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BioEnergy Research

, Volume 8, Issue 3, pp 910–921 | Cite as

Field Evaluation of Transgenic Switchgrass Plants Overexpressing PvMYB4 for Reduced Biomass Recalcitrance

  • Holly L. Baxter
  • Charleson R. Poovaiah
  • Kelsey L. Yee
  • Mitra Mazarei
  • Miguel RodriguezJr.
  • Olivia A. Thompson
  • Hui Shen
  • Geoffrey B. Turner
  • Stephen R. Decker
  • Robert W. Sykes
  • Fang Chen
  • Mark F. Davis
  • Jonathan R. Mielenz
  • Brian H. Davison
  • Richard A. Dixon
  • C. Neal StewartJr.
Article

Abstract

High biomass yields and minimal agronomic input requirements have made switchgrass, Panicum virgatum L., a leading candidate lignocellulosic bioenergy crop. Large-scale lignocellulosic biofuel production from such crops is limited by the difficulty to deconstruct cell walls into fermentable sugars: the recalcitrance problem. Our goal in this study was to assess the field performance of switchgrass plants overexpressing the switchgrass MYB4 (PvMYB4) transcription factor gene. PvMYB4 transgenic switchgrass can have great lignin reduction, which commensurately increases sugar release and biofuel production. Our results over two growing seasons showed that one transgenic event (out of eight) had important gains in both biofuel (32 % more) and biomass (63 % more) at the end of the second growing season relative to non-transgenic controls. These gains represent a doubling of biofuel production per hectare, which is the highest gain reported from any field-grown modified feedstock. In contrast to this transgenic event, which had relatively low ectopic overexpression of the transgene, five of the eight transgenic events planted did not survive the first field winter. The dead plants were all high-overexpressing events that performed well in the earlier greenhouse studies. Disease susceptibility was not compromised in any transgenic events over the field experiments. These results demonstrate the power of modifying the expression of an endogenous transcription factor to improve biofuel and biomass simultaneously, and also highlight the importance of field studies for “sorting” transgenic events. Further research is needed to develop strategies for fine-tuning temporal-spatial transgene expression in feedstocks to optimize desired phenotypes.

Keywords

MYB4 Field trial Lignocellulosic biofuel Switchgrass 

Notes

Acknowledgments

We thank Angela Ziebell, Erica Gjersing, Crissa Doeppke, Melvin Tucker, Logan Schuster, Kimberly Mazza, Melissa Glenn, and Kevin Cowley for their assistance with the cell wall characterization. We thank Reggie Millwood for his assistance with the USDA APHIS BRS permitting and adherence to regulations, Joshua Grant for preparing and propagating the plants for field planting, and Ben Wolfe, Marcus Laxton, Johnathan Branson, and the “UT field crew” for the general maintenance and applying fungicide in the field. We thank Arnold Saxton for his assistance with the field design and statistical analyses. This work was supported by funding from the BioEnergy Science Center. The BioEnergy Science Center is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Field research was also supported by University of Tennessee AgResearch and a USDA Hatch grant. 

