Abstract

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

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NCBI Reference Sequence

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Acknowledgements

We thank CCRC Analytical Services for glycosyl residue linkage analysis, W. Rottmann for leading the Populus transformation, L. Gunter for validation of Populus constructs, I. Gelineo-Albersheim for submission of BESC transformation pipeline file and, along with K. Hunt, for help in establishing the poplar greenhouse growth conditions, E. Chandler, R. Amos, and K. Engle for preparation of endopolygalacturonase, and B. Rockwell and C. Treager for assistance with cell wall isolation and extraction. We also thank B. Wolfe, M. Laxton, and the UT field staff for assistance with data collection and general field maintenance, and R. Millwood for assistance with the USDA APHIS BRS permit regulations. The work was primarily supported by BioEnergy Science Center grant DE-PS02-06ER64304, and partially by the Center for Bioenergy Innovation. The BioEnergy Science Center and the Center for Bioenergy Innovation are US Department of Energy Bioenergy Research Centers, supported by the Office of Biological and Environmental Research in the Department of Energy's Office of Science. The research was also partially funded by the Department of Energy Center Grant DE-SC0015662. The CCRC series of plant cell wall glycan-directed antibodies were generated with the support from the US National Science Foundation Plant Genome Program (grants DBI-0421683 and IOS-0923992).

Author information

Author notes

    • Yi-Ching Lee
    • , Ji-Yi Zhang
    • , Hema Ramanna
    • , Sivakumar Pattathil
    • , Robert W Sykes
    •  & Geoffrey B Turner

    Current addresses: National Synchrotron Radiation Research Center, Hsinchu Science Park, Hsinchu City, Taiwan (Y.-C.L.); Bayer CropScience Division, Research Triangle Park, North Carolina, USA (J.-Y.Z.); H-IVs-204, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India (H.R.); Mascoma LLC (Lallemand Inc.), Lebanon, New Hampshire, USA (S.P.); Nu Mark LLC, Richmond, Virginia, USA (G.B.T.).

Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.

    • Ajaya K Biswal
    • , Melani A Atmodjo
    • , Li Tan
    •  & Debra Mohnen
  2. Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA.

    • Ajaya K Biswal
    • , Melani A Atmodjo
    • , Ian M Black
    • , Sivakumar Pattathil
    • , Sushree S Mohanty
    • , David Ryno
    • , Li Tan
    • , Michael G Hahn
    •  & Debra Mohnen
  3. DOE-BioEnergy Science Center (BESC), Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.

    • Ajaya K Biswal
    • , Melani A Atmodjo
    • , Mi Li
    • , Holly L Baxter
    • , Chang Geun Yoo
    • , Yunqiao Pu
    • , Yi-Ching Lee
    • , Mitra Mazarei
    • , Ji-Yi Zhang
    • , Hema Ramanna
    • , Adam L Bray
    • , Zachary R King
    • , Peter R LaFayette
    • , Sivakumar Pattathil
    • , Bryon S Donohoe
    • , Sushree S Mohanty
    • , David Ryno
    • , Kelsey Yee
    • , Olivia A Thompson
    • , Miguel Rodriguez Jr.
    • , Alexandru Dumitrache
    • , Jace Natzke
    • , Kim Winkeler
    • , Cassandra Collins
    • , Xiaohan Yang
    • , Li Tan
    • , Robert W Sykes
    • , Erica L Gjersing
    • , Angela Ziebell
    • , Geoffrey B Turner
    • , Stephen R Decker
    • , Michael G Hahn
    • , Brian H Davison
    • , Michael K Udvardi
    • , Jonathan R Mielenz
    • , Mark F Davis
    • , Richard S Nelson
    • , Wayne A Parrott
    • , Arthur J Ragauskas
    • , C Neal Stewart Jr
    •  & Debra Mohnen
  4. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.

