Solid-state NMR of unlabeled plant cell walls: high-resolution structural analysis without isotopic enrichment

Background Multidimensional solid-state nuclear magnetic resonance (ssNMR) spectroscopy has emerged as an indispensable technique for resolving polymer structure and intermolecular packing in primary and secondary plant cell walls. Isotope (13C) enrichment provides feasible sensitivity for measuring 2D/3D correlation spectra, but this time-consuming procedure and its associated expenses have restricted the application of ssNMR in lignocellulose analysis. Results Here, we present a method that relies on the sensitivity-enhancing technique Dynamic Nuclear Polarization (DNP) to eliminate the need for 13C-labeling. With a 26-fold sensitivity enhancement, a series of 2D 13C–13C correlation spectra were successfully collected using the unlabeled stems of wild-type Oryza sativa (rice). The atomic resolution allows us to observe a large number of intramolecular cross peaks for fully revealing the polymorphic structure of cellulose and xylan. NMR relaxation and dipolar order parameters further suggest a sophisticated change of molecular motions in a ctl1 ctl2 double mutant: both cellulose and xylan have become more dynamic on the nanosecond and microsecond timescale, but the motional amplitudes are uniformly small for both polysaccharides. Conclusions By skipping isotopic labeling, the DNP strategy demonstrated here is universally extendable to all lignocellulose materials. This time-efficient method has landed the technical foundation for understanding polysaccharide structure and cell wall assembly in a large variety of plant tissues and species.

Table S1.Fit parameters of 13 C CP spectrum of wild-type (WT) sample.The peaks are classified in three groups according to their influence on the C4 region, as by integrating, interior and surface cellulose contribution is shown to respectively be 40.5% and 59.5% of the cellulose content.

δ [ppm]
Assignment   shows an enhancement factor of 44.The detailed experimental parameters have been listed in Table 2.

Figure S1 .
Figure S1.Lignin has increased methyl ether substitution in the double mutant.The spectra of wildtype (black) and ctl1 ctl2 double mutant (yellow) are normalized by the interior cellulose carbon 4 (i4) peak.The lignin methyl ethers (lignin -OMe) has a doubled intensity in ctl1 ctl2 but the lignin aromatics have a comparable intensity in both samples.

Figure S2 .
Figure S2.Additional dataset of samples prepared using different protocols.a, ctl1 ctl2 sample in the solvent of 13 C-depleted, d8-glycerol/D2O/H2O (60/30/10 vol%) has shown a 22-fold enhancement of sensitivity.b, the ctl1 ctl2 sample prepared using the matrix-free protocol (with only a few μL of D2O)

Figure S3 .
Figure S3.Timesaving by DNP on carbohydrate signals in unlabeled rice stems.Top row: The roomtemperature spectrum collected on a 400 MHz NMR gives a signal-to-noise (S/N) ratio of 37 for the highest peak after 43 h of measurement.Bottom row: 600 MHz/395 GHz DNP provides a S/N ratio of 618 after only 0.5 h of measurement.Cellulose peaks are well reserved, but intensity suppression has been observed for xylan signals, lignin methyl ether (-OMe) and small molecules (Glc; glucose).

Figure S4 .
Figure S4.DNP polarization is uniform across the cell wall.The microwave-on (MW on) and microwave-off (MW off) spectra are normalized by the interior cellulose carbon 4 peaks (i4) to compare the spectral pattern.The consistent spectral envelope clearly demonstrate that the polarization is uniform across the whole cell wall.

Figure S5 .
Figure S5.The experimental and simulated spectra have a good match.The 120 to 50 ppm regions of a, wild-type sample (left) and b, ctl1 ctl2 double mutant (right) are shown.All numerical parameters used to obtain the fits are summarized in TablesS1 and S2.Color code follows peak classification in these tables: i4 cellulose in red, s4 in magenta, close peaks in dark yellow, and others in grey.

Figure S6 .
Figure S6.1D cross sections of DNP-enabled 2D CHHC spectrum.Representative slices were extracted from the 2 ms CHHC spectrum of unlabeled wild-type rice stem.The 13 C FWHM linewidths and signal-tonoise (S/N) ratios are shown for the major peaks.

Figure S7 .
Figure S7.2D PDSD spectra of 13 C-labeled rice stems.The spectra are collected using a, 3 ms and b, 5 ms mixing times.Assignments of cellulose and xylan peaks are annotated on the spectra.

Figure S8 .
Figure S8.NMR relaxation curves of polysaccharides in unlabeled rice stems.The a, 13 C-T1 and b, 1 H-T1ρ data are plotted separately for wild-type and ctl1 ctl2 samples.Cellulose signals (red) generally exhibit faster relaxation than xylan peaks (blue).The exceptions in panel a only occur to the 62 ppm s6/x5 peak, which has mixed contribution from both cellulose and matrix polymers.

Table S2 . Fit parameters of 13 C CP spectrum of ctl1 ctl2 double mutant.
The peaks are classified in three groups according to their influence on C4 region, as by integrating, interior and surface cellulose contribution is shown to respectively be 44.6% and 55.4%of the cellulose content.

Table S3 . Peak numbers of INADEQUATE spectra shown in Fig. 4. Table S4. 13 C-T1 and 1 H-T1 relaxation times of cellulose and xylan in WT and ctl1 ctl2 samples.
The data is fit using single exponential equation  () =  −/ , where T could be T1 or T1.Error bars are standard deviations of the fitting parameters.CS: 13 C chemical shift.Unidentified (-).