Supplementary material

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References

  1. 1.
    McLaughlin SB, Walsh ME (1998) Evaluating environmental consequences of producing herbaceous crops for bioenergy. Biomass Bioenergy 14(4):317–324CrossRefGoogle Scholar
  2. 2.
    Schmer MR, Vogel KP, Mitchell RB, Perrin RK (2008) Net energy of cellulosic ethanol from switchgrass. Proc Natl Acad Sci U S A 105(2):464–469PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315(5813):804–807CrossRefPubMedGoogle Scholar
  4. 4.
    Jones L, Ennos AR, Turner SR (2001) Cloning and characterization of irregular xylem4 (irx4): a severely lignin-deficient mutant of Arabidopsis. Plant J 26(2):205–216CrossRefPubMedGoogle Scholar
  5. 5.
    Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 96(6):673–686CrossRefPubMedGoogle Scholar
  6. 6.
    Kumar P, Barrett DM, Delwiche MJ, Stroeve P (2009) Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind Eng Chem Res 48(8):3713–3729CrossRefGoogle Scholar
  7. 7.
    Poovaiah CR, Nageswara-Rao M, Soneji JR, Baxter HL, Stewart CN Jr (2014) Altered lignin biosynthesis using biotechnology to improve lignocellulosic biofuel feedstocks. Plant Biotechnol J. doi: 10.1111/pbi.12225 Google Scholar
  8. 8.
    Shen H, He X, Poovaiah CR, Wuddineh WA, Ma J, Mann DG, Wang H, Jackson L, Tang Y, Stewart CN Jr, Chen F, Dixon RA (2012) Functional characterization of the switchgrass (Panicum virgatum L.) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks. New Phytol 193(1):121–136Google Scholar
  9. 9.
    Shen H, Poovaiah CR, Ziebell A, Tschaplinski TJ, Pattathil S, Gjersing E, Engle NL, Katahira R, Pu Y, Sykes R, Chen F, Ragauskas AJ, Mielenz JR, Hahn MG, Davis M, Stewart CN Jr, Dixon RA (2013) Enhanced characteristics of genetically modified switchgrass (Panicum virgatum L.) for high biofuel production. Biotechnol Biofuels 6(1):71PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Pedersen JF, Vogel KP, Funnell DL (2005) Impact of reduced lignin on plant fitness. Crop Sci 45(3):812–819CrossRefGoogle Scholar
  11. 11.
    Baxter HL, Mazarei M, Labbe N, Kline LM, Cheng Q, Windham MT, Mann DGJ, Chunxiang F, Ziebell A, Sykes RW, Rodriguez M Jr, Davis MF, Mielenz JR, Dixon RA, Wang Z-Y, Stewart CN Jr (2014) Two‐year field analysis of reduced recalcitrance transgenic switchgrass. Plant Biotechnol J 12(7):914–924CrossRefPubMedGoogle Scholar
  12. 12.
    Moore KJ, Moser LE, Vogel KP, Waller SS, Johnson BE, Pedersen JF (1991) Describing and quantifying growth stages of perennial forage grasses. Agronomy J 83:1073–1077CrossRefGoogle Scholar
  13. 13.
    Shen H, Mazarei M, Hisano H, Escamilla-Treviño L, Fu C, Pu C, Rudis MR, Tang Y, Xiao X, Jackson L, Li G, Hernandez T, Chen F, Ragauskas AJ, Stewart CN, Wang Z-Y, Dixon RA (2013) A genomics approach to deciphering lignin biosynthesis in switchgrass. Plant Cell 25(11):4342–4361PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Sykes R, Yung M, Novaes E, Kirst M, Peter G, Davis M (2009) High-throughput screening of plant cell-wall composition using pyrolysis molecular beam mass spectroscopy. Biofuels. Springer, pp 169–183Google Scholar
  15. 15.
    Selig MJ, Tucker MP, Sykes RW, Reichel KL, Brunecky R, Himmel ME, Davis MF, Decker SR (2010) Biomass recalcitrance screening by integrated high throughput hydrothermal pretreatment and enzymatic saccharification. Ind Biotechnol 6:104–111CrossRefGoogle Scholar
  16. 16.
    Decker SR, Carlile M, Selig MJ, Doeppke C, Davis M, Sykes R, Turner G, Ziebell A (2012) Reducing the effect of variable starch levels in biomass recalcitrance screening. In: Himmel M (ed) Biomass conversion: methods and protocols. Springer, New York, pp 181–195CrossRefGoogle Scholar
  17. 17.
