title: Study of traits and recalcitrance reduction of field-grownCOMTdown-regulated switchgrass.
abstract: BACKGROUND
  The native recalcitrance of plants hinders the biomass conversion process using current biorefinery techniques. Down-regulation of the caffeic acidO-methyltransferase (COMT) gene in the lignin biosynthesis pathway of switchgrass reduced the thermochemical and biochemical conversion recalcitrance of biomass. Due to potential environmental influences on lignin biosynthesis and deposition, studying the consequences of physicochemical changes in field-grown plants without pretreatment is essential to evaluate the performance of lignin-altered plants. We determined the chemical composition, cellulose crystallinity and the degree of its polymerization, molecular weight of hemicellulose, and cellulose accessibility of cell walls in order to better understand the fundamental features of why biomass is recalcitrant to conversion without pretreatment. The most important is to investigate whether traits and features are stable in the dynamics of field environmental effects over multiple years.
  
  
  RESULTS
  Field-grownCOMTdown-regulated plants maintained both reduced cell wall recalcitrance and lignin content compared with the non-transgenic controls for at least 3 seasons. The transgenic switchgrass yielded 35-84% higher total sugar release (enzymatic digestibility or saccharification) from a 72-h enzymatic hydrolysis without pretreatment and also had a 25-32% increase in enzymatic sugar release after hydrothermal pretreatment. TheCOMT-silenced switchgrass lines had consistently lower lignin content, e.g., 12 and 14% reduction for year 2 and year 3 growing season, respectively, than the control plants. By contrast, the transgenic lines had 7-8% more xylan and galactan contents than the wild-type controls. Gel permeation chromatographic results revealed that the weight-average molecular weights of hemicellulose were 7-11% lower in the transgenic than in the control lines. In addition, we found that silencing ofCOMTin switchgrass led to 20-22% increased cellulose accessibility as measured by the Simons' stain protocol. No significant changes were observed on the arabinan and glucan contents, cellulose crystallinity, and cellulose degree of polymerization between the transgenic and control plants. With the 2-year comparative analysis, both the control and transgenic lines had significant increases in lignin and glucan contents and hemicellulose molecular weight across the growing seasons.
  
  
  CONCLUSIONS
  The down-regulation ofCOMTin switchgrass resulting in a reduced lignin content and biomass recalcitrance is stable in a field-grown trial for at least three seasons. Among the determined affecting factors, the reduced biomass recalcitrance of theCOMT-silenced switchgrass, grown in the field conditions for two and three seasons, was likely related to the decreased lignin content and increased biomass accessibility, whereas the cellulose crystallinity and degree of its polymerization and hemicellulose molecular weights did not contribute to the reduction of recalcitrance significantly. This finding suggests that lignin down-regulation in lignocellulosic feedstock confers improved saccharification that translates from greenhouse to field trial and that lignin content and biomass accessibility are two significant factors for developing a reduced recalcitrance feedstock by genetic modification.
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; BioSciences Division, ORNL, Oak Ridge, TN USA ; UT-ORNL Joint Institute for Biological Sciences, Oak Ridge, TN USA.
authors.forename: Mi
authors.initials: M
authors.lastname: Li
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; BioSciences Division, ORNL, Oak Ridge, TN USA ; UT-ORNL Joint Institute for Biological Sciences, Oak Ridge, TN USA.
authors.forename: Yunqiao
authors.initials: Y
authors.lastname: Pu
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; BioSciences Division, ORNL, Oak Ridge, TN USA ; UT-ORNL Joint Institute for Biological Sciences, Oak Ridge, TN USA.
authors.forename: Chang Geun
authors.initials: CG
authors.lastname: Yoo
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), Golden, CO USA.
authors.forename: Erica
authors.initials: E
authors.lastname: Gjersing
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), Golden, CO USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Decker
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), Golden, CO USA.
authors.forename: Crissa
authors.initials: C
authors.lastname: Doeppke
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory (NREL), Golden, CO USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Shollenberger
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; BioSciences Division, ORNL, Oak Ridge, TN USA ; UT-ORNL Joint Institute for Biological Sciences, Oak Ridge, TN USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Tschaplinski
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; BioSciences Division, ORNL, Oak Ridge, TN USA ; UT-ORNL Joint Institute for Biological Sciences, Oak Ridge, TN USA.
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Engle
authors.affiliation: National Bioenergy Center, NREL, Golden, CO USA.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Sykes
authors.affiliation: National Bioenergy Center, NREL, Golden, CO USA.
authors.forename: Mark F
authors.initials: MF
authors.lastname: Davis
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; Department of Plant Sciences, University of Tennessee, Knoxville, TN USA.
authors.forename: Holly L
authors.initials: HL
authors.lastname: Baxter
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; Department of Plant Sciences, University of Tennessee, Knoxville, TN USA.
authors.forename: Mitra
authors.initials: M
authors.lastname: Mazarei
authors.affiliation: Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK USA.
authors.forename: Chunxiang
authors.initials: C
authors.lastname: Fu
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; BioDiscovery Institute and Department of Biological Sciences, University of North Texas, Denton, TX USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Dixon
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK USA.
authors.forename: Zeng-Yu
authors.initials: ZY
authors.lastname: Wang
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; Department of Plant Sciences, University of Tennessee, Knoxville, TN USA.
authors.forename: C
authors.initials: C
authors.lastname: Neal Stewart
authors.affiliation: BioEnergy Science Center (BESC), Oak Ridge National Laboratory (ORNL), Oak Ridge, TN USA ; BioSciences Division, ORNL, Oak Ridge, TN USA ; UT-ORNL Joint Institute for Biological Sciences, Oak Ridge, TN USA ; Department of Chemical and Biomolecular Engineering & Department of Forestry, Wildlife and Fisheries, University of Tennessee, Knoxville, TN USA.
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Ragauskas
keywords: Biomass recalcitrance; Caffeic acid O-methyltransferase; Cellulose accessibility; Enzymatic hydrolysis; Lignin; Switchgrass