PvMYB4 binds to the AC-I, AC-II and AC-III elements of monolignol pathway genes and down-regulates these genes in vivo. Ectopic overexpression of PvMYB4 in transgenic switchgrass resulted in reduced lignin content and ester-linked p-CA : FA ratio, reduced plant stature, increased tillering and an approx. threefold increase in sugar release efficiency from cell wall residues. • We describe an alternative strategy for reducing recalcitrance in switchgrass by manipulating the expression of a key transcription factor instead of a lignin biosynthetic gene. PvMYB4-OX transgenic switchgrass lines can be used as potential germplasm for improvement of lignocellulosic feedstocks and provide a platform for further understanding gene regulatory networks underlying switchgrass cell wall recalcitrance.

[1]  Jerome L. Myers,et al.  INTEGRATED ANALYSIS I , 2013 .

[2]  R. Dixon,et al.  Selective lignin downregulation leads to constitutive defense response expression in alfalfa (Medicago sativa L.). , 2011, The New phytologist.

[3]  R. Dixon,et al.  Transcriptional networks for lignin biosynthesis: more complex than we thought? , 2011, Trends in plant science.

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

[5]  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.

[6]  R. Dixon,et al.  Mutation of WRKY transcription factors initiates pith secondary wall formation and increases stem biomass in dicotyledonous plants , 2010, Proceedings of the National Academy of Sciences.

[7]  E. Grotewold,et al.  ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux. , 2010, The Plant journal : for cell and molecular biology.

[8]  R. Zhong,et al.  Evolutionary conservation of the transcriptional network regulating secondary cell wall biosynthesis. , 2010, Trends in plant science.

[9]  R. Dixon,et al.  Distinct cinnamoyl CoA reductases involved in parallel routes to lignin in Medicago truncatula , 2010, Proceedings of the National Academy of Sciences.

[10]  Mark E. Borsuk,et al.  Biomass Production in Switchgrass across the United States: Database Description and Determinants of Yield , 2010 .

[11]  R. Wu,et al.  Complete Switchgrass Genetic Maps Reveal Subgenome Collinearity, Preferential Pairing and Multilocus Interactions , 2010, Genetics.

[12]  Xu Li,et al.  The Growth Reduction Associated with Repressed Lignin Biosynthesis in Arabidopsis thaliana Is Independent of Flavonoids[C] , 2010, Plant Cell.

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

[14]  R. Dixon,et al.  A Bioinformatic Analysis of NAC Genes for Plant Cell Wall Development in Relation to Lignocellulosic Bioenergy Production , 2009, BioEnergy Research.

[15]  G. Heath,et al.  Environmental and sustainability factors associated with next-generation biofuels in the U.S.: what do we really know? , 2009, Environmental science & technology.

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

[17]  P. Puigdoménech,et al.  The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana , 2009, Plant Molecular Biology.

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

[19]  R. Zhong,et al.  MYB58 and MYB63 Are Transcriptional Activators of the Lignin Biosynthetic Pathway during Secondary Cell Wall Formation in Arabidopsis[C][W] , 2009, The Plant Cell Online.

[20]  Jasmyn Pangilinan,et al.  Comparative Genomics in Switchgrass Using 61,585 High‐Quality Expressed Sequence Tags , 2008 .

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

[22]  J. Grima-Pettenati,et al.  Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis , 2008, Journal of experimental botany.

[23]  M. Somleva,et al.  Production of polyhydroxybutyrate in switchgrass, a value-added co-product in an important lignocellulosic biomass crop. , 2008, Plant biotechnology journal.

[24]  M. Ohme-Takagi,et al.  AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. , 2008, The Plant journal : for cell and molecular biology.

[25]  R. Dixon,et al.  Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa): effects on lignin composition in specific cell types. , 2008, The New phytologist.

[26]  Markus Pauly,et al.  Cell-wall carbohydrates and their modification as a resource for biofuels. , 2008, The Plant journal : for cell and molecular biology.

[27]  Jean-Michel Claverie,et al.  Phylogeny.fr: robust phylogenetic analysis for the non-specialist , 2008, Nucleic Acids Res..

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

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

[30]  E. Blancaflor,et al.  Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2. , 2007, The New phytologist.

[31]  Jinfeng Zhao,et al.  SAD2, an Importin β-Like Protein, Is Required for UV-B Response in Arabidopsis by Mediating MYB4 Nuclear Trafficking[W] , 2007, The Plant Cell Online.

[32]  J. Grima-Pettenati,et al.  Molecular characterization of EgMYB1, a putative transcriptional repressor of the lignin biosynthetic pathway , 2007 .

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

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

[35]  Christian Mazza,et al.  Statistical significance of quantitative PCR , 2007, BMC Bioinformatics.

[36]  Danny E Akin.  Grass lignocellulose , 2007, Applied biochemistry and biotechnology.

[37]  Nathan M. Springer,et al.  Allelic variation and heterosis in maize: how do two halves make more than a whole? , 2007, Genome research.

[38]  David K. Johnson,et al.  Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production , 2007, Science.

[39]  K. Shinozaki,et al.  NAC Transcription Factors, NST1 and NST3, Are Key Regulators of the Formation of Secondary Walls in Woody Tissues of Arabidopsis[W][OA] , 2007, The Plant Cell Online.