    • Mi Li
    • , Chang Geun Yoo
    • , Yunqiao Pu
    • , Kelsey Yee
    • , Olivia A Thompson
    • , Miguel Rodriguez Jr.
    • , Alexandru Dumitrache
    • , Jace Natzke
    • , Xiaohan Yang
    • , Brian H Davison
    • , Jonathan R Mielenz
    •  & Arthur J Ragauskas
  5. UT-ORNL Joint Institute for Biological Sciences, Oak Ridge, Tennessee, USA.

    • Mi Li
    • , Chang Geun Yoo
    • , Yunqiao Pu
    •  & Arthur J Ragauskas
  6. Department of Plant Sciences, University of Tennessee, Knoxville, Tennessee, USA.

    • Holly L Baxter
    • , Mitra Mazarei
    •  & C Neal Stewart Jr
  7. Noble Research Institute, Ardmore, Oklahoma, USA.

    • Yi-Ching Lee
    • , Ji-Yi Zhang
    • , Hema Ramanna
    • , Michael K Udvardi
    •  & Richard S Nelson
  8. Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, Georgia, USA.

    • Adam L Bray
    • , Zachary R King
    • , Peter R LaFayette
    •  & Wayne A Parrott
  9. National Renewable Energy Laboratory, Golden, Colorado, USA.

    • Bryon S Donohoe
    • , Robert W Sykes
    • , Erica L Gjersing
    • , Angela Ziebell
    • , Geoffrey B Turner
    • , Stephen R Decker
    •  & Mark F Davis
  10. ArborGen, Inc., Ridgeville, South Carolina, USA.

    • Kim Winkeler
    •  & Cassandra Collins
  11. Department of Plant Biology, University of Georgia, Athens, Georgia, USA.

    • Michael G Hahn
  12. Department of Chemical and Biomolecular Engineering & Department of Forestry, Wildlife, and Fisheries, University of Tennessee, Knoxville, Tennessee, USA.

    • Arthur J Ragauskas

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Contributions

A.K.B. participated in all aspects of the study, including line selection, plant phenotyping, expression study, tissue handling and distribution, and cell wall analysis, and wrote the manuscript. M.A.A. performed molecular cloning and generated heterologous expression constructs for poplar and switchgrass genes; designed and performed heterologous expression and enzymatic activity assays; and wrote the manuscript. M.L., C.G.Y., and Y.P. performed the cellulose analyses. H.L.B., M.M., and C.N.S. carried out and analyzed the switchgrass field study. Y.-C.L., J.-Y.Z., and H.R. carried out molecular cloning and production of RNAi plasmid for switchgrass and rice. I.M.B. performed some of the cell wall analyses. A.L.B., Z.R.K., and P.R.L. performed switchgrass and rice transformations and propagated transgenic plants. S.P. and M.G.H. performed and analyzed the glycome profiling analysis. B.S.D. performed the stereomicrograph measurement of switchgrass biomass water uptake. S.S.M. and D.R. participated in growth, sampling and analysis of the plants. K.Y., O.A.T., M.R., A.D., and J.N. carried out the ethanol fermentation analyses. K.W. carried out molecular cloning and production of the RNAi plasmid for poplar. C.C. performed Populus transformation and propagated transgenic plants. X.Y. contributed bioinformatic information for construction of poplar gene constructs. L.T. performed molecular cloning and produced heterologous expression construct of Arabidopsis gene. R.W.S. conducted high-throughput pyrolysis molecular beam mass spectrometry (py-MBMS) lignin assays. E.L.G. and A.Z. coordinated analysis of samples through the BioEnergy Science Center (BESC) high-throughput MBMS and saccharification pipelines. G.B.T. performed high-throughput recalcitrance pipeline through BESC. S.R.D. guided overall high-throughput saccharification pipeline through BESC and provided data analysis. W.A.P. guided the switchgrass and rice transformation pipeline and provided data analysis. M.K.U. guided cloning and production of RNAi vectors for switchgrass and rice. J.R.M. and B.H.D. guided the overall ethanol assay and interpreted the ethanol data. M.F.D. developed and provided leadership for the MBMS pipeline through BESC. R.S.N. directed the BESC transformation pipeline and coordinated analyses through the pipeline. A.J.R. coordinated and analyzed cellulose research. D.M. conceived of the study, coordinated the research, and contributed to the interpretation of results, and drafting and finalizing of the manuscript. All authors read and approved the final manuscript.

