Characterization and Elimination of Undesirable Protein Residues in Plant Cell Wall Materials for Enhancing Lignin Analysis by Solution-State Nuclear Magnetic Resonance Spectroscopy

12 páginas.-- 5 figuras.-- 5 tablas.-- 74 referenciasProtein polymers exist in every plant cell wall preparation, and they interfere with lignin characterization and quantification. Here, we report the structural characterization of the residual protein peaks in 2D NMR spectra in corn cob and kenaf samples and note that aromatic amino acids are ubiquitous and evident in spectra from various other plants and tissues. The aromatic correlations from amino acid residues were identified and assigned as phenylalanine and tyrosine. Phenylalanine's 3/5 correlation peak is superimposed on the peak from typical lignin p-hydroxyphenyl (H-unit) structures, causing an overestimation of the H units. Protein contamination also occurs when using cellulases to prepare enzyme lignins from virtually protein-free wood samples. We used a protease to remove the protein residues from the ball-milled cell walls, and we were able to reveal H-unit structures in lignins more clearly in the 2D NMR spectra, providing a better basis for their estimationThis work was supported by grants from the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DEFC02-07ER64494). We are grateful for Jane Marita for her various helpful discussions on this project. We also thank Steven Karlen for GC/MS experimental support and Kris Niemann (USDFRC) for assistance with nitrogen analysis.Peer reviewe

Monolignol ferulate transferase introduces chemically labile linkages into the lignin backbone

4 páginas.-- 4 figuras.-- 1 tabla.-- 27 referncias.--- El pdf. del Material Suplementarío tiene 29 páginas. Contiene: Materials and Methods.-- Supplementary Text.-- Figs. S1 to S9.-- Tables S1 to S4.-- 39 ReferencesRedesigning lignin, the aromatic polymer fortifying plant cell walls, to be more amenable to chemical depolymerization can lower the energy required for industrial processing. We have engineered poplar trees to introduce ester linkages into the lignin polymer backbone by augmenting the monomer pool with monolignol ferulate conjugates. Herein, we describe the isolation of a transferase gene capable of forming these conjugates and its xylem-specific introduction into poplar. Enzyme kinetics, in planta expression, lignin structural analysis, and improved cell wall digestibility after mild alkaline pretreatment demonstrate that these trees produce the monolignol ferulate conjugates, export them to the wall, and use them during lignification. Tailoring plants to use such conjugates during cell wall biosynthesis is a promising way to produce plants that are designed for deconstruction.Peer reviewe

Characterization of protein amino acid residues and a monolignol conjugate in whole plant cell walls by solution-state 2D NMR and their interference with authentic p-hydroxyphenyl (H) unit estimation

