Altered lignocellulose chemical structure and molecular assembly in CINNAMYL ALCOHOL DEHYDROGENASE-deficient rice

Abstract

Lignin is a complex phenylpropanoid polymer deposited in plant cell walls. Lignin has long been recognized as an important limiting factor for the polysaccharide-oriented biomass utilizations. To mitigate lignin-associated biomass recalcitrance, numerous mutants and transgenic plants that produce lignocellulose with reduced lignin contents and/or lignins with altered chemical structures have been produced and characterised. However, it is not fully understood how altered lignin chemistry affects the supramolecular structure of lignocellulose, and consequently, its utilization properties. Herein, we conducted comprehensive chemical and supramolecular structural analyses of lignocellulose produced by a rice cad2 mutant deficient in CINNAMYL ALCOHOL DEHYDROGENASE (CAD), which encodes a key enzyme in lignin biosynthesis. By using a solution-state two-dimensional NMR approach and complementary chemical methods, we elucidated the structural details of the altered lignins enriched with unusual hydroxycinnamaldehyde-derived substructures produced by the cad2 mutant. In parallel, polysaccharide assembly and the molecular mobility of lignocellulose were investigated by solid-state 13C MAS NMR, nuclear magnetic relaxation, X-ray diffraction, and Simon’s staining analyses. Possible links between CAD-associated lignin modifications (in terms of total content and chemical structures) and changes to the lignocellulose supramolecular structure are discussed in the context of the improved biomass saccharification efficiency of the cad2 rice mutant.

Understanding laccase/HBT-catalyzed grass delignification at the molecular level

“…In WEL pure a small peak was also observed at δ C 192 ppm, that was annotated as an aldehyde based on the presence of a correlation at δ C /δ H 192/9.8 in the corresponding HSQC spectrum (data not shown). 53 Nevertheless, as this HSQC correlation was also observed in WECEL pure control samples, and laccase/HBT treatment did not result in more than 1 aldehyde group per 100 aromatic rings, we concluded that aldehyde formation was not a main degradation pathway during LMS treatment. Remarkably, very clear correlations were observed that corresponded to S-ketones and S-acids (δ H 7.1-7.3), whereas the intensity of their G-analogues was very low.…”

Importance of Lignin Coniferaldehyde Residues for Plant Properties and Sustainable Uses

“…The use of Arabidopsis represents an ideal model to quickly investigate multiple genetic engineering strategies and validate analytical method-ologies, both transposable to agronomically relevant lignocellulosic feedstock species. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] In contrast to total G CHOH and S CHOH residue levels which could only be reduced or annulled compared to WT plants, total G CHO residue content varied by roughly threefold changes in either direction in Arabidopsis with specific genetic changes, namely increasing in the cad4xcad5 mutant and decreasing in the 4cl1x4cl2 mutant ( Figure 2A). Arabidopsis natural ecotype variant Wassilewskija (WS) presented~60 % more G CHO residues than the Columbia-0 (Col-0) ecotype, although their G CHOH and S CHOH residue amounts did not differ ( Figure S2).…”

“…[6,8] Similar increases of aldehyde residues in lignin by reducing CAD expression, using mutagenesis and transgenic approaches in poplar, tobacco, flax, brachypodium, switchgrass, rice and sorghum, have all showed either increased pulping, saccharification and/or biogas yields without affecting plant productivity. [5,[10][11][12][13][14][15][16][17][18] In fact, natural mutants in CADs thrive in the wild and have been readily identified, such as the CAD-null mutant of pine. [19,20] Natural mutants in CAD have also been selected and preferentially used in agriculture more than 100-years ago, like the Sekizaisou variety of mulberry trees, which improved both silkworm growth and silk quality when used for feed.…”

“…Nuclear magnetic resonance (NMR) analyses of lignin showed that G CHO content in lignin represented~7 % in pine, 4 % in spruce,~8 % in alfalfa and~6 % in rice, and reached 15 % in CAD-null pine,~19 % and~88 % respectively in the cad mutants of rice and alfalfa. [16,[18][19][20]28] Pyrolysis coupled to gas chromatography and mass spectroscopy (PyÀ GC/MS), which enables the quantitative measurement of G CHO , G CHOH and sinapyl alcohol (S CHOH ) residues, [29][30][31][32] showed that total G CHO content in lignin represented~9 % in spruce,~5 % in Arabidopsis,~2 % in eucalyptus and~0.2 % in poplar. [7,33,34] The content variability in lignin of G CHO , G CHOH and S CHOH residues was further examined using pyrolysis/GCÀ MS on a set of Arabidopsis thaliana mutants affected in one or several genes encoding for enzymes responsible of changing the C 6 and/or C 3 parts of lignin monomers ( Figure S1).…”

Initial pyrolysis mechanism and product formation of cellulose: An Experimental and Density functional theory(DFT) study

“…Biomass is a complex material that is mainly composed of crosslinked hemicellulose, cellulose, and lignin, along with extracts (tannins, fatty acids, and resins) and inorganic salts 2 . Numerous studies have indicated that cellulose is the most abundant organic linear polymer 3 . It is composed of D-glucose as a basic unit and is linked via β-O-4 glycosidic bonds.…”