[40]  C. Lapierre,et al.  Flavonoid Accumulation in Arabidopsis Repressed in Lignin Synthesis Affects Auxin Transport and Plant Growth , 2007, The Plant Cell Online.

[41]  C. Hawes,et al.  Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants , 2006, Nature Protocols.

[42]  T. Maes,et al.  Down-regulation of the maize and Arabidopsis thaliana caffeic acid O-methyl-transferase genes by two new maize R2R3-MYB transcription factors , 2006, Plant Molecular Biology.

[43]  H. Fukuda,et al.  A novel R2R3 MYB transcription factor NtMYBJS1 is a methyl jasmonate-dependent regulator of phenylpropanoid-conjugate biosynthesis in tobacco. , 2006, The Plant journal : for cell and molecular biology.

[44]  V. Lauvergeat,et al.  Characterization of a Grapevine R2R3-MYB Transcription Factor That Regulates the Phenylpropanoid Pathway1[W] , 2005, Plant Physiology.

[45]  K. Vogel,et al.  Analysis of expressed sequence tags and the identification of associated short tandem repeats in switchgrass , 2005, Theoretical and Applied Genetics.

[46]  J. Grima-Pettenati,et al.  EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis. , 2005, The Plant journal : for cell and molecular biology.

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

[48]  Addie Nina Olsen,et al.  NAC transcription factors: structurally distinct, functionally diverse. , 2005, Trends in plant science.

[49]  R. Parish,et al.  AtMYB32 is required for normal pollen development in Arabidopsis thaliana. , 2004, The Plant journal : for cell and molecular biology.

[50]  F. Sterky,et al.  MYB transcription factors are differentially expressed and regulated during secondary vascular tissue development in hybrid aspen , 2004, Plant Molecular Biology.

[51]  B. Peterlin,et al.  VP16 and Ubiquitin Binding of P-TEFb via Its Activation Domain and Ubiquitin Facilitates Elongation of Transcription of Target Genes , 2004, Current Biology.

[52]  J. Bell,et al.  Transgenic down-regulation of caffeic acid O-methyltransferase (COMT) led to improved digestibility in tall fescue (Festuca arundinacea). , 2004, Functional plant biology : FPB.

[53]  R. Sederoff,et al.  Characterisation of a pine MYB that regulates lignification. , 2003, The Plant journal : for cell and molecular biology.

[54]  B. Peterlin,et al.  A model of repression: CTD analogs and PIE-1 inhibit transcriptional elongation by P-TEFb. , 2003, Genes & development.

[55]  B. V. Conger,et al.  Agrobacterium‐Mediated Genetic Transformation of Switchgrass , 2002 .

[56]  V. R. Tolbert,et al.  High-value renewable energy from prairie grasses. , 2002, Environmental science & technology.

[57]  F. Ausubel,et al.  MAP kinase signalling cascade in Arabidopsis innate immunity , 2002, Nature.

[58]  J. Sheen.  Signal transduction in maize and Arabidopsis mesophyll protoplasts. , 2001, Plant physiology.

[59]  R. Stracke,et al.  The R2R3-MYB gene family in Arabidopsis thaliana. , 2001, Current opinion in plant biology.

[60]  C. Tonelli,et al.  Transcriptional repression by AtMYB4 controls production of UV‐protecting sunscreens in Arabidopsis , 2000, The EMBO journal.

[61]  J. Paz-Ares,et al.  More than 80R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[62]  Cathie Martin,et al.  The AmMYB308 and AmMYB330 Transcription Factors from Antirrhinum Regulate Phenylpropanoid and Lignin Biosynthesis in Transgenic Tobacco , 1998, Plant Cell.

[63]  K. Okada,et al.  The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl. , 1997, Genes & development.

[64]  M. Bevan,et al.  Two classes of cis sequences contribute to tissue-specific expression of a PAL2 promoter in transgenic tobacco. , 1995, The Plant journal : for cell and molecular biology.

[65]  G. Blissard,et al.  Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein , 1995, Journal of virology.

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

[67]  Yi Li,et al.  Generating yeast transcriptional activators containing no yeast protein sequences , 1991, Nature.

[68]  R. Dixon,et al.  Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[69]  Fred Winston,et al.  Methods in Yeast Genetics: A Laboratory Course Manual , 1990 .

[70]  Jun Ma,et al.  GAL4-VP16 is an unusually potent transcriptional activator , 1988, Nature.

[71]  J. Fry,et al.  A simple and general method for transferring genes into plants. , 1985, Science.

[72]  R. Dixon,et al.  Switchgrass (Panicum virgatum) possesses a divergent family of cinnamoyl CoA reductases with distinct biochemical properties. , 2010, The New phytologist.

[73]  F. Delsuc.  Comparative Genomics , 2010, Lecture Notes in Computer Science.

[74]  Y. Ge,et al.  Genetic transformation of switchgrass. , 2009, Methods in molecular biology.

[75]  Johan Trygg,et al.  Integrated analysis of transcript, protein and metabolite data to study lignin biosynthesis in hybrid aspen. , 2009, Journal of proteome research.

[76]  Jules Janick,et al.  Biomass yield stability of switchgrass cultivars. , 2002 .

[77]  J. Janick,et al.  Trends in new crops and new uses , 2002 .

[78]  Sarah J. Gurr,et al.  Molecular plant pathology : a practical approach , 1992 .