Evidence for Lignin-Carbohydrate Complexes from Studies of Transgenic Switchgrass and a Model Lignin-Pectin Composite

Riddhi S. Shah; Manjula Senanayake; Hong Hai Zhang; Yunqiao Pu; Ajaya K. Biswal; Sai Venkatesh Pingali; Brian Davison; Hugh O’Neill

doi:10.1021/acssuschemeng.3c04322

Evidence for Lignin-Carbohydrate Complexes from Studies of Transgenic Switchgrass and a Model Lignin-Pectin Composite

Riddhi S. Shah, Manjula Senanayake, Hong Hai Zhang, Yunqiao Pu, Ajaya K. Biswal, Sai Venkatesh Pingali, Brian Davison, Hugh O’Neill

Research output: Contribution to journal › Article › peer-review

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Abstract

Lignin-carbohydrate complexes (LCCs) form through interactions of lignin with plant cell wall polysaccharides and are thought to be a significant source of biomass recalcitrance. In this work, we investigated LCCs formed between lignin and pectin homogalacturonan (HG). The structural changes in HG deficient transgenic switchgrass (GAUT4-knockdown, GAUT4-KD) after hot water pretreatment were compared to wild-type plants using small-angle neutron scattering (SANS), which showed that there were ∼2.2-fold more lignin aggregates in GAUT4-KD biomass compared to the wild type. This demonstrated that decreased pectin resulted in more lignin redistribution and suggested that interactions between lignin and HG restrict lignin mobility in plant cell walls. To better understand the types of interactions between lignin and pectin, a model composite was prepared by polymerizing either protiated or partially deuterated coniferyl alcohol to form a dehydrogenation polymer (DHP) in the presence of HG. Small-angle X-ray scattering (SAXS) showed that the DHP and HG form a highly interconnected network structure that is not observed in a physical mixture of the individual polymers. Contrast matching SANS revealed the structure of DHP and HG in the composite and showed that the HG forms a swollen interconnected polymer network (power-law exponent, P = 1.5) interspersed with DHP particles (radius of gyration, R_g, 264 Å) that are composed of solvent-accessible DHP polymers (P = 2.3). Fourier transform infrared spectroscopy showed a unique ester absorption band in the DHP/HG composites. Solid-state nuclear magnetic resonance (NMR) analysis also supports interactions between DHP and HG. Overall, this study provides new insights into the relationship between primary and secondary cell wall polymers during cell wall synthesis and how LCCs formed between pectin and lignin could represent a previously unrecognized source of biomass recalcitrance. This knowledge may help develop new approaches to modulate cell wall properties to improve biofuel and bioproduct production.

Original language	English
Pages (from-to)	15941-15950
Number of pages	10
Journal	ACS Sustainable Chemistry and Engineering
Volume	11
Issue number	44
DOIs	https://doi.org/10.1021/acssuschemeng.3c04322
State	Published - Nov 6 2023

