Solution-state 2D NMR Characterization Of Plant Cell Wall Material

The plant cell wall,also known as lignocellulose,is an irreplaceable renewable energy source,mainly composed of cellulose,hemicellulose,and lignin.In order to fully utilize the potential of lignocellulosic biomass as a renewable resource for fuels and chemicals,it is necessary to further understand the chemical and molecular structures within the biomass,and how these structures are formed and transformed during the biosynthesis process.Therefore,these tasks require in-depth and rapid analysis techniques and ensure the integrity of plant cell wall characteristics.On the basis of not causing(or rarely)structural changes,nuclear magnetic resonance(NMR)analysis of plant cell walls is suitable for rapid and in-depth structural characterization of lignocellulosic biomass.In this paper,the two-dimensional hydrocarbon related nuclear magnetic resonance(2D HSQC NMR)of poplar and pine cell wall materials was optimized in detail,and the plant cell wall materials were separated and characterized under the same optimization conditions,which proved the method is feasible and accurate.The optimized method was used to characterize the structure of the cell wall material of flue-cured tobacco plants,the structural composition of the cell wall material of flue-cured tobacco was explored,and the influence of different experimental conditions on the structure of flue-cured tobacco was determined.The main research work of this article is as follows:First,the plant samples(poplar,pine)were ball milled and dissolved in deuterated dimethyl sulfoxide(DMSO-d6).The structure of plant cell wall material was characterized by 2D HSQC NMR.The chemical shifts of the components are assigned in the nuclear magnetic spectrum.The spectrum of plant cell wall material on 2D HSQC NMR can be divided into three regions:lignin aromatic region(?H 6.0-8.0 ppm,?c 100-150 ppm),aliphatic region(lignin side chain region and polysaccharide region,?H 2.5-6.0 ppm,?C 50-90 ppm)and polysaccharide anomeric carbon region(?H 3.5-6.0 ppm,?C 50-90 ppm).There are syringyl(S),guaiacyl(G),and p-hydroxyphenyl(H)signals in the aromatic region of the 2D HSQC map of plants.Four types of lignin linkages in the aliphatic region include ?-aryl ether(?-O-4),phenylcoumarin(?-5),resinol alcohol(?-?)and dibenzodioxocin(5-5/4-O-?)(not shown),polysaccharide anomeric carbon region contains[(1?4)-?-D-Glcp]and xylan[(1?4)-?-D-Xylp]signals.Therefore,the solution state two-dimensional NMR can well characterize the cell wall material of poplar and pine.Second,based on the investigation of a large number of literatures,the solvent and particle size are important factors that affect the plant cell wall material structure in two-dimensional nuclear magnetic spectrum.Deuterated dimethyl sulfoxide(DMSO-d6),deuterated dimethyl sulfoxide/deuterated pyridine(DMSO-d6/pyridine-d5)and deuterated dimethyl sulfoxide/deuterated hexamethylphosphoryl were chosen dissolve the plant cell wall(poplar,pine),and the richness and intensity of the peak signal under different solvents in DMSO-d6/HMPA-d18 were highest.The polysaccharide linkage LC? absented in DMSO-d6 and DMSO-d6/pyridine-d5 appeared in DMSO-d6/HMPA-d18.P-hydroxyphenyl(H),which appears only in the noise level of DMSO-d6/pyridine-d5,was well represented in DMSO-d6/HMPA-d18.Ball milling plant samples into different particle size ranges,the relationship between ball milling time and transmission rate(probability of particles with a particle size of<10 ?m in ball milling samples)can be obtained by scanning electron microscopy.The attribution(qualitative)and integral(semi-quantitative)of the peak signal in the NMR spectrum determine the optimal particle size,and it is expressed by the ball milling time.The results show that when the transmittance of poplar,pine,tobacco stems and tobacco leaf is 84.0%,94.24%,94.6%,91.9%,and the corresponding milling time is 6 h,the quality of the NMR spectrum is the highest.On the other hand,in order to prove the stability of the NMR experiment under optimized conditions,a two-dimensional NMR experiment was performed on the standard substance coniferyl alcohol.The results show that the chemical shift of coniferyl alcohol under DMSO-d6/HMPA-d18 is almost the same as that of DMSO-d6,so the addition of solvent HMPA-d18 will not cause the chemical environment of cell wall substances to change.In addition,in order to prove the accuracy of the NMR experiment under optimized conditions,the lignin,cellulose and hemicellulose of poplar were extracted separately and subjected to the NMR experiment under the same optimized conditions.The signal intensity of key components such as LA-S?,LC?,LC?,S,G,and H are almost the same in both maps,which proves that the two-dimensional nuclear magnetic analysis of plant cell walls is sufficient to characterize the properties of its substances.Third,the optimized conditions of the solution state two-dimensional nuclear magnetic resonance were applied to the characterization of flue-cured tobacco stems and leaf structure.Scanning electron microscopy results showed that the particle size range of the sample has stabilized,when ball milling time is 6 h.Under the optimized conditions,the 2D HSQC NMR spectrum results show that the tobacco shows abundant component information,such as lignin,Internal cellulose[(1?4)-?-D-Glcp],xylan[(1?4)-?-D-Xylp],etc.In addition,the fingerprint structure of the anomeric carbon region of polysaccharides,sucrose,arabinofuran residues(?-L-Araf),4-O-methyl-?-D-glucuronic acid(4-O-MeGlcA)appeared in the NMR spectrum.Therefore,NMR analysis under optimized conditions is suitable for tobacco structural analysis.