    Fu C, Mielenz JR, Xiao X, Ge Y, Hamilton CY, Rodriguez M Jr, Chen F, Foston M, Ragauskas A, Bouton J, Dixon RA, Wang ZY (2011) Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Proc Natl Acad Sci U S A 108(9):3803–3808PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Yee KL, Rodriguez M Jr, Thompson OA, Fu C, Wang Z-Y, Davison BH, Mielenz JR (2014) Consolidated bioprocessing of transgenic switchgrass by an engineered and evolved Clostridium thermocellum strain. Biotechnol Biofuels 7:75PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Mielenz JR, Bardsley JS, Wyman CE (2009) Fermentation of soybean hulls to ethanol while preserving protein value. Bioresour Technol 100:3532–3539CrossRefPubMedGoogle Scholar
  20. 20.
    Raman B, Pan C, Hurst GB, Rodriguez M Jr, McKeown CK, Lankford PK, Samatova NF, Mielenz JR (2009) Impact of pretreated switchgrass and biomass carbohydrates on Clostridium thermocellum ATCC 27405 cellulosome composition: a quantitative proteomic analysis. PLoS One 4(4):e5271PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Yee KL, Rodriguez M Jr, Tschaplinski TJ, Engle NL, Martin MZ, Fu C, Wang Z-Y, Hamilton-Brehm SD, Mielenz JR (2012) Evaluation of the bioconversion of genetically modified switchgrass using simultaneous saccharification and fermentation and a consolidated bioprocessing approach. Biotechnol Biofuels 5:81PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Shen H, Fu C, Xiao X, Ray T, Tang Y, Wang Z, Chen F (2009) Developmental control of lignification in stems of lowland switchgrass variety Alamo and the effects on saccharification efficiency. BioEnergy Res 2:233–245CrossRefGoogle Scholar
  23. 23.
    Casler MD, Buxton DR, Vogel KP (2002) Genetic modification of lignin concentration affects fitness of perennial herbaceous plants. Theor Appl Genet 104(1):127–131CrossRefPubMedGoogle Scholar
  24. 24.
    Fu C, Xiao X, Xi Y, Ge Y, Chen F, Bouton J, Dixon RA, Wang Z-Y (2011) Downregulation of cinnamyl alcohol dehydrogenase (CAD) leads to improved saccharification efficiency in switchgrass. BioEnergy Res 4(3):153–164CrossRefGoogle Scholar
  25. 25.
    Fu C, Sunkar R, Zhou C, Shen H, Zhang J-Y, Matts J, Wolf J, Mann DGJ, Stewart CN, Tang Y, Wang Z-Y (2012) Overexpression of miR156 in switchgrass (Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production. Plant Biotechnol J 10:443–452PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Saathoff AJ, Sarath G, Chow EK, Dien BS, Tobias CM (2011) Downregulation of cinnamyl-alcohol dehydrogenase in switchgrass by RNA silencing results in enhanced glucose release after cellulase treatment. PLoS One 6(1):e16416PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Xu B, Escamilla‐Treviño LL, Sathitsuksanoh N, Shen Z, Shen H, Percival Zhang Y-H, Dixon RA, Zhao B (2011) Silencing of 4‐coumarate: coenzyme A ligase in switchgrass leads to reduced lignin content and improved fermentable sugar yields for biofuel production. New Phytol 192(3):611–625CrossRefPubMedGoogle Scholar
  28. 28.
    Jung JH, Fouad WM, Vermerris W, Gallo M, Altpeter F (2012) RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass. Plant Biotechnol J 10(9):1067–1076CrossRefPubMedGoogle Scholar
  29. 29.
    Bonawitz ND, Chapple C (2013) Can genetic engineering of lignin deposition be accomplished without an unacceptable yield penalty? Curr Opin Biotechnol 24(2):336–343CrossRefPubMedGoogle Scholar
  30. 30.
    Moura JCMS, Bonine CAV, De Oliveira Fernandes Viana J, Dornelas MC, Mazzafera P (2010) Abiotic and biotic stresses and changes in the lignin content and composition in plants. J Integr Plant Biol 52(4):360–376CrossRefPubMedGoogle Scholar
  31. 31.
    Stout AT, Davis AA, Domec J-C, Yang C, Shi R, King JS (2014) Growth under field conditions affects lignin content and productivity in transgenic Populus trichocarpa with altered lignin biosynthesis. Biomass Bioenergy 68:228–239CrossRefGoogle Scholar
  32. 32.
    Jung JH, Vermerris W, Gallo M, Fedenko JR, Erickson JE, Altpeter F (2013) RNA interference suppression of lignin biosynthesis increases fermentable sugar yields for biofuel production from field‐grown sugarcane. Plant Biotechnol J 11(6):709–716CrossRefPubMedGoogle Scholar
  33. 33.