Funding

This work was supported by the Genomic Science Program, Office of Biological and Environmental Research (OBER), U.S. Department of Energy (DOE), under Contract FWP ERKP752. d -Coniferyl alcohol was synthesized using the capabilities of the Center for Nanophase Materials Sciences (CNMS) supported by the Office of Basic Energy Sciences (BES), DOE. SANS studies of the DHP–pectin composites were performed using the Bio-SANS instrument of the Center for Structural Molecular Biology (FWP ERKP291), an OBER Structural Biology Resource. The authors thank Dr. Changwoo Do for his assistance with the SANS studies of switchgrass using the EQ-SANS instrument at the Spallation Neutron Source. A.K.B. was supported by the OBER funded Center for Bioenergy Innovation (CBI) managed by the Oak Ridge National Laboratory (ORNL). The authors thank Professor Debra Mohnen, Complex Carbohydrate Center, University of Georgia, for scientific discussions and providing materials for the project. GAUT4-KD and control switchgrass samples were provided by the CBI. This research used resources at the High Flux Isotope Reactor and Spallation Neutron Source, a DOE Office of Science User Facility operated by the ORNL. ORNL is operated by UT-Battelle, LLC, under Contract DE-AC05-00OR22725 with the U.S. Department of Energy. 5 This work was supported by the Genomic Science Program, Office of Biological and Environmental Research (OBER), U.S. Department of Energy (DOE), under Contract FWP ERKP752. d5-Coniferyl alcohol was synthesized using the capabilities of the Center for Nanophase Materials Sciences (CNMS) supported by the Office of Basic Energy Sciences (BES), DOE. SANS studies of the DHP-pectin composites were performed using the Bio-SANS instrument of the Center for Structural Molecular Biology (FWP ERKP291), an OBER Structural Biology Resource. The authors thank Dr. Changwoo Do for his assistance with the SANS studies of switchgrass using the EQ-SANS instrument at the Spallation Neutron Source. A.K.B. was supported by the OBER funded Center for Bioenergy Innovation (CBI) managed by the Oak Ridge National Laboratory (ORNL). The authors thank Professor Debra Mohnen, Complex Carbohydrate Center, University of Georgia, for scientific discussions and providing materials for the project. GAUT4-KD and control switchgrass samples were provided by the CBI. This research used resources at the High Flux Isotope Reactor and Spallation Neutron Source, a DOE Office of Science User Facility operated by the ORNL. ORNL is operated by UT-Battelle, LLC, under Contract DE-AC05-00OR22725 with the U.S. Department of Energy.

Funders	Funder number
Center for Structural Molecular Biology	FWP ERKP291
U.S. Department of Energy	FWP ERKP752
Office of Science
Basic Energy Sciences
Biological and Environmental Research
Oak Ridge National Laboratory	DE-AC05-00OR22725, GAUT4-KD

Keywords

lignin−carbohydrate complexes
neutron scattering
pectin−lignin composite
plant cell wall polysaccharides
transgenic switch grass

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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title = "Evidence for Lignin-Carbohydrate Complexes from Studies of Transgenic Switchgrass and a Model Lignin-Pectin Composite",

abstract = "Lignin-carbohydrate complexes (LCCs) form through interactions of lignin with plant cell wall polysaccharides and are thought to be a significant source of biomass recalcitrance. In this work, we investigated LCCs formed between lignin and pectin homogalacturonan (HG). The structural changes in HG deficient transgenic switchgrass (GAUT4-knockdown, GAUT4-KD) after hot water pretreatment were compared to wild-type plants using small-angle neutron scattering (SANS), which showed that there were ∼2.2-fold more lignin aggregates in GAUT4-KD biomass compared to the wild type. This demonstrated that decreased pectin resulted in more lignin redistribution and suggested that interactions between lignin and HG restrict lignin mobility in plant cell walls. To better understand the types of interactions between lignin and pectin, a model composite was prepared by polymerizing either protiated or partially deuterated coniferyl alcohol to form a dehydrogenation polymer (DHP) in the presence of HG. Small-angle X-ray scattering (SAXS) showed that the DHP and HG form a highly interconnected network structure that is not observed in a physical mixture of the individual polymers. Contrast matching SANS revealed the structure of DHP and HG in the composite and showed that the HG forms a swollen interconnected polymer network (power-law exponent, P = 1.5) interspersed with DHP particles (radius of gyration, Rg, 264 {\AA}) that are composed of solvent-accessible DHP polymers (P = 2.3). Fourier transform infrared spectroscopy showed a unique ester absorption band in the DHP/HG composites. Solid-state nuclear magnetic resonance (NMR) analysis also supports interactions between DHP and HG. Overall, this study provides new insights into the relationship between primary and secondary cell wall polymers during cell wall synthesis and how LCCs formed between pectin and lignin could represent a previously unrecognized source of biomass recalcitrance. This knowledge may help develop new approaches to modulate cell wall properties to improve biofuel and bioproduct production.",

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author = "Shah, {Riddhi S.} and Manjula Senanayake and Zhang, {Hong Hai} and Yunqiao Pu and Biswal, {Ajaya K.} and Pingali, {Sai Venkatesh} and Brian Davison and Hugh O{\textquoteright}Neill",

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T1 - Evidence for Lignin-Carbohydrate Complexes from Studies of Transgenic Switchgrass and a Model Lignin-Pectin Composite

AU - Shah, Riddhi S.

