Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy
Tracking enzyme localization and following the local biochemical modification of the substrate should help explain the recalcitrance of lignocellulosic plant cell walls to enzymatic degradation. Time-lapse studies using conventional imaging require enzyme labeling and following the biochemical modif...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2018-02-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | http://journal.frontiersin.org/article/10.3389/fpls.2018.00200/full |
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| author | Marie-Françoise Devaux Frédéric Jamme William André Brigitte Bouchet Camille Alvarado Sylvie Durand Paul Robert Luc Saulnier Estelle Bonnin Fabienne Guillon |
| author_facet | Marie-Françoise Devaux Frédéric Jamme William André Brigitte Bouchet Camille Alvarado Sylvie Durand Paul Robert Luc Saulnier Estelle Bonnin Fabienne Guillon |
| author_sort | Marie-Françoise Devaux |
| collection | DOAJ |
| description | Tracking enzyme localization and following the local biochemical modification of the substrate should help explain the recalcitrance of lignocellulosic plant cell walls to enzymatic degradation. Time-lapse studies using conventional imaging require enzyme labeling and following the biochemical modifications of biopolymers found in plant cell walls, which cannot be easily achieved. In the present work, synchrotron facilities have been used to image the enzymatic degradation of lignocellulosic biomass without labeling the enzyme or the cell walls. Multichannel autofluorescence imaging of the protein and phenolic compounds after excitation at 275 nm highlighted the presence or absence of enzymes on cell walls and made it possible to track them during the reaction. Image analysis was used to quantify the fluorescence intensity variations. Consistent variations in the enzyme concentration were found locally for cell cavities and their surrounding cell walls. Microfluidic FT-IR microspectroscopy allowed for time-lapse tracking of local changes in the polysaccharides in cell walls during degradation. Hemicellulose degradation was found to occur prior to cellulose degradation using a Celluclast® preparation. Combining the fluorescence and FT-IR information yielded the conclusion that enzymes did not bind to lignified cell walls, which were consequently not degraded. Fluorescence multiscale imaging and FT-IR microspectroscopy showed an unexpected variability both in the initial biochemical composition and the degradation pattern, highlighting micro-domains in the cell wall of a given cell. Fluorescence intensity quantification showed that the enzymes were not evenly distributed, and their amount increased progressively on degradable cell walls. During degradation, adjacent cells were separated and the cell wall fragmented until complete degradation. |
| first_indexed | 2024-12-11T19:44:39Z |
| format | Article |
| id | doaj.art-f4d96f92be484f989306daeda85107ef |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-11T19:44:39Z |
| publishDate | 2018-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-f4d96f92be484f989306daeda85107ef2022-12-22T00:52:56ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2018-02-01910.3389/fpls.2018.00200290313Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared MicrospectroscopyMarie-Françoise Devaux0Frédéric Jamme1William André2Brigitte Bouchet3Camille Alvarado4Sylvie Durand5Paul Robert6Luc Saulnier7Estelle Bonnin8Fabienne Guillon9UR1268 Biopolymères Interactions et Assemblages, Institut National de la Recherche Agronomique Pays de la Loire, Nantes, FranceSynchrotron SOLEIL, Gif-Sur-Yvette, FranceSynchrotron SOLEIL, Gif-Sur-Yvette, FranceUR1268 Biopolymères Interactions et Assemblages, Institut National de la Recherche Agronomique Pays de la Loire, Nantes, FranceUR1268 Biopolymères Interactions et Assemblages, Institut National de la Recherche Agronomique Pays de la Loire, Nantes, FranceUR1268 Biopolymères Interactions et Assemblages, Institut National de la Recherche Agronomique Pays de la Loire, Nantes, FranceUR1268 Biopolymères Interactions et Assemblages, Institut National de la Recherche Agronomique Pays de la Loire, Nantes, FranceUR1268 Biopolymères Interactions et Assemblages, Institut National de la Recherche Agronomique Pays de la Loire, Nantes, FranceUR1268 Biopolymères Interactions et Assemblages, Institut National de la Recherche Agronomique Pays de la Loire, Nantes, FranceUR1268 Biopolymères Interactions et Assemblages, Institut National de la Recherche Agronomique Pays de la Loire, Nantes, FranceTracking enzyme localization and following the local biochemical modification of the substrate should help explain the recalcitrance of lignocellulosic plant cell walls to enzymatic degradation. Time-lapse studies using conventional imaging require enzyme labeling and following the biochemical modifications of biopolymers found in plant cell walls, which cannot be easily achieved. In the present work, synchrotron facilities have been used to image the enzymatic degradation of lignocellulosic biomass without labeling the enzyme or the cell walls. Multichannel autofluorescence imaging of the protein and phenolic compounds after excitation at 275 nm highlighted the presence or absence of enzymes on cell walls and made it possible to track them during the reaction. Image analysis was used to quantify the fluorescence intensity variations. Consistent variations in the enzyme concentration were found locally for cell cavities and their surrounding cell walls. Microfluidic FT-IR microspectroscopy allowed for time-lapse tracking of local changes in the polysaccharides in cell walls during degradation. Hemicellulose degradation was found to occur prior to cellulose degradation using a Celluclast® preparation. Combining the fluorescence and FT-IR information yielded the conclusion that enzymes did not bind to lignified cell walls, which were consequently not degraded. Fluorescence multiscale imaging and FT-IR microspectroscopy showed an unexpected variability both in the initial biochemical composition and the degradation pattern, highlighting micro-domains in the cell wall of a given cell. Fluorescence intensity quantification showed that the enzymes were not evenly distributed, and their amount increased progressively on degradable cell walls. During degradation, adjacent cells were separated and the cell wall fragmented until complete degradation.http://journal.frontiersin.org/article/10.3389/fpls.2018.00200/fullspectral imagingmaize stem cellscellulose and hemicellulose degradationligninimage analysis |
| spellingShingle | Marie-Françoise Devaux Frédéric Jamme William André Brigitte Bouchet Camille Alvarado Sylvie Durand Paul Robert Luc Saulnier Estelle Bonnin Fabienne Guillon Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy Frontiers in Plant Science spectral imaging maize stem cells cellulose and hemicellulose degradation lignin image analysis |
| title | Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy |
| title_full | Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy |
| title_fullStr | Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy |
| title_full_unstemmed | Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy |
| title_short | Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy |
| title_sort | synchrotron time lapse imaging of lignocellulosic biomass hydrolysis tracking enzyme localization by protein autofluorescence and biochemical modification of cell walls by microfluidic infrared microspectroscopy |
| topic | spectral imaging maize stem cells cellulose and hemicellulose degradation lignin image analysis |
| url | http://journal.frontiersin.org/article/10.3389/fpls.2018.00200/full |
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