The in situ characterization of select plant cell wall components through NMR spectroscopy and X-ray diffraction

In situ High-Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy and in situ Wide-Angle X-ray Scattering (WAXS) have proven to be valuable tools in the study of plant biochemistry and morphology. The in situ technique is beneficial in that it offers the advantage of being able to examine molecular ordering, composition, and structure within intact cell walls without altering the nature of the cell wall construct. Both wild-type and mutant Arabidopsis thaliana tissues were subjected to in situ HR-MAS NMR and WAXS analyses to ascertain differences occurring in cell wall polysaccharides and lignin as a result of certain mutations affecting a putative endo-(1→4)-beta- D-glucanase. 1D 1H HR-MAS NMR spectra indicated an overall decrease in lignin content for shoot tissues of mutants with decreased gene expression. For the root tissues of A. thaliana mutants with decreased gene expression, 2D 1H-1H COSY and 1H-13C HMQC spectra revealed a reduction in the rhamnogalacturonan I component of pectin. In situ WAXS analysis determined that changes were occurring in the abundance and organization of the crystalline cellulose as a result of the genomic mutations. In particular, one mutant plant, a carbohydrate-binding module overexpressing mutant, demonstrated the greatest variation from the native plant in that it displayed a more orderly microfibril arrangement about the fiber axis as well as a greater cellulose crystallite size. In situ HR-MAS NMR analysis was also used to investigate differences in poplar hybrid cell wall constituents occurring as a result of inoculation with the endophytic bacterium Enterobacter sp. 638. 1D 1H and 2D 1H- 1H TOCSY and 1H-13C HMQC HR-MAS NMR spectra revealed that older immature leaf tissues from Enterobacter sp. 638 inoculated plants exhibited an overall increase in lignin content with a concomitant decrease in dihydroconiferyl alcohol lignin monomers. The in situ HR-MAS NMR and WAXS techniques utilized in this work have proven to be rapid methods in identifying plant cell wall alterations arising from selective modifications. As both cellulose and lignin are desirable feedstocks for bio-derived energy, the alterations made to both the Arabidopsis and poplar plants provided valuable information pertinent to the area of biofuels.;Key Words: In situ; HR-MAS; NMR; WAXS; Arabidopsis thaliana; Poplar; Endophytic Bacteria; APE; Cell Wall; Cellulose; Pectin; RG-I; Lignin; Endoglucanase; CBM...