    Tu Y, Rochfort S, Liu Z, Ran Y, Griffith M, Badenhorst P, Louie GV, Bowman ME, Smith KF, Noel JP, Mouradov A, Spangenberg G (2010) Functional analyses of caffeic acid O-methyltransferase and cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne). Plant Cell 22(10):3357–3373PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Chuck GS, Tobias C, Sun L, Kraemer F, Li CL, Dibble D, Arora R, Bragg JN, Vogel JP, Singh S, Simmons BA, Pauly M, Hake S (2011) Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switchgrass. Proc Natl Acad Sci U S A 108(42):17550–17555PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Yang F, Mitra P, Zhang L, Prak L, Verhertbruggen Y, Kim J-S, Sun L, Zheng K, Tang K, Auer M, Scheller HV, Loqué D (2013) Engineering secondary cell wall deposition in plants. Plant Biotechnol J 11(3):325–335PubMedCentralCrossRefPubMedGoogle Scholar
  36. 36.
    Tschaplinski TJ, Standaert RF, Engle NL, Martin MZ, Sangha AK, Parks JM, Smith JC, Samuel R, Jiang N, Pu Y, Ragauskas AJ, Hamilton CY, Fu C, Wang ZY, Davison BH, Dixon RA, Mielenz JR (2012) Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog. Biotechnol Biofuels 5(1):71PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Zhao Q, Dixon RA (2014) Altering the cell wall and its impact on plant disease: from forage to bioenergy. Annu Rev Plant Physiol Plant Mol Biol 52:62–91Google Scholar
  38. 38.
    Kaur H, Shaker K, Heinzel N, Ralph J, Galis I, Baldwin IT (2012) Environmental stresses of field growth allow cinnamyl alcohol dehydrogenase-deficient Nicotiana attenuata plants to compensate for their structural deficiencies. Plant Physiol, pp-112Google Scholar
  39. 39.
    Bi C, Chen F, Jackson L, Gill BS, Li W (2011) Expression of lignin biosynthetic genes in wheat during development and upon infection by fungal pathogens. Plant Mol Biol Rep 29(1):149–161CrossRefGoogle Scholar
  40. 40.
    Uppalapati SR, Serba DD, Ishiga Y, Szabo LJ, Mittal S, Bhandari HS, Bouton JH, Mysore KS, Saha MC (2013) Characterization of the rust fungus, Puccinia emaculata, and evaluation of genetic variability for rust resistance in switchgrass populations. BioEnergy Res 6(2):458–468CrossRefGoogle Scholar
  41. 41.
    Baxter HL, Stewart CN Jr (2013) Effects of altered lignin biosynthesis on phenylpropanoid metabolism and plant stress. Biogeosciences 4:635–650Google Scholar
  42. 42.
    Maury S, Delaunay A, Mesnard F, Cronier D, Chabbert B, Geoffroy P, Legrand M (2010) O-methyltransferase(s)-suppressed plants produce lower amounts of phenolic vir inducers and are less susceptible to Agrobacterium tumefaciens infection. Planta 232:975–986CrossRefPubMedGoogle Scholar
  43. 43.
    Quentin M, Allasia V, Pegard A et al. Imbalanced lignin biosynthesis promotes the sexual reproduction of homothallic oomycete pathogens. PLoS Pathol 5, e1000264Google Scholar
  44. 44.
    Ghazvini H, Tekauz A (2007) Virulence diversity in the population of Bipolaris sorokiniana. Plant Dis 91(7):814–821CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Holly L. Baxter
    • 1
    • 5
  • Charleson R. Poovaiah
    • 1
    • 5
  • Kelsey L. Yee
    • 2
    • 5
  • Mitra Mazarei
    • 1
    • 5
  • Miguel RodriguezJr.
    • 2
    • 5
  • Olivia A. Thompson
    • 2
    • 5
  • Hui Shen
    • 3
    • 5
  • Geoffrey B. Turner
    • 4
    • 5
  • Stephen R. Decker
    • 4
    • 5
  • Robert W. Sykes
    • 4
    • 5
  • Fang Chen
    • 3
    • 5
  • Mark F. Davis
    • 4
    • 5
  • Jonathan R. Mielenz
    • 2
    • 5
  • Brian H. Davison
    • 2
    • 5
  • Richard A. Dixon
    • 3
    • 5
  • C. Neal StewartJr.
    • 1
    • 5
  1. 1.Department of Plant SciencesUniversity of TennesseeKnoxvilleUSA
  2. 2.Biosciences DivisionOak Ridge National LaboratoryOak RidgeUSA
  3. 3.Department of Biological SciencesUniversity of North TexasDentonUSA
  4. 4.National Renewable Energy LaboratoryGoldenUSA
  5. 5.BioEnergy Science Center (BESC)Oak Ridge National LaboratoryOak RidgeUSA

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