Competing interests

The strategy to produce improved biomass as described in this paper has been included in a patent application. C. C. and K.W. are employees of ArborGen Inc., a global provider of conventional and next-generation plantation tree seedling products for the forestry industry.

Corresponding author

Correspondence to Debra Mohnen.

Integrated supplementary information

Supplementary figures

  1. 1.

    Phylogenetic tree of GAUT Protein Family and gene model, RNAi construct, and relative transcript abundance of GAUT4 in switchgrass, rice and poplar knockdown (KD) lines.

  2. 2.

    Saccharification yield from poplar control and PdGAUT4-KD lines, and lignin content and S/G ratio from switchgrass and poplar control and KD lines.

  3. 3.

    Plant morphology and dry matter accumulation in greenhouse-grown switchgrass PvGAUT4-KD lines and disease severity in field-grown lines.

  4. 4.

    Growth and relative water content of poplar control and PdGAUT4-KD lines.

  5. 5.

    HG:GalAT activity of Arabidopsis (AtGAUT4), poplar (PdGAUT4), and switchgrass (PvGAUT4) recombinant GAUT4 transiently expressed in N. benthamiana.

  6. 6.

    Glycome profiling of switchgrass biomass from WT and PvGAUT4-KD lines.

  7. 7.

    Glycome profiling of poplar biomass from WT and PdGAUT4-KD lines.

  8. 8.

    Transmission electron microscopy of immunogold-labeled switchgrass stem cross-sections.

  9. 9.

    Mass of cell wall alcohol insoluble residue (AIR), each sequential AIR extract, and the insoluble pellet from (A) switchgrass R1 stage tillers and (B) poplar stem biomass from control (WT only for switchgrass; WT and vector control for poplar) and GAUT4-KD lines.

  10. 10.

    Physical assessment of switchgrass WT and PvGAUT4-KD biomass.

  11. 11.

    Characteristics of cellulose extracted from switchgrass WT and PvGAUT4-KD lines.

  12. 12.

    Microscopic analysis of poplar wood phloem and xylem tissue.

  13. 13.

    Size of individual xylem vessel and fiber cells from PdGAUT4-KD and WT debarked and depithed stem wood tissue.

  14. 14.

    Field design of the 3-year field-trial of switchgrass WT and PvGAUT4-KD lines.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–14

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Tables and Supplementary Notes

    Supplementary Table 1 and Supplementary Notes 1–6

Excel files

  1. 1.

    Supplementary Table 2

    Glycosyl residue composition of cell wall extracts from tillers of switchgrass WT and PvGAUT4-KD plants

  2. 2.

    Supplementary Table 3

    Glycosyl residue composition and total carbohydrate in cell wall extracts from WT and PvGAUT4-KD lines

  3. 3.

    Supplementary Table 4

    Glycosyl residue composition of cell wall extracts from stems of P. deltoides WT, vector control and PdGAUT4-KD plants

  4. 4.

    Supplementary Table 5

    Glycosyl residue composition and total carbohydrate of cell wall extracts from WT and PdGAUT4-KD lines (AB23.2,AB23.5, AB23.12, AB23.14)

  5. 5.

    Supplementary Table 6

    Glycosyl linkage analysis of fractionated cell walls from switchgrass WT and PvGAUT4-KD lines

  6. 6.

    Supplementary Table 7

    Glycosyl linkage analysis of fractionated cell walls from P.deltoides WT and PdGAUT4-KD lines.

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https://doi.org/10.1038/nbt.4067

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