Resumen de la comunicación presentada en el 255th National Meeting and Exposition of the American-Chemical-Society (ACS) - Nexus of Food, Energy, and Water (New Orleans), LA 18-22, march 2018Protein polymers exist in every plant cell wall preparation, and they interfere with lignin characterization and quantification. The current solution-state 2D NMR technique for whole plant cell wall structural profiling provides detailed information regarding cell walls. However, certain components intrude on lignin structural analysis. Here we report the structural characterization of the residual protein peaks in 2D NMR spectra in corn cob and kenaf samples, and note that aromatic amino acids are ubiquitous and evident in spectra from various other plants and tissues. The aromatic correlations from amino acid residues were identified and assigned as phenylalanine and tyrosine. Phenylalanine’s 3/5 correlation peak is superimposed on the peak from typical lignin p-hydroxyphenyl (H-unit) structures, causing an overestimation of the H units. Similarly, protein residues in many species also result in incorrect values for lignin analyses by the Klason method. Protein contamination also occurs when using cellulases to prepare enzyme lignins from virtually protein-free wood samples. We used a protease to remove the protein residues from the ballmilled cell walls, and we were able to reveal H-unit structures in lignins more clearly in the 2D NMR spectra, providing a better basis for their estimation. We also recently characterized a new monolignol conjugate, ML-benzoate (BA), in the cell wall samples of leaf tissues from Canary Island date palm (Phoenix canariensis). These NMR correlations have not been observed from other plant families including commelinid monocotyledons, such as the Poaceae (grasses, e.g., maize, rice, brachypodium). The presence of lignin-bound benzoates BA in the palms (Arecaceae) is very distinguishable. However, benzoate’s 3/5 correlation peak is virtually superimposed on the peak from typical lignin p-hydroxyphenyl (H-unit) structures, also causing an overestimation of the H units.Protein polymers exist in every plant cell wall preparation, and they interfere with lignin characterization and quantification. The current solution-state 2D NMR technique for whole plant cell wall structural profiling provides detailed information regarding cell walls. However, certain components intrude on lignin structural analysis. Here we report the structural characterization of the residual protein peaks in 2D NMR spectra in corn cob and kenaf samples, and note that aromatic amino acids are ubiquitous and evident in spectra from various other plants and tissues. The aromatic correlations from amino acid residues were identified and assigned as phenylalanine and tyrosine. Phenylalanine’s 3/5 correlation peak is superimposed on the peak from typical lignin p-hydroxyphenyl (H-unit) structures, causing an overestimation of the H units. Similarly, protein residues in many species also result in incorrect values for lignin analyses by the Klason method. Protein contamination also occurs when using cellulases to prepare enzyme lignins from virtually protein-free wood samples. We used a protease to remove the protein residues from the ballmilled cell walls, and we were able to reveal H-unit structures in lignins more clearly in the 2D NMR spectra, providing a better basis for their estimation. We also recently characterized a new monolignol conjugate, ML-benzoate (BA), in the cell wall samples of leaf tissues from Canary Island date palm (Phoenix canariensis). These NMR correlations have not been observed from other plant families including commelinid monocotyledons, such as the Poaceae (grasses, e.g., maize, rice, brachypodium). The presence of lignin-bound benzoates BA in the palms (Arecaceae) is very distinguishable. However, benzoate’s 3/5 correlation peak is virtually superimposed on the peak from typical lignin p-hydroxyphenyl (H-unit) structures, also causing an overestimation of the H units.Peer reviewe

Protein amino acid residues and a new monolignol conjugate in lignins and their interference with p-hydroxyphenyl (H) unit estimation - Póster presentación

4 páginas.-- 3 figuras.-- 7 referencias.-- Poster presentación en el 15th European Workshop on Lignocellulosics and Pulp June 26-29, 2018 Aveiro, PortugalWe elucidated the detailed structures of the residual protein peaks, phenylalanine and tyrosine, in 2D NMR spectra from corn cob and kenaf samples. Phenylalanine¿s 3/5 correlation peak is superimposed on the peak from typical lignin p-hydroxyphenyl (H-unit) structures, causing an overestimation of the H units. We used a protease to remove the protein residues from the ballmilled cell walls. Additionally, we also identified a new monolignol conjugate, ML-benzoate (BA), in the cell wall samples of leaf tissues from Canary Island date palm (Phoenix canariensis) and also a small amount from macaúba (Acrocomia aculeata (Jacq.) Lodd. ex Mart.) endocarp (and a trace in the stem) along with stilbenes, also causing an overestimation of the H units.This work was supported by grants from the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494), and Spanish projects CTQ2014-60764-JIN and AGL2017-83036-R (financed by Agencia Estatal de Investigación, AEI, and Fondo Europeo de Desarrollo Regional, FEDER).Peer Reviewe

Protein amino acid residues and a new monolignol conjugate in lignins and their interference with p-hydroxyphenyl (H) unit estimation

Póster presentado en el Lignin Gordon Research Conference Towards Viable Solutions for Lignin Valorization August 5 - 10, 2018, (Easton, MA), USPeer reviewe

BdCESA7, BdCESA8, and BdPMT Utility Promoter Constructs for Targeted Expression to Secondary Cell-Wall-Forming Cells of Grasses.

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase

Misregulation of p-coumaroyl-CoA:monolignol transferase in Brachypodium distachyon

International audienc

p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta