Solid-state NMR investigations of cellulose structure and interactions with matrix polysaccharides in plant primary cell walls

Abstract

Until recently, the 3D architecture of plant cell walls was poorly understood due to the lack of high-resolution techniques for characterizing the molecular structure, dynamics, and intermolecular interactions of the wall polysaccharides in these insoluble biomolecular mixtures. We introduced multidimensional solid-state NMR (SSNMR) spectroscopy, coupled with (13)C labelling of whole plants, to determine the spatial arrangements of macromolecules in near-native plant cell walls. Here we review key evidence from 2D and 3D correlation NMR spectra that show relatively few cellulose-hemicellulose cross peaks but many cellulose-pectin cross peaks, indicating that cellulose microfibrils are not extensively coated by hemicellulose and all three major polysaccharides exist in a single network rather than two separate networks as previously proposed. The number of glucan chains in the primary-wall cellulose microfibrils has been under active debate recently. We show detailed analysis of quantitative (13)C SSNMR spectra of cellulose in various wild-type (WT) and mutant Arabidopsis and Brachypodium primary cell walls, which consistently indicate that primary-wall cellulose microfibrils contain at least 24 glucan chains.

Keywords: Arabidopsis; Brachypodium; cellulose microfibrils; dynamics; intermolecular contact; multidimensional correlation NMR..

Fig. 1.

2D ¹³C-¹³C J-INADEQUATE spectra of never-dried Arabidopsis cell walls at room temperature, correlating double-quantum (DQ) and single-quantum (SQ) ¹³C chemical shifts. The spectra were measured at 400, 600, and 900 MHz. Insets magnify the C1 region of the spectra to indicate the resolution enhancement by higher magnetic fields. The bottom row amplifies the C2–C4 region of arabinose, where high magnetic fields significantly improve the resolution of multiple forms of arabinose.

Fig. 2.

2D ¹³C-¹³C PDSD spectra of plant cell walls measured with 1.5 s spin diffusion mixing. (A) Intact Arabidopsis cell wall at 20 °C. (B) HG-depleted Arabidopsis cell wall at −20 °C. (C) Brachypodium cell wall at −20 °C. Cellulose–pectin cross peaks are observed in both intact and HG-depleted Arabidopsis cell walls, and cellulose–GAX cross peaks are detected in the Brachypodium sample.

Fig. 3.

(A) 2D ¹³C-¹³C PDSD spectrum of Brachypodium cell wall with a 3.0 s mixing. The spectrum was measured at 20 °C with a short ¹H-¹³C CP contact time of 35 μs to suppress the signals of mobile GAX. (B) Representative cross sections of interior cellulose (black) and surface cellulose (orange). The different intensity patterns indicate that ¹³C magnetization has not equilibrated between interior and surface cellulose. The difference spectra (purple), obtained after normalizing the two cross sections by the sC4 peak, correspond to core cellulose chains that are inaccessible to the surface. (C) Illustration of the cellulose microfibril structure, where interior cellulose consists of a surface-bound fraction and a core fraction. (D) The two types of interior cellulose chains have slightly different C6 chemical shifts.

Fig. 4.

2D ¹³C-¹³C PDSD spectrum of Arabidopsis cell walls with a 1.5 s mixing time. The spectrum was measured at −20 °C under 9kHz MAS. (A) 2D spectrum. (B) Representative cellulose cross sections of interior and surface cellulose exhibit different intensity patterns. The difference spectra (purple) were obtained after normalizing the two spectra by the sC4 peak. The surface cellulose cross section has contribution from Ara and XyG backbone, but the difference spectra mainly show signals of interior cellulose. (C) C1 and C6 regions of the cellulose cross sections and the difference spectra. Core cellulose C1 shows two peaks at 105.5 ppm and 104.1 ppm, and core cellulose C6 (cC6) exhibits a 0.3 ppm downfield shift from the average interior cellulose C6 (iC6) and 0.6 ppm downfield shift from the surface-bound interior cellulose (bC6).

Fig. 5.

1D quantitative ¹³C DP spectra of ¹³C-labelled primary cell walls at ambient temperature. All spectra were measured with recycle delays of 15 to 25 s, except for the xxt1xxt2xxt5 sample, which was measured with recycle delays of 10 s. (A) Spectra of grass cell walls with negligible amounts of XyG. Two grasses, Brachypodium distachyon (top) and Poa annua (bottom), were measured. The Ara C1 (AC1) and interior cellulose C4 (iC4) peaks are highlighted in green and red, respectively. The mixed peaks of surface cellulose C4 and Ara C2 and C4 are shaded in grey. The integrated intensities were used to calculate the surface:interior cellulose ratio (s:i). Grass has a small s:i ratio of 1.3–1.4, indicating at least 24 glucan chains (see Fig. 6). (B) A triple mutant of Arabidopsis thaliana with negligible XyG. (C) Intact (top) and digested (bottom) Arabidopsis cell walls. (D) WT and CESA mutant of Arabidopsis. The integration regions are 111.8–107.2 ppm for AC1, 92.0–86.8 ppm for iC4, and 86.8–80.4 ppm for the mixed peak of sC4 and matrix polysaccharides. The boundary of the mixed peak changed to 81.0 ppm for the xxt1xxt2xxt5 mutant cell wall to avoid overlap with a strong pectin peak at 79.6 ppm.

Fig. 6.

Number of glucan chains in cellulose microfibrils as a function of the s:i ratio. The minimum number of glucan chains for s:i values of 1.1, 1.2, 1.3, 1.4, and 1.5 are 30, 29, 28, 24, and 30, respectively (filled circles). (B) Representative cellulose microfibril cross sections with different s:i ratios. For each model, glucan chains from core cellulose (magenta), surface-bound cellulose (red), and surface cellulose (orange) are depicted. Structural models with 18 or fewer chains correspond to s:i ratios of 2.0 or higher and lack core cellulose, which are inconsistent with the experimental data.