AU - Senanayake, Manjula

AU - Zhang, Hong Hai

AU - Pu, Yunqiao

AU - Biswal, Ajaya K.

AU - Pingali, Sai Venkatesh

AU - Davison, Brian

AU - O’Neill, Hugh

PY - 2023/11/6

Y1 - 2023/11/6

N2 - Lignin-carbohydrate complexes (LCCs) form through interactions of lignin with plant cell wall polysaccharides and are thought to be a significant source of biomass recalcitrance. In this work, we investigated LCCs formed between lignin and pectin homogalacturonan (HG). The structural changes in HG deficient transgenic switchgrass (GAUT4-knockdown, GAUT4-KD) after hot water pretreatment were compared to wild-type plants using small-angle neutron scattering (SANS), which showed that there were ∼2.2-fold more lignin aggregates in GAUT4-KD biomass compared to the wild type. This demonstrated that decreased pectin resulted in more lignin redistribution and suggested that interactions between lignin and HG restrict lignin mobility in plant cell walls. To better understand the types of interactions between lignin and pectin, a model composite was prepared by polymerizing either protiated or partially deuterated coniferyl alcohol to form a dehydrogenation polymer (DHP) in the presence of HG. Small-angle X-ray scattering (SAXS) showed that the DHP and HG form a highly interconnected network structure that is not observed in a physical mixture of the individual polymers. Contrast matching SANS revealed the structure of DHP and HG in the composite and showed that the HG forms a swollen interconnected polymer network (power-law exponent, P = 1.5) interspersed with DHP particles (radius of gyration, Rg, 264 Å) that are composed of solvent-accessible DHP polymers (P = 2.3). Fourier transform infrared spectroscopy showed a unique ester absorption band in the DHP/HG composites. Solid-state nuclear magnetic resonance (NMR) analysis also supports interactions between DHP and HG. Overall, this study provides new insights into the relationship between primary and secondary cell wall polymers during cell wall synthesis and how LCCs formed between pectin and lignin could represent a previously unrecognized source of biomass recalcitrance. This knowledge may help develop new approaches to modulate cell wall properties to improve biofuel and bioproduct production.

AB - Lignin-carbohydrate complexes (LCCs) form through interactions of lignin with plant cell wall polysaccharides and are thought to be a significant source of biomass recalcitrance. In this work, we investigated LCCs formed between lignin and pectin homogalacturonan (HG). The structural changes in HG deficient transgenic switchgrass (GAUT4-knockdown, GAUT4-KD) after hot water pretreatment were compared to wild-type plants using small-angle neutron scattering (SANS), which showed that there were ∼2.2-fold more lignin aggregates in GAUT4-KD biomass compared to the wild type. This demonstrated that decreased pectin resulted in more lignin redistribution and suggested that interactions between lignin and HG restrict lignin mobility in plant cell walls. To better understand the types of interactions between lignin and pectin, a model composite was prepared by polymerizing either protiated or partially deuterated coniferyl alcohol to form a dehydrogenation polymer (DHP) in the presence of HG. Small-angle X-ray scattering (SAXS) showed that the DHP and HG form a highly interconnected network structure that is not observed in a physical mixture of the individual polymers. Contrast matching SANS revealed the structure of DHP and HG in the composite and showed that the HG forms a swollen interconnected polymer network (power-law exponent, P = 1.5) interspersed with DHP particles (radius of gyration, Rg, 264 Å) that are composed of solvent-accessible DHP polymers (P = 2.3). Fourier transform infrared spectroscopy showed a unique ester absorption band in the DHP/HG composites. Solid-state nuclear magnetic resonance (NMR) analysis also supports interactions between DHP and HG. Overall, this study provides new insights into the relationship between primary and secondary cell wall polymers during cell wall synthesis and how LCCs formed between pectin and lignin could represent a previously unrecognized source of biomass recalcitrance. This knowledge may help develop new approaches to modulate cell wall properties to improve biofuel and bioproduct production.

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Evidence for Lignin-Carbohydrate Complexes from Studies of Transgenic Switchgrass and a Model Lignin-Pectin Composite

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Keywords

UN SDGs

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